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nasdaq:bmrn
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BioMarin Pharmaceutical
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Dec 27th, 2005 12:00AM
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Nov 17th, 2000 12:00AM
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https://www.uspto.gov?id=US06979563-20051227
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Attenuation of tumor growth, metastasis and angiogenesis
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A highly purified and specific glycosaminoglycan degrading enzyme, chondroitinase AC, and to a lesser extent, chondroitinase B, can be used in the treatment of metastatic cancers and in other disorders characterized by angiogenesis. The enzymatic removal of chondroitin sulfates A and C, and to a lesser extent, chondroitin sulfate B, from cell surfaces directly decreases the ability of tumor cells to invade blood vessels and thus prevents the formation of metastatic, or secondary tumors; inhibits tumor cell growth; and decreases angiogenesis by inhibiting both endothelial cell proliferation and capillary formation. Decreasing the formation of new blood vessels into the tumor in turn decreases the potential for tumor growth, and further decreases the ability of tumor cells to invade the bloodstream. These effects are opposite to the prometastatic effects of tumor-secreted heparanase.
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6979563
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1. A method to decrease angiogenesis comprising administering to a site in an individual in need of treatment thereof for an established disorder requiring angiogenesis an effective amount of a purified chondroitinase enzyme to decrease angiogenesis at the site, wherein the decrease in angiogenesis is measured as a decrease in endothelial cell proliferation or a decrease in the formation of capillary-like structures.
2. The method of claim 1 wherein the enzyme is selected from the group consisting of chondroitinase AC from Flavobacterium heparinum, chondroitinase B from Flavobacterium heparinum, a chrondroitin sulfate degrading enzyme from Bacteroides species, a chrondroitin sulfate degrading enzyme from Proteus vulgaris, a chrondroitin sulfate degrading enzyme from Microcossus, a chrondroitin sulfate degrading enzyme from Vibrio species, a chrondroitin sulfate degrading enzyme from Arthrobacter aurescens, and combinations thereof wherein these enzymes are expressed from recombinant nucleotide sequences in bacteria.
3. The method of claim 1 wherein the enzyme is a mammalian enzyme.
4. The method of claim 1 wherein the enzyme is a chrondroitinase AC.
5. The method of claim 1 wherein the chondroitinase is chondroitinase AC.
6. The method of claim 1 wherein the enzyme is administered to an individual having cancer as evidenced by palpable tumors.
7. The method of claim 6 wherein the cancer is a solid tumor and the enzyme is chondroitinase AC.
8. The method of claim 1 wherein the individual has a disorder in which angiogenesis is involved, the disorder being selected from the group consisting of rheumatoid arthritis: psoriasis; ocular angiogenic disease, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; Crohn's disease, atherosclerosis, scleroderma, hypertrophic scarring, adhesions, cirrhosis of the liver, pulmonary fibrosis following acute respiratory distress syndrom or other pulmonary fibrosis of the newborn, endometriosis, polyposis, obesity, uterine fibroids, prostatic hypertrophy, and amyloidosis.
9. The method of claim 1 wherein the enzyme is administered systemically.
10. The method of claim 1 wherein the enzyme is administered locally at or adjacent a site in need of treatment.
11. The method of claim 1 wherein the enzyme is administered in a controlled and/or sustained release formulation.
12. The method of claim 7 wherein the chondroitinase is administered in a dosage in the range of 0.1 to 250 IU chondroitinase AC/tumor for tumors in the size range from 20 mm3 to 15 cm3.
13. The method of claim 1 wherein the enzyme is administered in combination with another active agent selected from the group consisting of antibiotics, cytokine cytotoxic agents, and anti-inflammatories.
14. The method of claim 7 wherein the enzyme is administered after excision of the tumor.
15. The method of claim 9 wherein the enzyme is administered by a route selected from the group consisting of intravenous, intra-cranial, and depo.
16. The method of claim 9 wherein the enzyme is administered using an infusion pump.
17. The method of claim 1 wherein the enzyme is chondroitinase B.
18. The method of claim 8 wherein the enzyme is chondroitinase B.
19. The method of claim 1 wherein the individual has a disorder in which angiogenesis is involved, the disorder being selected from the group consisting of disease of excessive or abnormal stimulation of endothelial cells, diseases that have angiogenesis as a pathologic consequence, and scarring following transplantation.
20. The method of claim 1 wherein the enzyme is administered topically.
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This claims priority to U.S. Ser. No. 60/165,957 filed Nov. 17, 1999, entitled “Attenuation of Tumor Growth, Metastasis and Angiogenesis” by Elizabeth M. Denholm, Yong-Qing Lin, and Paul J. Silver.
BACKGROUND OF THE INVENTION
The present invention is a method and formulations using chondroitinase AC and chondroitinase B, glycosaminoglycan degrading enzymes, to inhibit tumor cell growth, metastasis and angiogenesis, and thereby to treat or prevent certain cancers.
Proteoglycans on the cell surface and in the extracellular matrix contain variable glycosaminoglycan chains, which include heparan sulfate and chondroitin sulfates A, B, or C. While some proteoglycans contain only one type of glycosaminoglycan, others contain a mixture of heparan and chondroitin sulfates (Jackson et. al., Physiol. Rev. 71:481–530, 1991). Extracellular proteoglycans form a structural framework for cells and tissues, and together with cell-associated proteoglycans, have major functions in regulating cell adhesion, migration, and proliferation. Disruption of the normal synthesis and function of proteoglycans is thought to have an important role in tumor cell metastasis.
Tumor metastasis is the process by which malignant cells from a tumor spread throughout the body and develop into multiple secondary tumors (Lida et. al. Sem. Cancer Biol. 7:155–162, 1996; Meyer and Hart Eur. J. Cancer 34:214–221, 1998). In order to spread to other parts of the body, tumor cells must escape from the primary or original tumor, enter the blood stream or lymphatic system, and from there invade the tissue of other organs, where they multiply and form new tumors. Escape from the primary tumor and invasion into other organs is a complex multi-step process. Metastasis involves changes in tumor cell adhesion and motility, secretion of proteolytic enzymes, chemoattractants, and proteoglycans. In addition to these tumor cell activities, angiogenesis, or the formation of new blood vessels, is also a vital step in the metastatic process (Folkman Nature Medicine 1:27–31, 1995).
The involvement of different types of glycosaminoglycans in tumor cell metastasis has been investigated. Heparan sulfates on the cell surface appear to inhibit cell motility (Culp et. al. J. Cell Biol. 79:788–801, 1978). Heparan sulfates in the extracellular matrix act to impede cell movement through the formation of a tight network with other matrix components. Tumor cells can secrete a glycosaminoglycan-degrading enzyme, heparanase, which cleaves heparan sulfates and enhances escape from the tumor and promotes metastasis (Culp, et al. J. Cell Biol. 79:788–801, 1978; Nakajima et. al. Science 220:611–613, 198).
In contrast, chondroitin sulfates have never been linked to an enhancement of motility of both endothelial and tumor cells (Culp et. al. 1978). When formation of chondroitin sulfate proteoglycans is inhibited by treating cells with -xylosides, motility, migration and the ability to invade matrix material are inhibited (Henke et. al., J. Clin. Invest. 97:2541–2552, 1996; Faassen et. al., J. Cell Biol. 116:521–531, 1992 and Trochan et. al. Int. J. Cancer 66:664–668, 1996). Removal of chondroitin sulfates from the cell surface with chondroitinase ABC also decreases cell motility (Faassen et. al., 1992); however the effects of this enzyme on invasion or metastasis, or on angiogenesis are not known.
It is an object of the present invention to provide methods for treating or preventing tumor growth, metastasis or angiogenesis.
It is a further object of the present invention to provide formulations for treating or preventing tumor growth, metastasis or angiogenesis.
SUMMARY OF THE INVENTION
A highly purified and specific glycosaminoglycan degrading enzyme, chondroitinase AC, and to a lesser extent, chondroitinase B, can be used in the treatment of metastatic cancers. The enzymatic removal of chondroitin sulfates A and C, and to a lesser extent, chondroitin sulfate B, from tumor cell surfaces effectively A) decreases their ability to proliferate when stimulated by oncogenic growth factors, B) decreases the ability of tumor cells to invade blood vessels and thus prevents the formation of metastatic, or secondary tumors, and C) decreases angiogenesis by inhibiting both endothelial cell proliferation and capillary formation. Decreasing the formation of new blood vessels into the tumor in turn decreases the potential for tumor growth, and further decreases the ability of tumor cells to invade the bloodstream. These anti-metastatic effects of chondroitinases are opposite to the pro-metastatic effects of tumor secreted-heparanases.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are graphs of the release of sulfated glycosaminoglycans from human SK-MEL melanoma cells, following treatment with Flavobacterium heparinum derived Chondroitinase AC. FIG. 1A is the release of 35S-glycosaminoglycans after treatment with the indicated concentration (control., 0.1, 1 and 2 IU/ml) of enzyme for one hr. FIG. 1B is the release of 35S-glycosaminoglycans after treatment with 1.0 IU/ml of enzyme for the indicated time, zero, 5, 15 30 and 60 minutes. Data are the cpm/well of 35S-glycosaminoglycans released by enzyme treatment or by medium along (control), mean±sem of representative experiments performed in quadruplicate.
FIG. 2 is a graph of the dose-dependent effects (0, 1.0, and 10 IU/ml) of Flavobacterium heparinum derived Chondroitinase AC on the invasion of SK-MEL melanoma and HT-1080 fibrosarcoma cells into Matrigel™. Data are expressed as the number cells migrated through the filters and Matrigel™ and are the number of cells counted in ten 400× microscopic fields. Each bar represents the mean±sem of three experiments performed in duplicate.
FIG. 3 is a graph of the dose-dependent effects of Flavobacterium heparinum derived Chondroitinase AC (ChAC) (0, 0.1, 1.0, 5.0, and 10 IU/ml) on melanoma cell proliferation in response to 10% serum. Data are the mean number of cells/well, 48 hrs after treatment of SK-MEL cells with either ChAC or medium alone (control). Each bar represents the mean±sem of four experiments performed in triplicate.
FIG. 4 is a graph of the dose-dependent effects of Flavobacterium heparinum derived Chondroitinase AC on the proliferation of endothelial cells in response to 20 ng/ml of vascular endothelial growth factor. Data are the mean±sem of five experiments performed in quadruplicate.
FIG. 5 is a graph of the dose-dependent effects of Flavobacterium heparinum derived Chondroitinase AC on angiogenesis within Matrigel™. Data are the number of capillary-like structures (CLS) present per 100× field. Each bar represents the mean±sem of five experiments performed in duplicate.
FIG. 6 is a graph of comparison of the effects of Flavobacterium heparinum derived Chondroitinase AC, and Chondroitinase B, and the combination of Chondroitinase AC and B on tumor cell proliferation, tumor cell invasion, endothelial proliferation and angiogenesis. The effects of 1.0 IU/ml or 5.0 IU/ml (endothelial proliferation) of Chondroitinase AC and Chondroitinase B, on these cellular activities were determined as described in FIGS. 2 through 5. Data are expressed as the % Inhibition, determined by comparing the responses of untreated and chondroitinase treated cells. Each bar represents the mean±sem of five experiments for each activity.
FIG. 7 is a graph of the effects of Flavobacterium heparinum derived Chondroitinase AC (0.1 to 10 IU/ml) and Chondroitinase B (1.0 IU/ml) on melanoma and endothelial cell apoptosis. Data are expressed as % control, determined by comparing the activity of chondroitinase treated cells with that of untreated controls (100%). The apoptosis-inducer, Genistein (40 mg/ml) was used as a positive control. Each bar represents the mean±sem of five experiments performed in duplicate.
FIG. 8 is a graph of the effects of Flavobacterium heparinum derived Chondroitinase AC on tumor growth in vivo in mice. Mice were implanted subcutaneously with cells of a mouse Lewis lung carcinoma at Day 0. Animals were injected, directly into the tumor, on days 7, 8, 9, 11, and 13 with either 55 IU of chondroitinase AC or with a similar volume of saline. Animals were sacrificed and tumor size was measured on the indicated days. Data are shown as the tumor size in mm2, and are the mean±sem of 10 mice per group. The asterisks indicate a statistical difference between groups; * indicates p=0.035, and **indicates<.005.
DETAILED DESCRIPTION OF THE INVENTION
Events in the metastasis of, growth of, and angiogenesis within cancerous tumors can be inhibited by the use of one or more highly purified glycosaminoglycan degrading enzymes derived from various sources, but most preferably from Flavobacterium heparinum. Glycosaminoglycans, including chondroitin sulfates A, B or C, and heparan sulfate, are the sulfated polysaccharide components of proteoglycans located on cell surfaces, where they act as co-receptors in interactions between cell determinant proteins and extracellular matrix components such as hyaluronic acid and collagens; and in the extracellular space where they form the structure of the extracellular matrix and serve as a supporting and organizational structure of tissues and organs.
Chondroitin sulfates have been found to be associated with a cell adhesion molecule, CD44, which is important in tumor cell invasion. The biological activities of CD44 have been linked to the chondroitin sulfates on this protein (Faassen, et. al. J. Cell Science 105:501–511, 1993). Antibodies to CD44 inhibit formation of metastatic tumors in vivo (Zawadzki et. al. Int. J. Cancer 75:919–924, 1998), and inhibit endothelial cell migration and formation of capillary like structures in vitro (Henke et. al. 1996 and Trochan et. al. 1996).
The combination of data from studies on the effects of inhibiting chondroitin sulfates and from studies on the effects of anti-CD44 antibodies, all lead to the conclusion that chondroitin sulfates play a vital role in both tumor cell as well as endothelial cell growth and vessel formation (angiogenesis). This role for chondroitin sulfates in angiogenesis is relevant to its role in both sustained growth of tumors and tumor metastasis, since formation of new blood vessels is vital in supplying nutrients to a growing tumor and in providing a pathway by which invasive tumor cells travel to distant organs and form secondary tumors.
The Chondroitinase AC and chondroitinase B described in the examples are glycosaminoglycan degrading enzymes from Flavobacterium heparinum. These enzymes remove and degrade glycosaminoglycans from proteoglycans, and thereby modulate the interactions involved in tumor cell invasion and proliferation, as well as the processes involved in endothelial capillary formation and proliferation. Chondroitinase AC and chondroitinase B regulate tumor cell growth and metastasis by: i) cleaving chondroitin sulfate proteoglycans from cell surfaces; ii) reducing the invasive capacity of tumor cells by degrading chondroitin sulfate GAGs linked to CD44; iii) decreasing endothelial cell proliferation and capillary formation and thereby reducing the supply of nutrients to the tumor and reducing tumor cell access to the bloodstream; and iv) directly inhibiting growth factor-dependent proliferation of tumors.
Enzyme Formulations
Enzymes
Glycosaminoglycans are unbranched polysaccharides consisting of alternating hexosamine and hexuronic residues which carry sulfate groups in different positions. This class of molecules can be divided into three families according to the composition of the disaccharide backbone. These are: heparin/heparan sulfate [HexA-GlcNAc(SO4)]; chondroitin sulfate [HexA-GalNAc]; and keratan sulfate [Gal-GlcNAc].
Representative glycosaminoglycan degrading enzymes include heparinase 1 from Flavobacterium heparinum, heparinase 2 from Flavobacterium heparinum, heparinase 3 from Flavobacterium heparinum, chondroitinase AC from Flavobacterium heparinum, and chondroitinase B from Flavobacterium heparinum, heparinase from Bacteroides strains, heparinase from Flavobacterium Hp206, heparinase from Cytophagia species, chondroitin sulfate degrading enzymes from Bacteroides species, chondroitin sulfate degrading enzymes from Proteus vulgaris, chondroitin sulfate degrading enzymes from Microcossus, chondroitin sulfate degrading enzymes from Vibrio species, chondroitin sulfate degrading enzymes from Arthrobacter aurescens, these enzymes expressed from recombinant nucleotide sequences in bacteria and combinations thereof. Other enzymes which degrade glycosaminoglycans are present in mammalian cells and include heparanases, arylsulfatase B, N-acetylgalactosamine-6-sulfatase, and iduronate sulfatase.
The chondroitin sulfate family includes seven sub-types designated unsulfated chondroitin sulfate, oversulfated chondroitin sulfate and chondroitin sulfates A–E which vary in the number and position of their sulfate functional groups. Additionally, chondroitin sulfate B, also referred to as dermatan sulfate, differs in that iduronic acid is the predominant residue in the alternative hexuronic acid position.
Chondroitin sulfates A, B and C are the predominant forms found in mammals and may be involved in the modulation of various biological activities including cell differentiation, adhesion, enzymatic pathways and hormone interactions. The presence of chondroitin sulfate proteoglycans is elevated in the later stages of cell growth in response to tissue and vessel damage, as reported by Yeo, et al., Am. J. Pathol. 138:1437–1450, 1991, Richardson and Hatton, Exp. Mol. Pathol. 58:77–95, 1993 and Forrester, et al., J. Am. Coll. Cardiol. 17:758–769, 1991. Chondroitin sulfates also have been associated with events involved in the progression of vascular disease and lipoprotein uptake as described by Tabas, et al., J. Biol. Chem., 268(27):20419–20432, 1993.
Chondroitinases have been isolated from several bacterial species: Flavobacterium heparinum, Aeromonas sp., Proteus vulgaris, Aurebacterium sp. and Bacillus thetaiotamicron (Linhardt et. al., 1986; Linn et. al., J. Bacteriol. 156:859–866, 1983; Michelacci et. al., Biochim. Biophys. Acta. 923:291–201, 1987; and Sato et. al., Agric. Biol. Chem. 50:1057–1059, 1986). PCT/US95/08560 “Chondroitin Lyase Enzymes” by Ibex Technologies R and D, Inc., et al. describes methods for purification of naturally produced chondroitinases, especially separation of chondroitinase AC from chondroitinase B, as well as expression and purification of recombinant chondroitinases. Mammalian enzymes which degrade chondroitin sulfates include arylsulfatase B, N-acetylgalactosamine-6-sulfatase, and iduronate sulfatase.
Formulations
Pharmaceutical compositions are prepared using the glycosaminoglycan degrading enzyme as the active agent to inhibit tumor growth or angiogenesis based on the specific application. Application is either topical, localized, or systemic. Any of these formulations may also include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically or pharmaceutically effective agents which do not exert a detrimental effect on the glycosaminoglycan degrading enzyme or cells. For treatment of tumors, the composition may include a cytotoxic agent which selectively kills the faster replicating tumor cells, many of which are known and clinically in use.
For topical application, the glycosaminoglycan degrading enzyme is combined with a carrier so that an effective dosage is delivered, based on the desired activity, at the site of application. The topical composition can be applied to the skin for treatment of diseases such as psoriasis. The carrier may be in the form of an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick. A topical composition for treatment of eye disorders consists of an effective amount of glycosaminoglycan degrading enzyme in a ophthalmically acceptable excipient such as buffered saline, mineral oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol solutions, or liposomes or liposome-like products.
Compositions for local or systemic administration, for example, into a tumor, will generally include an inert diluent. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
For directed internal topical applications, for example for treatment of solid tumors, resection sites, or hemorrhoids, the composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
The glycosaminoglycan degrading enzyme can also be administered in combination with a biocompatible polymeric implant which releases the glycosaminoglycan degrading enzyme over a controlled period of time at a selected site. Examples of preferred biodegradable polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polyesters such as polylactic acid, polyglycolic acid, polyethylene vinyl acetate, and copolymers and blends thereof. Examples of preferred non-biodegradable polymeric materials include ethylene vinyl acetate copolymers.
Other Therapeutic Agents which can be Administered in Combination
The glycosaminoglycan degrading enzymes can be administered alone or in combination with other treatments. For example, the enzymes can be administered with antibiotics, cytokines, and anti-inflammatories such as cortisone, and/or other types of angiogenic inhibitors. Other combinations will be apparent to those skilled in the art. In some embodiments, the enzymes are administered with a barrier, such as methylcellulose or other polymeric material, either topically at the time of surgery or incorporated into the barrier, which is inserted at the time of surgery.
Methods of Treatment
Disorders
A variety of disorders to be treated. In the principal embodiment, the glycosaminoglycan degrading enzymes chondroitinase AC and chondroitinase B are used to inhibit formation, growth and/or metastasis of tumors, especially solid tumors. Examples of tumors including carcinomas, adenocarcinomas, lympohomas, sarcomas, and other solid tumors, as described in U.S. Pat. No. 5,945,403 to Folkman, et al., solid tumors; blood born tumors such as leukemias; tumor metastasis; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas. Other disorders involving angiogenesis including rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; disease of excessive or abnormal stimulation of endothelial cells, including intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids, and diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori), can also be treated. Angiogenic inhibitors can be used to prevent or inhibit adhesions, especially intra-peritoneal or pelvic adhesions such as those resulting after open or laproscopic surgery, and burn contractions. Other conditions which should be beneficially treated using the angiogenesis inhibitors include prevention of scarring following transplantation, cirrhosis of the liver, pulmonary fibrosis following acute respiratory distress syndrome or other pulmonary fibrosis of the newborn, implantation of temporary prosthetics, and adhesions after surgery between the brain and the dura. Endometriosis, polyposis, cardiac hypertrophyy, as well as obesity, may also be treated by inhibition of angiogenesis. These disorders may involve increases in size or growth of other types of normal tissue, such as uterine fibroids, prostatic hypertrophy, and amyloidosis.
Angiogenesis, the proliferation and migration of endothelial cells that result in the formation of new blood vessels, is an essential event in a wide variety of normal and pathological processes. For example, angiogenesis plays a critical role in embryogenesis, wound healing, psoriasis, diabetic retinopathy, and tumor formation, as reported by Folkman, J. Angiogenesis and its inhibitors. In: V. T. DeVita, S. Hellman and S. A. Rosenberg (eds.). Important Advances in Oncology, pp. 42–62, (J. B. Lippincott Co., Philadelphia, 1985); Brem, H., et al., Brain tumor angiogenesis. In: P. L. Komblith and M. D. Walker (eds.), Advances in Neuro-Oncology, pp. 89–101. (Future Publishing Co., Mount Kisco, N.Y. 1988); Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med., 285; 1182–1186 (1971); and Folkman, J. Successful treatment of an angiogenic disease. N. Engl. J. Med., 320: 1211–1212 (1989).
Identification of several agents that inhibit tumor angiogenesis has provided a conceptual framework for the understanding of angiogenesis in general. The inhibition of angiogenesis by certain steroids and heparin derivatives, reported by Folkman, J., et al., Science 221: 719 (1983); and Murray, J. B., et al., J. Biol. Chem., 261: 4154–4159 (1986); led to studies elucidating the crucial role of remodeling of the extracellular matrix in angiogenesis. These agents apparently prevent angiogenesis by specifically disrupting the deposition and cross-linking of collagen, as reported by Ingber, D., and Folkman, J. Lab. Invest., 59: 44–51 (1989).
Other studies on inhibition of angiogenesis have highlighted the importance of enzyme mediated remodeling of the extracellular matrix in capillary growth and proliferation (Folkman, J., et al., Science 221: 719–725 (1983); Ingber, D., et al. Lab. Invest. 59: 44–51 (1989); Folkman, J., et al., Science 243: 1490–1493 (1989); Krum, R., et al., Science 230: 1375–1378 (1985); Ingber, D., et al., Endocrinol. 119: 1768–1775 (1986); and Ingber, D., et al., J. Cell. Biol. 109: 317–330 (1989)).
Methods of Administration
The composition can be administered systemically using any of several routes, including intravenous, intra-cranial, subcutaneous, orally, or by means of a depot. The composition can be administered by means of an infusion pump, for example, of the type used for delivering insulin or chemotherapy to specific organs or tumors, or by injection.
Chondroitinase AC and chondroitinase B can be injected using a syringe or catheter directly into a tumor or at the site of a primary tumor prior to or after excision; or systemically following excision of the primary tumor.
The enzyme formulations are administered topically or locally as needed. For prolonged local administration, the enzymes may be administered in a controlled release implant injected at the site of a tumor. For topical treatment of a skin condition, the enzyme formulation may be administered to the skin in an ointment or gel.
Effective Dosage
An effective dosage can be determined by the amount of enzyme activity units (IU) per tumor. An expected effective dosage range includes 0.1 to 250 IU/tumor for expected tumor sizes ranging from 20 mm3 to 15 cm3.
The present invention will be further understood by reference to the following non-limiting examples.
EXAMPLE 1
Enzyme Substrate Specificity
Chondroitinase B (no EC number) and chondroitinase AC (EC 4.2.2.5) are native enzymes of Flavobacterium heparinum and can also be recombinantly expressed in this same bacterium (Gu et. al., Biochem. J. 312:569–577 (1995)). Specific activity and substrate specificity were determined for each enzyme, using a kinetic spectrophotometric assay, performed essentially as described by Gu et al. (1995). In these assays, enzyme concentrations were 0.25 IU/ml and substrate concentrations were 0.5 mg/ml (chondroitin sulfate B and chondroitin sulfate AC) or 0.75 mg/ml (heparan sulfate). The specific activities of the enzymes were: 97 IU/mg for Chondroitinase B and 221 IU/mg for chondroitinase AC.
The substrate specificity of ultra-purified Chondroitinase B and AC were determined by testing the ability of the enzymes to degrade chondroitin sulfate B, chondroitin sulfate A, chondroitin sulfate C, and heparan sulfate. As shown in Table 1, both enzymes were active towards the corresponding sulfated glycosaminoglycan, with 0.2% or less activity against any of the other glycosaminoglycans. These results confirm the substrate specificity of the purified Chondroitinase B and Chondroitinase AC used in this application.
TABLE 1
Comparative Enzymatic Activities Against Glycosaminoglycans
Substrate
Enzyme
CSB
CSA
CSC
HS
Chondroitinase B
IU/ml
399
0.04
0.03
0.92
(relative activity)
(100)
(0.01)
(0.01)
(0.230
Chondroitinase AC
IU/ml
0.604
1238
735
2.2
(relative activity)
(0.05)
(100)
(59)
(0.18)
Enzyme activities are shown as IU/ml with each substrate, and as the relative activity towards each substrate. Relative activity was determined after assigning 100% for the preferred substrate (CSB for chondroitinase B, CSA for chondroitinase AC. CSB=chondroitin sulfate B; CSA=chondroitin sulfate A; CSC=chondroitin sulfate C; HS=heparan sulfate). Substrate concentrations were 500 mg/ml (CSB, CSA, CSC) or 750 g/ml (HS).
EXAMPLE 2
Removal of Glycosaminoglycans from Cells
The effectiveness of the chondroitinase AC in removing sulfated glycosaminoglycans from cells was examined using cells with glycosaminoglycans labeled by incubation with Na35SO4 (Dupont, NEN). Human melanoma cells (SK-MEL) were plated at a density of 6×104 cells/well in 24 well plates, in MEM with 10% serum and 25 mCi/ml of Na235SO4, and incubation continued for 2.5 days. The medium was removed and cells rinsed 2× with MEM then treated with Chondroitinase AC as indicated. Medium was removed and radioactivity determined. The release of sulfated glycosaminoglycans from cells by enzyme was expressed as cpm/well.
Cells were exposed to 0.1, 1.0 or 2.0 IU/ml of Chondroitinase AC, at 37 C. for 1 hour. As shown in FIG. 1A, maximal release of sulfated GAGs by chondroitinase AC was achieved with 1.0 IU/ml of enzyme. Further experiments were done in which SK cells were treated with 1.0 IU/ml of chondroitinase AC for 5 to 60 minutes. FIG. 1B illustrates that the release of sulfated glycosaminoglycans from SK cells was also dependent on the length of time that they were exposed to chondroitinase AC.
Other experiments were done to identify the radiolabelled glycosaminoglycans released into the medium after chondroitinase AC treatment of cells. Cells were treated with 1.0 IU/ml of chondroitinase AC for 1 hour at 37° C., after which glycosaminoglycans in the medium were precipitated with Cetavalon (Aldrich Chemicals, St. Louis, Mo.) and analyzed with agarose gel electrophoresis (Volpi, Carbohydrate Res. 247:263–278, 1993). The 35S-glycosaminoglycans released in to the medium were identified as disaccharide fragments of chondroitin sulfate based on the distance migrated into the agarose gels. As measured from the wells, migration distances into the gels for glycosaminoglycan standards were: 25 mm for heparan sulfate, 31 mm for dermatan sulfate, 37 mm for chondroitin sulfate, and 10 mm for fragments of chondroitin sulfate prepared by digestion with chondroitinase AC. The 35S-glycosaminoglycans released from cells migrated 10 mm into the gels.
EXAMPLE 3
Effects on Tumor Cell Invasion
The effects of Chondroitinase AC on tumor cell invasion were assessed in an in vitro assay. Two human cell lines were used: SK-MEL-2, a melanoma and HT-1080, a fibrosarcoma, both obtained from the ATCC in Manassas, Va. Each cell line was grown to a density of approximately 4×105 cells/well, in MEM with 10% serum. Cells were rinsed with PBS, then treated with the indicated concentration of Chondroitinase AC in serum free medium for one hour at 37 C. Following enzyme treatment, cells were rinsed with serum free medium, removed from dishes by trypsinization and resuspended in medium containing 1% serum containing the indicated concentration of chondroitinase AC.
The invasion assay was performed in 8 mm pore polycarbonate filter cell culture inserts (Falcon, Franklin Lakes, N.J.). Insert filters were pre-coated with 25 μg of Matrigel (Collaborative Biochemicals, Cambridge, Mass.) in serum free medium. Coated filters were dried overnight and equilibrated with serum free medium for 1 hr prior to use. Fifty thousand tumor cells in medium with 1% BSA were placed on top of the filters, and fibroblast conditioned medium (prepared as described by Jin-inchi et. al., Cancer Res. 50:6731–6737, 1990) was placed below the filter as a chemoattractant. Invasion assays were incubated for 16 hrs. at 37° C., after which cells remaining on the top of the filters were removed. Filters were then stained using the Diff-Quik™ staining set (Baxter, Miami, Fla.). Invasion was assessed as the number of cells which migrated through matrix material (Matrigel™), to the underside of the filters. For each filter, 10 fields were counted at 400×. All samples were run in duplicate. Controls consisted of cells treated with medium alone.
Invasion of the melanoma cells (SK-MEL) was inhibited by 32% and 38% following treatment of cells with 1.0 and 10.0 IU/ml of chondroitinase AC, as shown in FIG. 2. Invasion of fibrosarcoma cells (HT-1080) was also inhibited by chondroitinase AC. Chondroitinase AC at concentrations of 1.0 and 10 IU/ml inhibited fibrosarcoma cell invasion by 27% and 40%, respectively, as shown in FIG. 2.
EXAMPLE 4
Effects on Tumor Cell Proliferation
Human melanoma cells (SK-MEL) were obtained from the ATCC, Manassas, Va. Cells were cultured in MEM containing 1% antibiotics and 10% serum. The proliferation assay was performed as described by Denholm and Phan, Am J Pathol. 134(2):355–63 (1989). Briefly, cells were plated in MEM with 10% serum; 24 hrs later medium was replaced with serum free medium, and incubation continued for an additional 24 hrs. Cells were then treated with either serum free MEM alone, or MEM containing 0.1 to 10 IU/ml of chondroitinase AC for 1 hour at 37° C. Following enzyme treatment, cells were rinsed 1× with MEM, then given MEM with 10% serum and incubated for 48 hrs. Controls for each experiment were: (negative) untreated cells incubated in serum free medium, and (positive) untreated cells incubated in MEM with 10% serum. The number of cells per well was quantified using the CyQuant™ assay method from Molecular Probes, Eugene, Oreg. Fluorescence/well was determined using a CytoFluor™ Series 4000 fluorescent plate reader (PerSeptive Biosystems) and cell numbers calculated from a standard curve. Experiments were performed to determine if treatment of SK-MEL melanoma cells with chondroitinase AC would have an effect on proliferation of these cells. Melanoma cell proliferation in response to 10% serum was inhibited by 45% with 10 IU/ml of chondroitinase AC, as shown by FIG. 3.
EXAMPLE 5
Effects on Endothelial Cell Proliferation
Endothelial cell proliferation assays were conducted essentially as those described in Example 4 for tumor cells, except that endothelial cells were plated at 1.5×104 cells/ml in MEM containing 10% serum. On Day 3 cells were treated with 0, 1 to 10 IU/ml of chondroitinase AC for 1 hr then rinsed with serum free medium and given fresh medium containing 20 ng/ml of VEGF. The number of cells/well was quantified 48 hrs later using the CyQuant™ assay as described in example 4. Chondroitinase AC treatment inhibited endothelial cell proliferation (FIG. 4) in a dose dependent manner. Endothelial cell proliferation was inhibited by 11 to 55% following treatment with 1.0 to 10 IU/ml of chondroitinase AC, respectively.
EXAMPLE 6
Effects on Angiogenesis
The effects of chondroitinase AC on angiogenesis were assessed in an in vitro system. Human endothelial cells (ATCC, Manassas, Va.) were grown in MEM with 10% serum. Cells were washed with PBS then treated with the indicated concentration of chondroitinase AC for 1 hr at 37 C. Following enzyme treatment, cells were washed, removed from dishes with trypsin, and resuspended in serum free medium to a concentration of 4×105 cells/ml. This endothelial cell suspension was mixed in a ratio of 1:1 with 2 mg/ml type I collagen (rat tail, Collaborative Biochemical Products), or in a ratio of 2:1 with 19 mg/ml growth factor-reduced Matrigel™. Ten ml of this cell suspension was added to the center of each well of a 48 well culture dish, and incubated for 30 mins at 37 C. Following formation, medium containing 2 mg/ml BSA and 20 ng/ml of VEGF (Peprotech, Rocky Hill, NJ) was added, with the indicated concentration of chondroitinase AC. Angiogenesis was assessed as the formation of Capillary-like Structures (CLS) after incubation for 3 days (collagen) or 6 days (Matrigel). To visualize and quantify the CLS, endothelial cells were labeled with 1 mM calcein AM (Molecular Probes Inc, Portland, Oreg.) for 30 mins. CLS were quantified by counting the number of CLS in 3, 100× fields.
Chondroitinase AC inhibited angiogenesis in a dose-dependent manner. Angiogenesis was inhibited by 46 and 72% following treatment with 1.0 and 10 IU/ml of chondroitinase AC, respectively (FIG. 5).
EXAMPLE 7
Effects on Multiple Cellular Activities
The effects of chondroitinase AC, chondroitinase B and the combination of chondroitinase AC and B, on endothelial and tumor cell activities were compared. Melanoma or endothelial cells were treated with either medium alone (controls), 1.0 IU/ml or 5.0 IU/ml of one or both of the chondroitinase enzymes for one hour at 37° C. The cellular activities examined were tumor cell proliferation, tumor cell invasion, endothelial proliferation and angiogenesis, which were assayed as described in the previous examples.
Each enzyme had significant inhibitory effects on all the activities assayed, when compared to untreated controls as shown by FIG. 6. For each activity assayed, chondroitinase AC was more effective than chondroitinase B. However, this difference was significant only in regards to tumor cell proliferation. Further more, treating cells with chondroitinase AC alone was as effective in inhibiting cellular activities, as was a combination of chondroitinase AC and chondroitinase B, as shown by FIG. 7.
EXAMPLE 8
Effects on Apoptosis
The effects of Chondroitinase AC on tumor cell and endothelial cell apoptosis were assessed. This was done to determine if the induction of apoptosis by Chondroitinase AC might be the mechanism by which Chondroitinase AC inhibits the multiple cellular activities in Example 7.
Melanoma or endothelial cells were treated with either medium alone (negative controls), 0.10 IU/ml to 10.0 IS/ml of Chondroitinase AC, or 1.0 IU/ml of Chondroitinase B, 48 hrs at 37° C. As a positive control, cells were incubated in parallel, with 40 μg/ml of Genistein, a known inducer of apoptosis. At the end of the incubation period, cells were lysed and assayed for caspase-3 activity, as a marker of apoptosis. Caspasc-3 assays were done using an assay kit from BioSource International.
Compared to untreated controls (100%) apoptosis was increased in both melanoma and endothelial cells (FIG. 7). Apoptosis (caspase-3 activity), was increased over that of controls by 64% in endothelial cells, and 150% in melanoma cells, following treatment with Chondroitinase AC. In comparison, Chondroitinase B did not significantly increase caspase-3 activity in melanoma cells, but did increase activity in endothelial cells 60% higher than that of controls. Genistein increased caspase activity of endothelial cells to 89% higher than controls, and of melanoma cells by 169% over controls.
EXAMPLE 9
Effects on Tumor Growth
The effects of Chondroitinase AC on tumor growth were assessed in mice. Mice (C57BL strain) weighed 20 to 25 g. Tumor cells were H-59, a sub-line of mouse Lewis lung carcinoma cells, as described by Brodt, Cancer Res. 46:2442–2448, 1986. Tumors were induced in mice, by the subcutaneous injection of 2×105 cells on day zero. Mice were palpitated daily for the appearance of tumors at the site of injection. Once tumors were palpable, mice were divided into two groups of 10 mice. Intra-tumor injections of either sterile saline (controls) or 55 IU of Chondroitinase AC (Treated) in saline, were done on Days 7,8,9, 11 and 13. Tumors were measured daily using calipers. In accordance with the animal protocol and regulations governing the use of animals in research, mice had to be sacrificed once tumor size reached 150 mm2. For this reason, mice in the control group were all terminated on Day 18.
Tumor growth in mice treated with Chondroitinase AC was significantly reduced, when compared to saline-treated controls (FIG. 8). Comparison of the mean tumor size in the two groups, showed that tumors in Chondroitinase AC treated mice were smaller than those in the controls at all times. In addition, there was no further growth of the tumors in Chondroitinase AC-treated animals between Day 18 and 24, at which time the experiment was terminated.
Modifications and variations of the methods and compositions described herein are intended to be encompassed by the following claims. The teachings of the foregoing references cited herein are specifically incorporated by reference.
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09715965
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biomarin enzymes, inc.
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USA
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B1
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Utility Patent Grant (no pre-grant publication) issued on or after January 2, 2001.
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Open
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435/183
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Apr 2nd, 2019 12:00AM
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Sep 21st, 2015 12:00AM
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https://www.uspto.gov?id=US10246707-20190402
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Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means
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The invention relates to a nucleic acid molecule that binds and/or is complementary to the nucleotide molecule having sequence 5′-GUGGCUAACAGAAGCU (SEQ ID NO 1) and to its use in a method for inducing skipping of exon 44 of the DMD gene in a DMD patient.
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10246707
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1. A single stranded antisense oligonucleotide 16 to 25 nucleotides in length that comprises a base sequence of the sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 16, SEQ ID NO: 20 to SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 34, SEQ ID NO: 41, and SEQ ID NO: 46, wherein the antisense oligonucleotide induces skipping of exon 44 of human dystrophin pre-mRNA, and comprises a modification.
2. The antisense oligonucleotide according to claim 1, wherein said antisense oligonucleotide comprises the base sequence of the sequence SEQ ID NO: 5.
3. The antisense oligonucleotide according to claim 1, comprising a 2′-O-alkyl phosphorothioate antisense oligonucleotide.
4. The antisense oligonucleotide according to claim 3, wherein said antisense oligonucleotide comprises a 2′-O-methyl phosphorothioate ribose.
5. A viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of an antisense oligonucleotide according to claim 1.
6. The antisense oligonucleotide of claim 1, wherein said modification comprises a modified backbone.
7. The antisense oligonucleotide according to claim 6, wherein the modified backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.
8. The antisense oligonucleotide according to claim 1, wherein said modification is selected from the group consisting of: phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid, and locked nucleic acid.
9. The antisense oligonucleotide according to claim 2, wherein the antisense oligonucleotide is a PMO.
10. The oligonucleotide of claim 1, wherein said oligonucleotide is conjugated to a ligand.
11. The oligonucleotide of claim 10, wherein said ligand is a peptide.
12. The antisense oligonucleotide according to claim 1, wherein said modification comprises a locked nucleic acid (LNA).
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12
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CROSS REFERENCE
This application is a continuation of U.S. application Ser. No. 12/992,218 filed Nov. 11, 2010, which is a continuation of PCT application No. PCT/NL2009/050258 filed May 14, 2009, which claims priority to European application No. EP 08156193.8 filed May 14, 2008, and to U.S. provisional application No. 61/128,010 filed May 15, 2008, the contents of each of which is incorporated by reference in their entirety.
The present specification is being filed with a Sequence Listing in Computer Readable Form (CFR), which is entitled 11808-325-999_SEQLIST.txt of 41,702 bytes in size and was created Oct. 8, 2018; the content of which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to the modulation of splicing of the human Duchenne Muscular Dystrophy gene.
BACKGROUND
Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the dystrophin gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
Several possible treatments have been investigated over the last 20 years, including myoblast-transplantation, DNA-targeted gene therapy, and antisense-mediated exon skipping (van Deutekom and van Ommen, (2003), Nat. Rev. Genet., 4(10):774-83). Antisense-mediated exon skipping aims at transforming out-of-frame mutations present in DMD patients into in-frame BMD-like mutations that result in synthesis of an at least partially functional dystrophin, which will prolong the viability of the muscles (Aartsma-Rus and van Ommen, (2007), RNA, 13(10): 1609-24).
Exon skipping can be induced by antisense oligonucleotides (AON) directed against the splice donor or splice acceptor site of a splice junction that are involved in the enzymatic process of exon joining, or against exon-internal sequences. In general, splice donor and splice acceptor sites comprise conserved sequences and targeting these sequences has the inevitable risk of co-targeting splice sites of additional exons from DMD or other gene transcripts.
Exon 44 of the DMD gene consists of 148 base pairs. Therapeutic skipping of exon 44 would restore the correct reading frame in DMD patients having deletions including but not limited to exons 03-43, 05-43, 06-43, 10-43, 13-43, 14-43, 17-43, 19-43, 28-43, 30-43, 31-43, 33-43, 34-43, 35-43, 36-43, 37-43, 38-43, 40-43, 41-43, 42-43, 43, 45, 45-54, and 45-68, or having a duplication of exon 44. Furthermore, for some DMD patients the mutations are such that the simultaneous skipping of one or more exons is required in addition to exon 44 skipping to restore the reading frame. Non-limiting examples of such mutations are nonsense point mutations in the flanking exons 43 or 45, requiring exon 43+44 skipping or exon 44+45 skipping respectively. The aforementioned mutations in total occur in about 6-8% of all DMD patients. The majority of resulting dystrophin proteins will be truncated in the central rod domain of the protein, leaving the essential N-terminal actin-binding domain and the C-terminal domain binding to dystrobrevin and syntrophin, and the β-dystroglycan-binding C-terminal cysteine-rich domain, intact.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention identifies four different regions in exon 44 that are particularly suited for inducing skipping of exon 44. The invention thus provides a method for modulating splicing of exon 44 of the DMD gene in a cell, the method comprising providing said cell with a molecule that binds to a nucleotide sequence comprising SEQ ID NO. 1: 5′-GUGGCUAACAGAAGCU; SEQ ID NO. 2: 5′-GGGAACAUGCUAAAUAC, SEQ ID NO. 3: 5′-AGACACAAAUUCCUGAGA, or SEQ ID NO. 4: 5′-CUGUUGAGAAA. This molecule preferably binds or is complementary to any of SEQ ID NO: 1, 2, 3, or 4 when SEQ ID NO:1, 2, 3, or 4 is present within exon 44 of the DMD pre-mRNA.
Throughout the application, the expression “inducing skipping” is synonymous of “modulating splicing”.
It was found that a molecule that binds to a nucleotide sequence comprising SEQ ID NO. 1: 5′-GUGGCUAACAGAAGCU; SEQ ID NO. 2: 5′-GGGAACAUGCUAAAUAC, SEQ ID NO. 3: 5′-AGACACAAAUUCCUGAGA, or SEQ ID NO. 4: 5′-CUGUUGAGAAA results in highly efficient skipping of exon 44 in cells provided with this molecule. Furthermore, none of the indicated sequences is derived from conserved parts of splice junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes. In addition, other (immuno)toxicity is preferably avoided by avoiding CpG pairs in the molecule that binds to a nucleotide sequence as defined herein above.
Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mRNA, with a molecule capable of interfering with sequences such as, for example, the splice donor or splice acceptor sequence required for allowing the enzymatic process of splicing, or that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included into the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
Certain methods of the invention will alleviate one or more characteristics of a myogenic cell or muscle cell of a DMD patient having deletions including, but not limited to, exons 03-43, 05-43, 06-43, 10-43, 13-43, 14-43, 17-43, 19-43, 28-43, 30-43, 31-43, 33-43, 34-43, 35-43, 36-43, 37-43, 38-43, 40-43, 41-43, 42-43, 43, 45, 45-54, and 45-68, or having a duplication of exon 44. Furthermore, the removal of a flanking exon, such as, for example, exon 43 or exon 45, because of a nonsense point mutation in the flanking exon, will result in an out of frame transcript. The additional skipping of exon 44, in combination with skipping of the flanking exon, will restore the reading frame of the DMD gene in myogenic cells or muscle cells of DMD patients. Non-limiting examples of such mutations are nonsense point mutations in the flanking exons 43 or 45, requiring exon 43+44 skipping or exon 44+45 skipping respectively.
In an embodiment, a method of the invention may also alleviate one or more characteristics of a myogenic cell or muscle cell of a strong BMD patient, to the characteristics of a mild BMD patient. The characteristics of a cell of a DMD or BMD patient include increased calcium uptake by muscle cells, increased collagen synthesis, altered morphology, altered lipid biosynthesis, increased oxidative stress, and/or damaged sarcolemma. Preferred embodiments of a method of the invention are later defined herein.
In one embodiment, a molecule as defined herein can be a compound molecule that binds and/or is complementary to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the indicated nucleotide sequence on a RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein incorporated by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein incorporated by reference. Binding to one of the specified SEQ ID NO: 1, 2, 3, or 4 sequence, preferably in the context of exon 44 of DMD may be assessed via techniques known to the skilled person. A preferred technique is gel mobility shift assay as described in EP 1 619 249. In a preferred embodiment, a molecule is said to bind to one of the specified sequences as soon as a binding of said molecule to a labelled sequence SEQ ID NO: 1, 2, 3 or 4 is detectable in a gel mobility shift assay. Alternatively or in combination with previous embodiment, a molecule is an oligonucleotide which is complementary or substantially complementary to SEQ ID NO:1, 2, 3, or 4 or part thereof as later defined herein. The term “substantially” complementary used in this context indicates that one or two or more mismatches may be allowed as long as the functionality, i.e. inducing skipping of exon 44, is still acceptable.
The invention provides a method for designing a molecule, preferably an oligonucleotide able to induce the skipping of exon 44 of the DMD gene. First said oligonucleotide is selected to bind to one of SEQ ID NO: 1, 2, 3, or 4 or parts thereof as earlier defined herein. Subsequently, in a preferred method at least one of the following aspects has to be taken into account for designing, improving said molecule any further:
The molecule does not contain a CpG,
The molecule does not contain a G-quartet motif,
The molecule has acceptable RNA binding kinetics and/or thermodynamic properties.
The presence of a CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (Dorn and Kippenberger, Curr Opin Mol Ther 2008 10(1) 10-20). This increased immunogenicity is undesired since it may induce the breakdown of muscle fibers. Immunogenicity may be assessed in an animal model by assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration in muscle biopsy of said animal. Immunogenicity may also be assessed in blood of an animal or of a human being treated with an oligonucleotide of the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said oligonucleotide using a standard immunoassay known to the skilled person. An increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said oligonucleotide using a standard immunoassay.
An oligonucleotide comprising a G-quartet motif has the tendency to form a quadruplex, a multimer or aggregate formed by the Hoogsteen base-pairing of four single-stranded oligonucleotides (Cheng and Van Dyke, Gene. 1997 Sep. 15; 197(1-2):253-60), which is of course not desired: as a result the efficiency of the oligonucleotide is expected to be decreased. Multimerisation or aggregation is preferably assessed by standard polyacrylamide non-denaturing gel electrophoresis techniques known to the skilled person. In a preferred embodiment, less than 20% or 15%, 10%, 7%, 5% or less of a total amount of an oligonucleotide of the invention has the capacity to multimerise or aggregate assessed using the assay mentioned above.
The invention allows designing an oligonucleotide with acceptable RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (www.unc.edu/˜cail/biotool/oligo/index.html) for single stranded RNA using the basic Tm and the nearest neighbour model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). If a Tm is too high, the oligonucleotide is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 65° C. and an acceptable free energy may be ranged between 15 and 45 kcal/mol.
The skilled person may therefore first choose an oligonucleotide as a potential therapeutic compound as binding and/or being complementary to SEQ ID NO:1, 2, 3, or 4 of exon 44 or parts thereof as defined herein. The skilled person may check that said oligonucleotide is able to bind to said sequences as earlier defined herein. Optionally in a second step, he may use the invention to further optimise said oligonucleotide by checking for the absence of CpG, the absence of a G-quartet motif, and/or by optimizing its Tm and/or free energy of the AON-target complex. He may try to design an oligonucleotide wherein no CpG and/or no G-quartet motif are present and/or wherein a more acceptable Tm and/or free energy are obtained by choosing a distinct sequence of exon 44 (for example SEQ ID NO:1, 2, 3, or 4) to which the oligonucleotide is complementary. Alternatively, if an oligonucleotide complementary to a given stretch within SEQ ID NO:1, 2, 3 or 4 of exon 44, comprises a CpG, a G-quartet motif and/or does not have an acceptable Tm and/or free energy, the skilled person may improve any of these parameters by decreasing the length of the oligonucleotide, and/or by choosing a distinct stretch within any of SEQ ID NO: 1, 2, 3, or 4 to which the oligonucleotide is complementary and/or by altering the chemistry of the oligonucleotide.
As an example, if one chooses SEQ ID NO:1, several oligonucleotides were designed which were found to bind this sequence: SEQ ID NO: 5, 41, and 46. The oligonucleotide comprising SEQ ID NO:5 was found to have the most optimal RNA binding kinetics and/or thermodynamic properties, such as the most optimal Tm. When we tested the functionality of these oligonucleotides to induce the skipping of exon 44, it was confirmed that an oligonucleotide comprising SEQ ID NO:5 is the most efficient of these four oligonucleotides. Each of these oligonucleotides is functional in the sense of the invention. However, an oligonucleotide comprising SEQ ID NO:5 is the most preferred oligonucleotide identified that binds and/or is complementary to SEQ ID NO:1.
At any step of the method, an oligonucleotide of the invention is preferably an olignucleotide, which is still able to exhibit an acceptable level of a functional activity. A functional activity of said oligonucleotide is preferably to induce the skipping of exon 44 of the DMD gene to a certain extent, to provide an individual with a functional dystrophin protein and/or mRNA and/or at least in part decreasing the production of an aberrant dystrophin protein and/or mRNA. Each of these features is later defined herein. Such functional activity may be measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell. The assessment of the functionality may be carried out at the mRNA level, preferably using RT-PCR. The assessment of the functionality may be carried out at the protein level, preferably using western blot analysis or immunofluorescence analysis of cross-sections. In a preferred embodiment, an oligonucleotide is said to induce skipping of exon 44 of a DMD gene, when tested in a muscle cell of a DMD patient, by RT-PCR, the exon 44 skipping percentage is of at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%.
In a preferred embodiment, such oligonucleotide is preferably a medicament. More preferably, said medicament is for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or a patient. As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. A patient is preferably intended to mean a patient having DMD or BMD or a patient susceptible to develop DMD or BMD due to his or her genetic background.
In the case of a DMD patient, an oligonucleotide used will preferably correct at least one of the DMD mutations as present in the DMD gene of said patient and therefore will preferably create a dystrophin that will look like a BMD dystrophin: said dystrophin will preferably be a functional dystrophin as later defined herein.
In the case of a BMD patient, an oligonucleotide as used will preferably correct at least one of the BMD mutations as present in the DMD gene of said patient and therefore will preferably create a, or more of a, dystrophin, which will be more functional than the dystrophin which was originally present in said BMD patient. Even more preferably, said medicament increases the production of a functional or more functional dystrophin protein and/or mRNA and/or at least in part decreases the production of an aberrant or less functional dystrophin protein and/or mRNA in an individual.
Preferably, a method of the invention increases production of a more functional dystrophin protein and/or mRNA and/or decreases the production of an aberrant or less functional dystrophin protein and/or mRNA in a patient, by inducing and/or promoting skipping of at least exon 44 of the DMD pre-mRNA as identified herein in one or more cells, preferably muscle cells of said patient. Increasing the production of a more functional dystrophin protein and/or mRNA and/or decreasing the production of an aberrant dystrophin protein and/or mRNA in a patient is typically applied in a DMD patient. Increasing the production of a more functional or functional dystrophin and/or mRNA is typically applied in a BMD patient.
Therefore a preferred method is a method, wherein in a patient or in one or more cells of said patient, production of a more functional or functional dystrophin protein and/or mRNA is increased and/or the production of an aberrant dystrophin protein and/or mRNA in said patient is decreased, wherein the level of said aberrant or more functional dystrophin protein and/or mRNA is assessed by comparison to the level of said dystrophin and/or mRNA in said patient at the onset of the method.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 47. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO: 47. In another embodiment, a functional dystrophin is a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a wild type dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC)(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144.). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a biopsy of a muscle suspected to be dystrophic, as known to the skilled person.
Individuals suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevents synthesis of the complete protein, i.e. a premature stop prevents the synthesis of the C-terminus of the protein. In Becker muscular dystrophy the dystrophin gene also comprises a mutation compared to the wild type but the mutation does typically not include a premature stop and the C-terminus of the protein is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as a wild type protein, although not necessarily the same amount of activity. In a preferred embodiment, a functional dystrophin protein means an in frame dystrophin gene. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO: 47. Exon-skipping for the treatment of DMD is preferably but not exclusively directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-domain shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated using an oligonucleotide as defined herein will be provided a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin comparable in functionality to a dystrophin from an individual having BMD: preferably said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
Decreasing the production of an aberrant dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional or less to non-functional or semi-functional dystrophin mRNA or protein. A non-functional pre-mRNA dystrophin is preferably leads to an out of frame dystrophin protein, which means that no dystrophin protein will be produced and/or detected. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
Increasing the production of a functional dystrophin in a patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional or in frame dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional or in frame dystrophin mRNA.
Increasing the production of a functional dystrophin in a patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
An increase or a decrease is preferably assessed in a muscular tissue or in a muscular cell of an individual or a patient by comparison to the amount present in said individual or patient before treatment with said molecule or composition of the invention. Alternatively, the comparison can be made with a muscular tissue or cell of said individual or patient, which has not yet been treated with said oligonucleotide or composition in case the treatment is local.
In a further aspect, there is provided a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or alleviate one or more characteristic(s) of a myogenic or muscle cell of said individual, the method comprising administering to said individual an oligonucleotide or a composition as defined herein.
There is further provided a method for enhancing, inducing or promoting skipping of an exon from a dystrophin pre-mRNA in a cell expressing said pre-mRNA in an individual suffering from Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the method comprising administering to said individual an oligonucleotide or a composition as defined herein. Further provided is a method for increasing the production of a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in a cell, said cell comprising a pre-mRNA of a dystrophin gene encoding an aberrant dystrophin protein, the method comprising providing said cell with an oligonucleotide or composition of the invention and allowing translation of mRNA produced from splicing of said pre-mRNA. In one embodiment, said method is performed in vivo, for instance using a cell culture. Preferably, said method is in vivo in said individual.
In this context, increasing the production of a functional dystrophin protein has been defined herein.
Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual using a molecule or a composition of the invention may be assessed by any of the following assays: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a molecule or composition of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602).
Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a characteristic of a muscle fiber relating to its function, integrity and/or survival, said characteristic being assessed on the patient self. Such characteristics may be assessed at the cellular, tissue level of a given patient. An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
An oligonucleotide as used herein preferably comprises an antisense oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied. Exon skipping interferes with the natural splicing processes occurring within a eukaryotic cell. In higher eukaryotes the genetic information for proteins in the DNA of the cell is encoded in exons which are separated from each other by intronic sequences. These introns are in some cases very long. The transcription machinery of eukaryotes generates a pre-mRNA which contains both exons and introns, while the splicing machinery, often already during the production of the pre-mRNA, generates the actual coding region for the protein by splicing together the exons present in the pre-mRNA.
Exon-skipping results in mature mRNA that lacks at least one skipped exon. Thus, when said exon codes for amino acids, exon skipping leads to the expression of an altered product. Technology for exon-skipping is currently directed towards the use of antisense oligonucleotides (AONs). Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy. The mdx mouse carries a nonsense mutation in exon 23. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. AONs directed to, respectively, the 3′ and/or 5′ splice sites of introns 22 and 23 in dystrophin pre-mRNA, have been shown to interfere with factors normally involved in removal of intron 23 so that also exon 23 was removed from the mRNA (Alter J, et al. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med 2006; 12(2):175-7, Lu Q L, et al. Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 6:6, Lu Q L, et al. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci USA 2005; 102(1):198-203, Mann C J, et al, Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. J Gene Med 2002; 4(6):644-54 or Graham I R, et al, Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays. J Gene Med 2004; 6(10):1149-58).
By the targeted skipping of a specific exon, a DMD phenotype is converted into a milder BMD phenotype. The skipping of an exon is preferably induced by the binding of AONs targeting exon-internal sequences. An oligonucleotide directed toward an exon internal sequence typically exhibits no overlap with non-exon sequences. It preferably does not overlap with the splice sites at least not insofar, as these are present in the intron. An oligonucleotide directed toward an exon internal sequence preferably does not contain a sequence complementary to an adjacent intron. Further provided is thus an oligonucleotide according to the invention, wherein said oligonucleotide, or a functional equivalent thereof, is for inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA produced from splicing of said pre-mRNA. An exon skipping technique is preferably applied such that the absence of an exon from mRNA produced from dystrophin pre-mRNA generates a coding region for a more functional—albeit shorter—dystrophin protein. In this context, inhibiting inclusion of an exon preferably means that the detection of the original, aberrant dystrophin mRNA and/or protein is decreased as earlier defined herein.
Within the context of the invention, a functional equivalent of an oligonucleotide preferably means an oligonucleotide as defined herein wherein one or more nucleotides have been substituted and wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by quantifying the amount of a functional dystrophin protein or by quantifying the amount of a functional dystrophin mRNA. A functional dystrophin protein (or a functional dystrophin mRNA) is herein preferably defined as being a dystrophin protein (or a dystrophin protein encoded by said mRNA) able to bind actin and members of the DGC protein. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR (m-RNA) or by immunofluorescence or Western blot analyses (protein). Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Such activity may be measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to an activity of a corresponding oligonucleotide of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
In a preferred embodiment, an oligonucleotide of the invention, which comprises a sequence that binds and/or is complementary to a sequence of exon 44 of dystrophin pre-mRNAas earlier defined herein is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99%, or even more preferably 100%. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of dystrophin pre-mRNA as defined herein. As an example, an oligonucleotide may comprise a sequence that is complementary to part of dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to the oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.
It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the oligonucleotide one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may, to some extent, be allowed, if under the circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridising to the complementary part. In this context, “sufficiently” preferably means that using a gel mobility shift assay as described in example 1 of EP 1 619 249, binding of an oligonucleotide is detectable. Optionally, said oligonucleotide may further be tested by transfection into muscle cells of patients. Skipping of the targeted exon may be assessed by RT-PCR (as described in EP 1 619 249). The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-)mRNA in the system. The risk that also one or more other pre-mRNA will be able to hybridise to the oligonucleotide decreases with increasing size of the oligonucleotide. It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain the capacity to hybridise and/or bind to the targeted region(s) in the pre-mRNA, can be used in the present invention. However, preferably at least the complementary parts do not comprise such mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides having such mismatches in one or more complementary regions. It is thought, that higher hybridisation strengths, (i.e. increasing number of interactions with the opposing strand) are favourable in increasing the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the complementarity is between 90 and 100%. In general this allows for 1 or 2 mismatch(es) in an oligonucleotide of 20 nucleotides or 1, 2, 3 or 4 mismatches in an oligonucleotide of 40 nucleotides, or 1, 2, 3, 4, 5 or 6 mismatches in an oligonucleotide of 60 nucleotides.
A preferred molecule of the invention comprises or consists of a nucleotide-based sequence that is antisense to a sequence selected from exon 44 of the DMD pre-mRNA. The sequence of the DMD pre-mRNA is preferably selected from SEQ ID NO 1: 5′-GUGGCUAACAGAAGCU; SEQ ID NO 2: 5′-GGGAACAUGCUAAAUAC, SEQ ID NO 3: 5′-AGACACAAAUUCCUGAGA, and SEQ ID NO 4: 5′-CUGUUGAGAAA.
A molecule of the invention is preferably an isolated molecule.
A molecule of the invention is preferably a nucleic acid molecule or a nucleotide-based molecule or an oligonucleotide or an antisense oligonucleotide which binds and/or is complementary to a sequence of exon 44 selected from SEQ ID NO:1, 2, 3 or 4.
A preferred molecule of the invention comprises or consists of from about 8 to about 60 nucleotides, more preferred from about 10 to about 50 nucleotides, more preferred from about 17 to about 40 nucleotides, more preferred from about 18 to about 30 nucleotides, more preferred from about 18 to about 24 nucleotides, most preferred about 20 nucleotides, such as 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides or 23 nucleotides.
A preferred molecule of the invention comprises or consists of from 8 to 60 nucleotides, more preferred from 10 to 50 nucleotides, more preferred from 17 to 40 nucleotides, more preferred from 18 to 30 nucleotides, more preferred from 21 to 60, more preferred from 22 to 55, more preferred from 23 to 53, more preferred from 24 to 50, more preferred from 25 to 45, more preferred from 26 to 43, more preferred from 27 to 41, more preferred from 28 to 40, more preferred from 29 to 40, more preferred from 18 to 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
In certain embodiments, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences depicted in Table 1A.
A molecule or nucleic acid molecule of the invention that binds and/or is complementary and/or is antisense to a nucleotide having nucleotide sequence: SEQ ID NO 1: 5′-GUGGCUAACAGAAGCU preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 5; SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO: 41 or SEQ ID NO: 46. A preferred molecule that targets this region of the DMD pre-mRNA comprises or consists of the antisense nucleotide sequence of SEQ ID NO:5, SEQ ID NO 41, or SEQ ID NO 46. Most preferred oligonucleotide comprises or consists of the antisense nucleotide sequence of SEQ ID NO:5.
In a more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence SEQ ID NO 5: 5′-UCAGCUUCUGUUAGCCACUG. It was found that this molecule is very efficient in modulating splicing of exon 44 of the DMD gene in muscle cells. This preferred molecule of the invention comprising SEQ ID NO:5 comprises from 21 to 60, more preferred from 22 to 55, more preferred from 23 to 53, more preferred from 24 to 50, more preferred from 25 to 45, more preferred from 26 to 43, more preferred from 27 to 41, more preferred from 28 to 40, more preferred from 29 to 40, or preferably comprises or consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
In another preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence SEQ ID NO 41 or 46. These preferred molecules of the invention comprising either SEQ ID NO: 41 or SEQ ID NO: 46 further comprise from 18 to 60, more preferred from 18 to 55, more preferred from 20 to 53, more preferred from 24 to 50, more preferred from 25 to 45, more preferred from 26 to 43, more preferred from 27 to 41, more preferred from 28 to 40, more preferred from 29 to 40, or preferably comprises or consists of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
In a further embodiment, a molecule of the invention that is antisense to SEQ ID NO 2: 5′-GGGAACAUGCUAAAUAC preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 35 or SEQ ID NO 36. These preferred molecules of the invention comprising either SEQ ID NO: 35 or SEQ ID NO: 36, further comprise from 17 to 60 nucleotides, more preferred from 18 to 30 nucleotides, more preferred from 21 to 60, more preferred from 22 to 55, more preferred from 23 to 53, more preferred from 24 to 50, more preferred from 25 to 45, more preferred from 26 to 43, more preferred from 27 to 41, more preferred from 28 to 40, more preferred from 29 to 40, more preferred from 18 to 24 nucleotides, or preferably comprises or consists of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
In yet a further embodiment, a molecule of the invention that is antisense to SEQ ID NO 3: 5′-AGACACAAAUUCCUGAGA preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 39 or SEQ ID NO 40. These preferred molecules of the invention comprising either SEQ ID NO: 39 or SEQ ID NO: 40 further comprise from 17 to 60 nucleotides, more preferred from 18 to 30 nucleotides, more preferred from 17 to 60, more preferred from 22 to 55, more preferred from 23 to 53, more preferred from 24 to 50, more preferred from 25 to 45, more preferred from 26 to 43, more preferred from 27 to 41, more preferred from 28 to 40, more preferred from 29 to 40, more preferred from 18 to 24 nucleotides, or preferably comprises or consists of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
In still a further embodiment, a molecule of the invention that is antisense to SEQ ID NO 4: 5′-CUGUUGAGAAA preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 37 or SEQ ID NO 38. These preferred molecules of the invention comprising either SEQ ID NO: 37 or SEQ ID NO: 38 further comprise from 11 to 60 nucleotides, more preferred from 11 to 30 nucleotides, more preferred from 11 to 60, or preferably comprises or consists of 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.
A nucleotide sequence of a molecule of the invention may contain RNA residues, or one or more DNA residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.
It is preferred that a molecule of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
It is further preferred that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative of. A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
In another embodiment, a nucleotide analogue or equivalent of the invention comprises one or more base modifications or substitutions. Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.
It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
A preferred antisense oligonucleotide according to the invention comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
A most preferred antisense oligonucleotide according to the invention comprises a 2′-O-methyl phosphorothioate ribose.
It will also be understood by a skilled person that different antisense oligonucleotides can be combined for efficiently skipping of exon 44. In a preferred embodiment, a combination of at least two antisense oligonucleotides are used in a method of the invention, such as two different antisense oligonucleotides, three different antisense oligonucleotides, four different antisense oligonucleotides, or five different antisense oligonucleotides.
An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably myogenic cells or muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
A preferred antisense oligonucleotide comprises a peptide-linked PMO.
An oligonucleotide of the invention may be indirectly administrated using suitable means known in the art. An oligonucleotide may for example be provided to an individual or a cell, tissue or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of a molecule as identified herein. A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 44 by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector (Goyenvalle A, et al. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 2004; 306(5702):1796-9, De Angelis F G, et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells. Proc Natl Acad Sci USA 2002; 99(14):9456-61 or Denti M A, et al. Chimeric adeno-associated virus/antisense U1 small nuclear RNA effectively rescues dystrophin synthesis and muscle function by local treatment of mdx mice. Hum Gene Ther 2006; 17(5):565-74) and the like. Also, plasmids, artificial chromosomes, plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from PolIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are PolIII driven transcripts. Preferably, in the form of a fusion transcript with an U1 or U7 transcript (see the same Goyenvalle A et al, De Angelis F G et al or Denti M A et al). Such fusions may be generated as described (Gorman L, et al, Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proc Natl Acad Sci USA 1998; 95(9):4929-34 or Suter D, et al, Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations. Hum Mol Genet 1999; 8(13):2415-23). The oligonucleotide may be delivered as is. However, the oligonucleotide may also be encoded by the viral vector. Typically, this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.
One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 44 of DMD.
A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of an antisense oligonucleotide of the invention for inducing skipping of exon 44 of the DMD gene.
Improvements in means for providing an individual or a cell, tissue, organ of said individual with an oligonucleotide and/or an equivalent thereof, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. An oligonucleotide and/or an equivalent thereof can be delivered as is to an individual, a cell, tissue or organ of said individual. When administering an oligonucleotide and/or an equivalent thereof, it is preferred that an oligonucleotide and/or an equivalent thereof is dissolved in a solution that is compatible with the delivery method. Muscle or myogenic cells can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient or transfection agentia that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a muscle cell. Preferred are excipients or transfection agentia capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agentia comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as antisense nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an oligonucleotide across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an oligonucleotide for use in the current invention to deliver it for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
In addition, an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, an oligonucleotide is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said oligonucleotide to a cell and/or enhancing its intracellular delivery. It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an oligonucleotide and a further adjunct compound as later defined herein.
A preferred oligonucleotide is for preventing or treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in an individual. An individual, which may be treated using an oligonucleotide of the invention may already have been diagnosed as having a DMD or a BMD. Alternatively, an individual which may be treated using an oligonucleotide of the invention may not have yet been diagnosed as having a DMD or a BMD but may be an individual having an increased risk of developing a DMD or a BMD in the future given his or her genetic background. A preferred individual is a human being.
If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.
Therefore, the invention also provides a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, or a viral-based vector expressing the antisense oligonucleotide according to the invention.
In a further aspect, there is provided a composition comprising an oligonucleotide as defined herein. Preferably, said composition comprises at least two distinct oligonucleotides as defined herein. More preferably, these two distinct oligonucleotides are designed to skip one or two or more exons. Multi-skipping is encompassed by the present invention, wherein an oligonucleotide of the invention inducing the skipping of exon 44 is used in combination with another oligonucleotide inducing the skipping of another exon. In this context, another exon may be exon 43, 45 or 52. Multi exon skipping has been already disclosed in EP 1 619 249. The DMD gene is a large gene, with many different exons. Considering that the gene is located on the X-chromosome, it is mostly boys that are affected, although girls can also be affected by the disease, as they may receive a bad copy of the gene from both parents, or are suffering from a particularly biased inactivation of the functional allele due to a particularly biased X chromosome inactivation in their muscle cells. The protein is encoded by a plurality of exons (79) over a range of at least 2.4 Mb. Defects may occur in any part of the DMD gene. Skipping of a particular exon or particular exons can, very often, result in a restructured mRNA that encodes a shorter than normal but at least partially functional dystrophin protein. A practical problem in the development of a medicament based on exon-skipping technology is the plurality of mutations that may result in a deficiency in functional dystrophin protein in the cell. Despite the fact that already multiple different mutations can be corrected for by the skipping of a single exon, this plurality of mutations, requires the generation of a series of different pharmaceuticals as for different mutations different exons need to be skipped. An advantage of an oligonucleotide or of a composition comprising at least two distinct oligonucleotide as later defined herein capable of inducing skipping of two or more exons, is that more than one exon can be skipped with a single pharmaceutical. This property is not only practically very useful in that only a limited number of pharmaceuticals need to be generated for treating many different DMD or particular, severe BMD mutations. Another option now open to the person skilled in the art is to select particularly functional restructured dystrophin proteins and produce compounds capable of generating these preferred dystrophin proteins. Such preferred end results are further referred to as mild phenotype dystrophins.
In a preferred embodiment, said composition being preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Such a pharmaceutical composition may comprise any pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. Each feature of said composition has earlier been defined herein.
If several oligonucleotides are used, concentration or dose already defined herein may refer to the total concentration or dose of all oligonucleotides used or the concentration or dose of each oligonucleotide used or added. Therefore in one embodiment, there is provided a composition wherein each or the total amount of oligonucleotide used is dosed in an amount ranged between 0.5 mg/kg and 10 mg/kg.
The invention further provides the use of an antisense oligonucleotide according to the invention, or a viral-based vector that expresses an antisense oligonucleotide according to the invention, for modulating splicing of the DMD mRNA. The splicing is preferably modulated in human myogenic cells or muscle cells in vitro. More preferred is that splicing is modulated in human myogenic cells or muscle cells in vivo.
A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for modulating skipping of exon 44 of the DMD mRNA.
The invention furthermore provides the use of an antisense oligonucleotide according to the invention, or of a viral-based vector expressing the antisense oligonucleotide according to the invention, for the preparation of a medicament for the treatment of a DMD or BMD patient.
Therefore in a further aspect, there is provided the use of a oligoucleotide or of a composition as defined herein for the manufacture of a medicament for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual. Each feature of said use has earlier been defined herein.
A treatment in a use or in a method according to the invention is at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an oligonucleotide, composition, compound or adjunct compound of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (i.e. dose), the formulation of said molecule. The frequency may be ranged between at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.
Dose ranges of oligonucleotide according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg.
In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 μM is used. Preferably, this range is for in vitro use in a cellular model such as muscular cells or muscular tissue. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. If several oligonucleotides are used, this concentration or dose may refer to the total concentration or dose of oligonucleotides or the concentration or dose of each oligonucleotide added. The ranges of concentration or dose of oligonucleotide(s) as given above are preferred concentrations or doses for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of oligonucleotide(s) used may further vary and may need to be optimised any further.
An oligonucleotide as defined herein for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD or BMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne and Becker muscular dystrophy have a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably, said cells comprise a gene encoding a mutant dystrophin protein. Preferably, said cells are cells of an individual suffering from DMD or BMD.
Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.
The expression “in vivo” as used herein may mean in a cellular system which may be isolated from the organism the cells derive from. Preferred cells are muscle cells. In vivo may also mean in a tissue or in a multicellular organism which is preferably a patient as defined herein. Through out the invention, in vivo is opposed to in vitro which is generally associated with a cell free system.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Each embodiment as identified herein may be combined together unless otherwise indicated.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C. Evaluation of AONs designed to induce the skipping of exon 44 from the DMD gene in transfected muscle cells from healthy control or a DMD patient with an exon 45 deletion.
(A) In differentiated muscle cells (myotubes) from a patient with an exon 45 deletion, all tested (transfected) AONs induced exon 44 skipping at a concentration of 150 nM, with PS188 (SEQ ID NO:5), PS190 (previously published as h44AON2; Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71), PS191 (SEQ ID NO: 39), PS193 (SEQ ID NO: 40), PS194 (SEQ ID NO: 38), and PS196 (SEQ ID NO: 43) demonstrating highest efficiencies (between 84% and 94%).
(B) The majority of AONs was also tested by transfection into healthy human control cells at 150 and 400 nM concentrations. The results are summarized in this column chart. PS188 (SEQ ID NO:5), PS190, PS191 (SEQ ID NO: 39), PS193 (SEQ ID NO: 40), PS194 (SEQ ID NO: 38), and PS196 (SEQ ID NO: 43) were confirmed to be most efficient in inducing exon 44 skipping. Note that the exon 44 skipping levels in patient cells are typically higher than in control cells as a result of the fact that, in contrast to healthy cells, in patient cells exon 44 skipping is frame-restoring and giving rise to a more functional and stable. No exon 44 skipping was observed in non-transfected muscle cells in all experiments (data not shown).
(C) Examples of PS197 (SEQ ID NO 44) and three additional AONs, PS199 (SEQ ID NO 36), PS200 (SEQ ID NO 41), and PS201 (SEQ ID NO 42), similarly tested in control muscle cells, at transfection concentrations 150 nM and 400 nM. The exon 44 skipping percentages varied between 1% (PS199) and 44% (PS200). M: DNA size marker (100 bp ladder).
FIGS. 2A-2B. Further evaluation of PS188 (SEQ ID NO:5) by transfection of human control muscle cells or peripheral blood mononuclear cells (PB-MNCs).
(A) Dose-response experiment. In human control muscle cells, PS188 showed increasing levels of exon 44 skipping at transfection doses increasing from 50 nM to 400 nM {in triplo), up to 45% at 400 nM.
(B) PB-MNCs of a healthy individual were transfected with 200 nM PS188. Despite the fact that dystrophin is only expressed at low levels in this type of cells, exon 44 skipping was clearly observed. These results confirm the efficiency of PS188 in inducing exon 44 skipping from the DMD gene. M: DNA size marker.
FIGS. 3A-3B. Further evaluation of PS188 (SEQ ID NO:5) by administration to transgenic hDMD mice expressing the full length human DMD gene, and to cynomolgus monkeys included in extensive toxicity studies.
(A) Following intramuscular injection of 2×40|ig PS188 into both gastrocnemius muscles (G1 and G2) of an hDMD mouse, exon 44 skipping was observed, albeit at low levels. This confirms the capacity of PS188 to induce human exon 44 skipping in muscle tissue in vivo. The low levels were expected given the fact that this mouse model has healthy muscle fibers typically showing lower levels of AON uptake when compared to dystrophic muscle fibers. NT: in non-treated hDMD muscle no exon 44 skipping was observed. M: DNA size marker
(B) In monkeys included in toxicity studies on PS188, exon 44 skipping was observed in peripheral blood mononuclear cells (PB-MNCs) after 1-hour intravenous infusions every fourth day for 29 days at a dose-level of 6 mg/kg PS188. No exon 44 skipping was observed in non-treated monkeys (NT). M: DNA size marker.
EXAMPLES
Example 1
Material and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program (Mathews et al., J Mol Biol 1999; 288(5): 911-40), on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software (rulai.cshl.edu/tools/ESE/) (Cartegni et al., Nucleic Acids Res 2003; 31(13): 3568-71), and on avoiding G-stretches of 3 or more nucleotides or CpG pairs. AONs (see Table 1) were synthesized by Eurogentec (Belgium) and Prosensa Therapeutics BV (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) or a DMD patient with an exon 45 deletion were processed as described previously (Aartsma-Rus et al. Hum Mol Genet 2003; 12(8): 907-14; Havenga et al. J Virol 2002; 76(9): 4612-20). For the screening of AONs, myotube cultures were transfected with 150 and/or 400 nM of each AON. Transfection reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) or a derivative (UNIFectylin, Prosensa Therapeutics BV, Netherlands) was used, with 2 μl ExGen500 or UNIFectylin per μg AON. A control AON with a fluorescein label was used to confirm optimal transfection efficiencies (typically over 90% fluorescent nuclei were obtained). RNA was isolated 24 to 48 hours after transfection as described (Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71). Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking exon 44 (Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71). PCR fragments were isolated from agarose gels (using the QIAquick Gel Extraction Kit (QIAGEN) for sequence verification (by the Leiden Genome Technology Center (LGTC) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), and ABI 3700 Sequencer (PE Applied Biosystems). For quantification, the PCR products were analyzed using the DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
A series of AONs targeting sequences within exon 44 were designed and tested both in healthy control and patient-derived myotube cultures, by transfection and subsequent RT-PCR and sequence analysis of isolated RNA. In myotubes derived from a DMD patient with a deletion of exon 45, specific exon 44 skipping was induced at 150 nM for every AON (PS187 to PS201) tested, with PS188 (SEQ ID NO:5), PS190 (previously published as h44AON2, Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71), PS191 (SEQ ID NO: 39), PS193 (SEQ ID NO: 40), PS194 (SEQ ID NO: 38), and PS196 (SEQ ID NO: 43) demonstrating highest levels of skipping (between 84% and 94% at 150 nM) (FIG. 1A).
Similar transfection experiments were done in control cells from a healthy individual. Percentages of exon 44 skipping were assessed and compared to those in the patient cell cultures (FIG. 1B). Inherent to nonsense-mediated RNA decay of the control transcript after exon 44 skipping, the control percentages were typically lower than those in the patient cells (see for instance results with PS197 in FIG. 1A (patient cells) vs FIG. 1C (control cells)).
Three additional AONs (PS199 (SEQ ID NO 36), PS200 (SEQ ID NO 41), and PS201 (SEQ ID NO. 42) were tested in control muscle cells, at concentrations of 150 nM and 400 nM. The exon 44 skipping percentages varied between 1% (PS199) and 44% (PS200) (FIG. 1C). Based on all transfection experiments, the AONs PS187, PS188, PS190, PS191, PS192, PS193, PS194, PS196 and PS200 were considered most efficient, and AONs PS189, PS197, PS198, PS199, and PS201 least efficient.
PS188 (SEQ ID NO 5) was further tested in dose-response experiments in healthy human control muscle cells, applying increasing doses from 50 to 400 nM in triplo. Increasing levels of exon 44 skipping were accordingly observed, up to 45% at 400 nMPS188 (FIG. 2A).
Example 2
Materials and Methods
A fresh healthy human control blood sample, collected in an EDTA tube, was layered on top of a HistoPaque gradient. Upon centrifugation, the second layer (of the four layers, from top to bottom) with the mononuclear cells was collected, washed, and centrifuged again. The cell pellet was resuspended in proliferation culturing medium and counted. In a 6-wells plate, 8×106 cells per well were plated and incubated at 37° C., 5% CO2 for 3 hrs. The cells were then transfected with 0 or 200 nM PS188 (SEQ ID NO:5; 2′OMePS RNA; Prosensa Therapeutics BV), in duplo, per dish. RNA was isolated 72 hrs after transfection, and analysed by RT-PCR analysis using DMD-gene specific primers flanking exon 44 (Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71). Sequence analysis (by the Leiden Genome Technology Center (LGTC) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), and ABI 3700 Sequencer (PE Applied Biosystems) was performed on isolated PCR products (using the QlAquick Gel Extraction Kit (QIAGEN) to confirm the specific exon 44 skipping on RNA level.
Results
In transfected peripheral blood mononuclear cells (PB-MNCs) from a healthy control individual, PS188 induced the production of a novel shorter transcript fragment when applied at 200 nM (FIG. 2B). This fragment was isolated an sequenced and confirmed due to the specific skipping of exon 44. In non-transfected PB-MNCs no exon 44 skipping was observed. These results indicate that PS188 is an efficient compound inducing human exon 44 skipping in vitro.
Example 3
Materials and Methods
Antisense Oligoribonucleotides (AONs).
Normal and mdx mice (Sicinski et al. (1989). Science 244: 1578-1580) were injected with the mouse-specific m46AON4 (van Deutekom et al. (2001) Hum Mol Genet 10: 1547-1554), whereas the hDMD mice with the human-specific PS196 (SEQ ID NO 43) or PS188 (SEQ ID NO 5). Both AONs contained a full-length phosphorothioate backbone and 2′-0-methyl modified ribose molecules (PS196: Eurogentec, Belgium; PS188: Prosensa Therapeutics BV).
Normal, Mdx and Transgenic hDMD Mice
Normal mice (C57Bl/6NCrL) and mdx mice (C57Bl/10ScSn-mthdJ) were obtained from Charles River Laboratories (The Netherlands). Transgenic hDMD mice were engineered in our own LUMC laboratories. Briefly, embryonic stem (ES) cells were genetically modified through fusions with yeast spheroplasts carrying a YAC of 2.7 Mb that contained the full-length (2.4 Mb) human DMD gene. This YAC was previously reconstructed by homologous recombination of smaller overlapping YACs in yeast (Den Dunnen et al. (1992). Hum Mol Genet 1: 19-28). ES-cells showing integration of one copy of the full-size YAC, as assessed by PFGE mapping, exon-PCR analysis across the entire gene, and metaphase FISH analysis, were then used to generate homozygous hDMD mice (′t Hoen et al., J. Biol. Chem. 2008). Transgenic hDMD mice do not appear to be physically affected by the genetic modification. Appropriate expression of the human DMD gene could be demonstrated in muscle, both at RNA and protein level. The engineering of these mice was authorised by the Dutch Ministry of Agriculture (LNV); project nr. VVA/BD01.284 (E21).
Administration of AONs.
The experiments on intramuscular AON-injections in mice were authorised by the animal experimental commission (UDEC) of the Medical Faculty of the Leiden University (project no. 00095, 03027). AONs were injected, either pure, or complexed to the cationic polymer polyethylenimine (PEI; ExGen 500 (20×), MBI Fermentas) at ratios of 1 ml PEI per nmol AON in a 5% w/v glucose solution, or to 15 nmol SAINT-18TM (Synvolux Therapeutics B.V., The Netherlands), according to the manufacturers' instructions. The SAINT-18TM delivery system is based on a cationic pyridinium head group and allows non-toxic delivery of antisense oligonucleotides. Mice were anaesthetised by intraperitoneal injection of a 1:1 (v/v) Hypnorm/Dormicum solution (Janssen Pharmaceutica, Belgium/Roche, The Netherlands). Pure AON (PS188) was administered in a final injection volume of 40 μl by intramuscular injection into both gastrocnemius muscles of the mice using a Hamilton syringe with a 22-Gauge needle. The mice received two injections of 40 μg at a 24 h interval. They were sacrificed at different time-points post-injection; for PS188-injected hDMD mice ten days after the last injection. Muscles were isolated and frozen in liquid nitrogen-cooled 2-methylbutane.
RT-PCR Analysis.
Muscle samples were homogenized in RNA-Bee solution (Campro Scientific, The Netherlands). Total RNA was isolated and purified according to the manufacturer's instructions. For cDNA synthesis with the reverse transcriptase C. therm polymerase or Transcriptor (Roche Diagnostics, The Netherlands), 300 ng of RNA was used in a 20·l reaction at 60° C. for 30 min, reverse primed with either mouse- or human-specific primers. First PCRs were performed with outer primer sets (flanking exons 43-45 for PS188-injected mice), for 20 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). One μl of this reaction (diluted 1:10) was then re-amplified using nested primer combinations in the exons directly flanking the target exon (exon 44 for PS188-injected mice), with 30 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). PCR products were analysed on 2% agarose gels. Skipping efficiencies were determined by quantification of PCR products using the DNA 1000 LabChip® Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, The Netherlands). Primer sets and sequences were described previously (Aartsma-Rus et al. (2002) Neuromuscul Disord 12 Suppl: S71.8, 17; van Deutekom et al. (2001) Hum Mol Genet 10: 1547-1554).
Sequence Analysis.
RT-PCR products were isolated from 2% agarose gels using the QIAquick Gel Extraction Kit (QIAGEN). Direct DNA sequencing was carried out by the Leiden Genome Technology Center (LGTC) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), and analyzed on an ABI 3700 Sequencer (PE Applied Biosystems).
MALDI-TOF Mass-Spectrometry.
RNA-Bee muscle homogenates were purified using a nucleic acid purification kit (Nucleic Acid Purification Kit for Sequazyme™ Pinpoint SNP Kit, Applied Biosystems) with 96 well spin plates (Applied Biosystems) following the manufacturer's instructions. Matrix solution (50 mg/ml 3-hydroxy picolinic acid and 25 mM dibasic ammonium citrate in 50% acetonitrile) was applied in 1 ml aliquots to an AnchorChip™ sample target (Bruker Daltonics, Germany) and air-dried. Samples were spotted in 0.5 ml aliquots onto the matrix crystals and air-dried. Mass determinations were performed on a Reflex III MALDI-TOF mass-spectrometer (Bruker Daltonics, Germany). Spectra were acquired in reflector mode and accumulated for approximately 900 laser shots. Samples of labelled and unlabelled m46AON4 were analyzed for comparison.
Results
Exon Skipping in Wild-Type Muscle
We first set up targeted exon skipping in mouse muscle in vivo and optimised different parameters of administration. Initial experiments were performed in wild type mice, and, while nonsense-mediated RNA decay will cause underestimation of the exon skipping efficiencies, the effect of the AONs was monitored on mRNA level only. We injected increasing dosages from 0.9 nmol to 5.4 nmol of each antisense oligonucleotide. RT-PCR analysis of total muscle RNA demonstrated the occurrence of a novel shorter transcript fragment in all samples injected. Sequence analysis confirmed the precise skipping of exon 44 in this product (data not shown).
Cross-sections of the contra-lateral injected muscles were analysed for dispersion and persistence of a fluorescein-labelled control AON. Following injection of pure AON, we observed fluorescent signals within some fibres for up to one week. At later time points only weak signals were observed, and mainly within the interstitial spaces. The use of PEI clearly enhanced both dispersion and persistence of the fluorescent signal, even after 3 weeks. However, it also induced fibre degeneration and monocyte infiltration absorbing most fluorescence. Using SAINT, most of the signal was detected in the interstitial spaces for up to one week, indicating that this reagent did not efficiently deliver the AON into the muscle fibres. Since the fluorescent signal may not correspond to the presence of intact and functional AONs, we performed MALDI-TOF mass-spectrometry of injected muscle samples. The analyses indicated that the fluorescent label was removed from the AON within 24 hours. The labelled AON was only detectable for up to two weeks when using PEI. The interstitial AONs were probably more vulnerable to degradation than the intracellular AONs. The unlabelled AON was observed for three to four weeks post-injection in all three series, but it may only be functional when present intracellularly, i.e. in the PEI series.
Human-Specific Exon Skipping in hDMD Muscle
Since the exon skipping strategy is a sequence-specific therapeutic approach, the ideal pre-clinical validation would be a target human DMD gene, in a mouse experimental background. We have engineered such transgenic, “humanised” DMD (hDMD) mice carrying an integrated and functional copy of the full-length human DMD gene. Expression of human dystrophin in hDMD mouse muscle was specifically detected by immunohistochemical analysis of cross-sections, using a human-specific antibody (MANDYS106). On muscle RNA level, RT-PCR analyses using either mouse- or human-specific primers demonstrated correct transcription of the human DMD gene. Furthermore, upon crossing with mdx mice, the hDMD construct showed to complement the dystrophic defect, as was assessed by histological and cDNA microarray analysis (′t Hoen et al., J. Biol. Chem. 2008). hDMD mice have healthy muscle fibers typically exhibiting a limited uptake of naked AONs. We injected the human-specific AON PS196 (SEQ ID NO 43) complexed to PEI, or PS188 (SEQ ID NO 5) without PEI, into the gastrocnemius muscles of the hDMD mice (2×40 μg injections within 24 hrs). At 7 to 10 days post-injection we clearly observed the skipping of the targeted exon 44 from the human DMD transcript (FIG. 3A). Although the human-specific AONs are highly homologous to the corresponding mouse sequences, with only 2 or 3 mismatches in the respective 20-mers, the mouse endogenous transcripts were not affected to any detectable level. PS188 induced exon 44 skipping, as confirmed by sequence analysis. No exon 44 skipping was observed in non-treated hDMD muscle. These results indicate that PS188 is an efficient compound inducing human exon 44 skipping in muscle tissue.
Example 4
Material and Methods
As part of an extensive toxicity program for PS188, non-fasted cynomolgus monkeys were treated by 1-hour intravenous infusion (5 mL/kg/h) every fourth day for 29 days at the dose-level of 6 mg/kg PS188 (SEQ ID NO 5; 2′OMePS RNA; Agilent Life Sciences, USA). The PS188 formulations were freshly prepared on each treatment day (on test days 1, 5, 9, 13, 17, 21, 25 and 29) shortly before initiation of the administration (as soon as possible before, at the most within one hour before start of administration). Formulations were prepared by dissolving PS188 in phosphate buffer; the purity and water content were taken into account as provided in the Certificate of Analysis of the drug substance. The amount of PS188 was adjusted to each animal's current body weight. The animals were sacrificed 96 hours after the last administration (day 33). Whole blood samples (10 ml) were collected in EDTA tubes, and (after overnight shipment at room temperature) layered on top of a HistoPaque gradient. Upon centrifugation, the second layer (of the four layers, from top to bottom) with the mononuclear cells was collected, washed, and centrifuged again. RNA was isolated from the resulting cell pellet and analysed by RT-PCR analysis using DMD-gene specific primers flanking exon 44 (Aartsma-Rus et al. Neuromuscul Disord 2002; 12 Suppl: S71). Sequence analysis (by the Leiden Genome Technology Center (LGTC) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), and ABI 3700 Sequencer (PE Applied Biosystems) was performed on isolated PCR products (using the QlAquick Gel Extraction Kit (QIAGEN) to confirm the specific exon 44 skipping on RNA level.
Results
In monkeys treated by 1-hour intravenous infusions every fourth day for 29 days at the dose-level of 6 mg/kg PS188, exon 44 skipping was observed in peripheral blood mononuclear cells (FIG. 3B), despite the fact that these cells express only low levels of dystrophin. The human and monkey DMD sequence targeted by PS188 is in fact 100% identical. No exon 44 skipping was observed in non-treated monkeys. These results indicate that PS188 is an efficient compound inducing exon 44 skipping in vivo.
TABLE 1A
Table 1 Antisense oligonucleotide sequences.
1 (PS188)
UCAGCUUCUGUUAGCCACUG
SEQ ID NO 5
2
UUCAGCUUCUGUUAGCCACU
SEQ ID NO 6
3
UUCAGCUUCUGUUAGCCACUG
SEQ ID NO 7
4
UCAGCUUCUGUUAGCCACUGA
SEQ ID NO 8
5
UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 9
6
UCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 10
7
UUCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 11
8
UCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 12
9
UUCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 13
10
UCAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 14
11
UUCAGCUUCUGUUAGCCACUGAUA
SEQ ID NO 15
12
UCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 16
13
UUCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 17
14
UCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 18
15
UUCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 19
16
CAGCUUCUGUUAGCCACUG
SEQ ID NO 20
17
CAGCUUCUGUUAGCCACUGAU
SEQ ID NO 21
18
AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 22
19
CAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 23
20
AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 24
21
CAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 25
22
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 26
23
CAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 27
24
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 28
25
CAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 29
26
AGCUUCUGUUAGCCACUGAU
SEQ ID NO 30
27
GCUUCUGUUAGCCACUGAUU
SEQ ID NO 31
28
GCUUCUGUUAGCCACUGAUUA
SEQ ID NO 32
29
GCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 33
30
GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 34
31 (PS 192)
CCAUUUGUAUUUAGCAUGUUCCC
SEQ ID NO 35
32 (PS 199)
AGAUACCAUUUGUAUUUAGC
SEQ ID NO 36
33 (PS 187)
GCCAUUUCUCAACAGAUCU
SEQ ID NO 37
34 (PS 194)
GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 38
35 (PS191)
AUUCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 39
36 (PS 193)
UCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 40
37 (PS 200)
GUUCAGCUUCUGUUAGCC
SEQ ID NO 41
38 (PS 201)
CUGAUUAAAUAUCUUUAUAU C
SEQ ID NO 42
TABLE 1B
39 (PS 196)
GCCGCCAUUUCUCAACAG
SEQ ID NO 43
40 (PS 197)
GUAUUUAGCAUGUUCCCA
SEQ ID NO 44
41 (PS 198)
CAGGAAUUUGUGUCUUUC
SEQ ID NO 45
42 (PS 189)
UCUGUUAGCCACUGAUUAAAU
SEQ ID NO 46
SEQ. ID NO: 47
MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRR
LLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTD
IVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQS
TRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRL
EHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSI
EAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP
RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDR
YQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAH
QGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEK
QSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQV
QQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR
WANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTT
GFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVT
QKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVT
TVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWI
TRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSA
QALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNNII
AFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSG
LQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKE
LQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLG
ELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRW
KKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPAL
GDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRL
ETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQA
EEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVI
AQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSY
LEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQI
RILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSA
QETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHE
ISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQR
LQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEV
EMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEK
CLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKAT
QKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSR
AEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKED
VLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRF
AAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFN
KDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALK
DLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERK
IKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIE
SKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQ
ALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSK
KTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWR
RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQ
TVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKR
LEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVK
LLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWI
KVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLE
IYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKR
KLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVV
TKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVM
VGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEART
IITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLG
QARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALK
LLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFP
LDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGE
IEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKS
LNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQ
KQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPREL
PPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ
EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLK
ENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQL
HEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPK
MTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQH
NLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNV
YDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRL
GLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLD
WMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHF
NYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFR
TKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSH
DDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSL
NQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLK
QQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQ
ILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQP
MLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNT
PGKPMREDTM
homo sapiens DMD amino acid sequence
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14859598
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Aug 29th, 2017 12:00AM
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Oct 23rd, 2014 12:00AM
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https://www.uspto.gov?id=US09745576-20170829
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RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders
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The current invention provides an improved oligonucleotide and its use for treating, ameliorating, preventing, delaying and/or treating a human cis-element repeat instability associated genetic neuromuscular or neurodegenerative disorder.
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9745576
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1. A 2′-O-methyl phosphorothioate oligoribonucleotide consisting of the base sequence (XYG)7, wherein each X is 5-methyl cytosine and each Y is uracil (SEQ ID NO: 2) or wherein each X is cytosine and each Y is 5-methyluracil (SEQ ID No:3).
2. An oligoribonucleotide according to claim 1, wherein said oligoribonucleotide is single stranded.
3. A composition comprising an oligoribonucleotide according to claim 1.
4. A composition according to claim 3, said composition comprising at least one excipient for enhancing the targeting and/or delivery of said composition to a tissue and/or cell and/or into a tissue and/or cell.
5. A method for treating a subject having a human cis-element repeat instability associated genetic disorder by administering an oligoribonucleotide according to claim 2 to said subject.
6. The method of claim 5, wherein said subject has the human cis-element repeat instability associated genetic disorder-Huntington Disease.
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6
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CROSS REFERENCE
This application is a continuation application of International Application No. PCT/NL2013/050306 filed Apr. 23, 2013, which claims priority to U.S. Provisional Application No. 61/636,914 filed Apr. 23, 2012 and EP Patent Application No. 12165139.2 filed Apr. 23, 2012, all of the contents of which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
The attached Sequence Listing is hereby incorporated by reference.
FIELD
The invention relates to the field of human genetics, more specifically neuromuscular disorders. The invention in particular relates to the use of antisense oligonucleotides (AONs) with improved characteristics enhancing clinical applicability as further defined herein.
BACKGROUND OF THE INVENTION
Neuromuscular diseases are characterized by impaired functioning of the muscles due to either muscle or nerve pathology (myopathies and neuropathies). The neuropathies are characterized by neurodegeneration and impaired nerve control leading to problems with movement, spasticity or paralysis. Examples include Huntington's disease (HD), several types of spinocerebellar ataxia (SCA), Friedreich's ataxia (FA), Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). A subset of neuropathies is caused by a cis-element repeat instability. For instance, HD is caused by a triplet (CAG)n repeat expansion in exon 1 of the HTT gene. Expansion of these repeats results in expansion of a glutamine stretch at the N-terminal end of the 348 kDa cytoplasmic huntingtin protein. Huntingtin has a characteristic sequence of 6 to 29 glutamine amino acid residues in the normal form; the mutated huntingtin causing the disease has more than 38 residues. The continuous expression of mutant huntingtin molecules in neuronal cells results in the formation of large protein deposits which eventually give rise to cell death, especially in the frontal lobes and the basal ganglia (mainly in the caudate nucleus). The severity of the disease is generally proportional to the number of extra residues. AONs specifically targeting the expanded CAG repeats (such as PS57 (CUG)7 as a 2′-O-methyl phosphorothioate RNA; SEQ ID NO:1 Evers et al.) can be applied to effectively reduce mutant huntingtin transcript and (toxic) protein levels in HD patient-derived cells. For treatment of neuropathies, systemically administered AONs need to pass the blood brain barrier. Thus, there is a need for optimization of oligochemistry allowing and/or exhibiting improved brain delivery.
The myopathies include genetic muscular dystrophies that are characterized by progressive weakness and degeneration of skeletal, heart and/or smooth muscle. Examples of myopathies are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), and myotonic dystrophy type 2 (DM2). DM1 and DM2 are both also caused by cis-element repeat instability; DM1 by a trinucleotide (CTG)n repeat expansion in the 3′ untranslated region of exon 15 in the DMPK gene, and DM2 by a tetranucleotide (CCTG)n repeat expansion in the DM2/ZNF9 gene. Also here, AONs specifically targeting the expanded repeats, such as PS58, (CAG)7, a 2′-O-methyl phosphorothioate RNA for DM1 (Mulders et al.), have been shown to efficiently induce the specific degradation of the (toxic) expanded repeat transcripts. In contrast to DMD where the gene defect is associated with increased permeability of the muscle fiber membranes for small compounds as AONs, for most other myopathies an enhanced AON distribution to and uptake by muscle tissue is essential to obtain a therapeutic effect. Thus, also here there is a need for optimization of oligochemistry allowing and/or exhibiting improved muscle delivery.
The particular characteristics of a chosen chemistry at least in part affect the delivery of an AON to the target transcript: administration route, biostability, biodistribution, intra-tissue distribution, and cellular uptake and trafficking. In addition, further optimization of oligonucleotide chemistry is conceived to enhance binding affinity and stability, enhance activity, improve safety, and/or to reduce cost of goods by reducing length or improving synthesis and/or purification procedures. Multiple chemical modifications have become generally and/or commercially available to the research community (such as 2′-O-methyl RNA and 5-substituted pyrimidines and 2,6-diaminopurines), whereas most others still present significant synthetic effort to obtain. Especially preliminary encouraging results have been obtained using 2′-O-methyl phosphorothioate RNA containing modifications on the pyrimidine and purine bases as identified herein.
In conclusion, to enhance the therapeutic applicability of AONs for treating human cis-element repeat instability associated genetic disorders as exemplified herein, there is a need for AONs with further improved characteristics.
DESCRIPTION OF THE INVENTION
Oligonucleotide
In a first aspect, the invention provides an oligonucleotide comprising 2′-O-methyl RNA nucleotide residues, having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, and comprising one or more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases; or an oligonucleotide consisting of 2′-O-methyl RNA nucleotide residues and having a backbone wherein all phosphate moieties are replaced by phosphorothioate moieties, and comprising one or more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases, for use as a medicament for treating human cis-element repeat instability associated genetic disorders.
In the context of the invention, “backbone” is used to identify the chain of alternating ribose rings and internucleoside linkages, to which the nucleobases are attached. The term “linkage” is used for the connection between two ribose units (i.e. “internucleoside linkage”), which is generally a phosphate moiety. Thus, an oligonucleotide having 10 nucleotides may contain 9 linkages, linking the 10 ribose units together. Additionally, there may be one or more last linkage(s) present at one or both sides of the oligonucleotide, which is only connected to one nucleotide. The terms “linkage” and “internucleoside linkage” are also meant to indicate such a pendant linkage. At least one of the linkages in the backbone of the oligonucleotide according to the invention consists of a phosphorothioate moiety, linking two ribose units. Thus, at least one of the naturally occurring 3′ to 5′ phosphodiester moieties present in RNA is replaced by a phosphorothioate moiety.
Within the context of the invention, “a” in each of the following expressions means “at least one”: a 2′-O-methyl RNA nucleotide residue, a 2′-O-methyl RNA residue, a phosphorothioate moiety, a 2′-O-methyl phosphorothioate RNA residue, a 5-methylpyrimidine base, a 5-methylcytosine base, a 5-methyluracil base, a thymine base, a 2,6-diaminopurine base.
Preferably, the oligonucleotide according to the invention is an oligonucleotide with less than 37 nucleotides. Said oligonucleotide may have 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. Such oligonucleotide may also be identified as an oligonucleotide having from 12 to 36 nucleotides.
Accordingly, an oligonucleotide of the invention, comprising a 2′-O-methyl RNA nucleotide residue having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, comprises less than 37 nucleotides (i.e. it comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides) and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.
Accordingly, an oligonucleotide of the invention, consisting of 2′-O-methyl RNA nucleotide residues and having a backbone wherein all phosphate moieties are replaced by phosphorothioate, and comprises less than 34 nucleotides (i.e. it comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides) and a 5-methylpyrimidine and/or a 2,6-diaminopurine base.
In a preferred embodiment, the oligonucleotide of the invention comprises a 2′-O-methyl phosphorothioate RNA nucleotide residue, or consists of 2′-O-methyl phosphorothioate RNA nucleotide residues. Such oligonucleotide comprises a 2′-O-methyl RNA residue, which is connected through a phosphorothioate linkage to the next nucleotide in the sequence. This next nucleotide may be, but not necessarily, another 2′-O-methyl phosphorothioate RNA nucleotide residue. Alternatively, such oligonucleotide consists of 2′-O-methyl phosphorothioate RNA nucleotide residues, wherein all nucleotides comprise a 2′-O-methyl moiety and a phosphorothioate moiety. Preferably, such oligonucleotide consists of 2′-O-methyl phosphorothioate RNA nucleotide residues. Such chemistry is known to the skilled person. Throughout the application, an oligonucleotide comprising a 2′-O-methyl RNA residue and a phosphorothioate linkage may be replaced by an oligonucleotide comprising a 2′-O-methyl phosphorothioate RNA nucleotide residue or an oligonucleotide comprising a 2′-O-methyl phosphorothioate RNA residue. Throughout the application, an oligonucleotide consisting of 2′-O-methyl RNA residues linked by or connected through phosphorothioate linkages or an oligonucleotide consisting of 2′-O-methyl phosphorothioate RNA nucleotide residues may be replaced by an oligonucleotide consisting of 2′-O-methyl phosphorothioate RNA.
In addition, an oligonucleotide of the invention comprises at least one base modification that increases binding affinity to target strands, increases melting temperature of the resulting duplex of said oligonucleotide with its target, and/or decreases immunostimulatory effects, and/or increases biostability, and/or improves biodistribution and/or intra-tissue distribution, and/or cellular uptake and trafficking. In an embodiment, an oligonucleotide of the invention comprises a 5-methylpyrimidine and/or a 2,6-diaminopurine base. A 5-methylpyrimidine base is selected from a 5-methylcytosine and/or a 5-methyluracil and/or a thymine, in which thymine is identical to 5-methyluracil. Where an oligonucleotide of the invention has two or more such base modifications, said base modifications may be identical, for example all such modified bases in the oligonucleotide are 5-methylcytosine, or said base modifications may be combinations of different base modifications, for example the oligonucleotide may have one or more 5-methylcytosines and one or more 5-methyluracils.
In a preferred embodiment, an oligonucleotide of the invention (i.e. an oligonucleotide comprising 2′-O-methyl RNA nucleotide residues, having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, and comprising one or more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases; or an oligonucleotide consisting of 2′-O-methyl RNA nucleotide residues and having a backbone wherein all phosphate moieties are replaced by phosphorothioate moieties, and comprising one or more 5-methylpyrimidine and/or one or more 2,6-diaminopurine bases) is such that it does not comprise a 2′-deoxy 2′-fluoro nucleotide (i.e. 2′-deoxy 2′-fluoro-adenosine, -guanosine, -uridine and/or -cytidine). Such oligonucleotide comprising a 2′-fluoro (2′-F) nucleotide has been shown to be able to recruit the interleukin enhancer-binding factor 2 and 3 (ILF2/3) and is thereby able to induce exon skipping in the targeted pre-mRNA (Rigo F, et al, WO2011/097614). In the current invention, the oligonucleotide used preferably does not recruit such factors and/or the oligonucleotide of the invention does not form heteroduplexes with RNA that are specifically recognized by the ILF2/3. The mechanism of action of the oligonucleotide of the current invention is assumed to be distinct from the one of an oligonucleotide with a 2′-F nucleotide: the oligonucleotide of the invention is expected to primarily induce the specific degradation of the (toxic) expanded repeat transcripts.
‘Thymine’ and ‘5-methyluracil’ may be interchanged throughout the document. In analogy, 2,6-diaminopurine is identical to 2-aminoadenine and these terms may be interchanged throughout the document.
The term “base modification” or “modified base” as identified herein refers to the modification of a naturally occurring base in RNA (i.e. pyrimidine or purine base) or to the de novo synthesis of a base. This de novo synthesized base could be qualified as “modified” by comparison to an existing base.
An oligonucleotide of the invention comprising a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine base means that at least one of the cytosine nucleobases of said oligonucleotide has been modified by substitution of the proton at the 5-position of the pyrimidine ring with a methyl group (i.e. a 5-methylcytosine), and/or that at least one of the uracil nucleobases of said oligonucleotide has been modified by substitution of the proton at the 5-position of the pyrimidine ring with a methyl group (i.e. a 5-methyluracil), and/or that at least one of the adenine nucleobases of said oligonucleotide has been modified by substitution of the proton at the 2-position with an amino group (i.e. a 2,6-diaminopurine), respectively. Within the context of the invention, the expression “the substitution of a proton with a methyl group in position 5 of the pyrimidine ring” may be replaced by the expression “the substitution of a pyrimidine with a 5-methylpyrimidine,” with pyrimidine referring to only uracil, only cytosine or both. Likewise, within the context of the invention, the expression “the substitution of a proton with an amino group in position 2 of adenine” may be replaced by the expression “the substitution of an adenine with a 2,6-diaminopurine.” If said oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more cytosines, uracils, and/or adenines, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more cytosines, uracils and/or adenines respectively have been modified this way. Preferably all cytosines, uracils and/or adenines have been modified this way or replaced by 5-methylcytosine, 5-methyluracil and/or 2,6-diaminopurine, respectively. No need to say that the invention could only be applied to oligonucleotides comprising at least one cytosine, uracil, or adenine, respectively, in their sequence.
We discovered that the presence of a 5-methylcytosine, 5-methyluracil and/or a 2,6-diaminopurine in an oligonucleotide of the invention has a positive effect on at least one of the parameters or an improvement of at least one parameters of said oligonucleotides. In this context, parameters may include: binding affinity and/or kinetics, silencing activity, biostability, (intra-tissue) distribution, cellular uptake and/or trafficking, and/or immunogenicity of said oligonucleotide, as explained below.
Binding affinity and kinetics depend on the AON's thermodynamic properties. These are at least in part determined by the melting temperature of said oligonucleotide (Tm; calculated with e.g. the oligonucleotide properties calculator (http://www.unc.edu/˜cail/biotool/oligo/index.html or http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/) for single stranded RNA using the basic Tm and the nearest neighbor model), and/or the free energy of the oligonucleotide-target exon complex (using RNA structure version 4.5 or RNA mfold version 3.5). If a Tm is increased, the exon skipping activity typically increases, but when a Tm is too high, the AON is expected to become less sequence-specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give preferred ranges for each of these parameters.
An activity of an oligonucleotide of the invention is to inhibit the formation of a mutant protein and/or silence or reduce or decrease the quantity of a disease-associated or disease-causing or mutant transcript containing an extended or unstable number of repeats in a cell of a patient, in a tissue of a patient and/or in a patient as explained later herein. An oligonucleotide of the invention comprising or consisting of a 2′-O-methyl phosphorothioate RNA and a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine base is expected to be able to silence or reduce or decrease the quantity of said transcript more efficiently than what an oligonucleotide comprising or consisting of a 2′-O-methyl phosphorothioate RNA but without any 5-methylcytosine, without any 5-methyluracil and without any 2,6-diaminopurine base will do. This difference in terms of efficiency may be of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%. The reduction or decrease may be assessed by Northern Blotting or (semi-) quantitative RT-PCR for transcript levels (preferably as carried out in the experimental part) or by Western blotting for protein levels. An oligonucleotide of the invention may first be tested in the cellular system like patient-derived fibroblasts as described in Example 1.
Biodistribution and biostability are preferably at least in part determined by a validated hybridization ligation assay adapted from Yu et al., 2002. In an embodiment, plasma or homogenized tissue samples are incubated with a specific capture oligonucleotide probe. After separation, a DIG-labeled oligonucleotide is ligated to the complex and detection followed using an anti-DIG antibody-linked peroxidase. Non-compartmental pharmacokinetic analysis is performed using WINNONLIN software package (model 200, version 5.2, Pharsight, Mountainview, Calif.). Levels of AON (ug) per mL plasma or mg tissue are monitored over time to assess area under the curve (AUC), peak concentration (Cmax), time to peak concentration (Tmax), terminal half life and absorption lag time (tlag). Such a preferred assay has been disclosed in the experimental part.
AONs may stimulate an innate immune response by activating the Toll-like receptors (TLR), including TLR9 and TLR7 (Krieg et al., 1995). The activation of TLR9 typically occurs due to the presence of non-methylated CG sequences present in oligodeoxynucleotides (ODNs), by mimicking bacterial DNA which activates the innate immune system through TLR9-mediated cytokine release. The 2′-O-methyl modification is however suggested to markedly reduce such possible effect. TLR7 has been described to recognize uracil repeats in RNA (Diebold et al., 2006).
Activation of TLR9 and TLR7 result in a set of coordinated immune responses that include innate immunity (macrophages, dendritic cells (DC), and NK cells)(Krieg et al., 1995; Krieg, 2000). Several chemo- and cytokines, such as IP-10, TNFα, IL-6, MCP-1 and IFNα (Wagner, 1999; Popovic et al., 2006) have been implicated in this process. The inflammatory cytokines attract additional defensive cells from the blood, such as T and B cells. The levels of these cytokines can be investigated by in vitro testing. In short, human whole blood is incubated with increasing concentrations of AONs after which the levels of the cytokines are determined by standard commercially available ELISA kits. A decrease in immunogenicity preferably corresponds to a detectable decrease of concentration of at least one of the cytokines mentioned above by comparison to the concentration of corresponding cytokine in an assay in a cell treated with an oligonucleotide comprising at least one 5-methylcytosine and/or 5-methyluracil, and/or 2,6-diaminopurine compared to a cell treated with a corresponding oligonucleotide having no 5-methylcytosines, 5-methyluracils, or 2,6-diaminopurines.
Accordingly, a preferred oligonucleotide of the invention has an improved parameter, such as an acceptable or a decreased immunogenicity and/or a better biodistribution and/or acceptable or improved RNA binding kinetics and/or thermodynamic properties by comparison to a corresponding oligonucleotide consisting of a 2′-O-methyl phosphorothioate RNA without a 5-methylcytosine, without a 5-methyluracil and without a 2,6-diaminopurine. Each of these parameters could be assessed using assays known to the skilled person or preferably as disclosed herein.
Below other chemistries and modifications of the oligonucleotide of the invention are defined. These additional chemistries and modifications may be present in combination with the chemistry already defined for said oligonucleotide, i.e. the presence of a 5-methylcytosine, a 5-methyluracil and/or a 2,6-diaminopurine, and the oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA.
A preferred oligonucleotide of the invention comprises or consists of an RNA molecule or a modified RNA molecule. In a preferred embodiment, an oligonucleotide is single stranded. The skilled person will understand that it is however possible that a single stranded oligonucleotide may form an internal double stranded structure. However, this oligonucleotide is still named a single stranded oligonucleotide in the context of this invention. A single stranded oligonucleotide has several advantages compared to a double stranded siRNA oligonucleotide: (i) its synthesis is expected to be easier than two complementary siRNA strands; (ii) there is a wider range of chemical modifications possible to enhance uptake in cells, a better (physiological) stability and to decrease potential generic adverse effects; (iii) siRNAs have a higher potential for non-specific effects (including off-target genes) and exaggerated pharmacology (e.g. less control possible of effectiveness and selectivity by treatment schedule or dose) and (iv) siRNAs are less likely to act in the nucleus and cannot be directed against introns.
In addition to the modifications described above, the oligonucleotide of the invention may comprise further modifications such as different types of nucleic acid nucleotide residues or nucleotides as described below. Different types of nucleic acid nucleotide residues may be used to generate an oligonucleotide of the invention. Said oligonucleotide may have at least one backbone modification (internucleoside linkage and/or sugar modification) and/or at least one base modification compared to an RNA-based oligonucleotide.
A base modification includes a modified version of the natural purine and pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, and thymine), such as hypoxanthine (e.g. inosine), orotic acid, agmatidine, lysidine, pseudouracil, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g. 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in U.S. Pat. No. 6,683,173 (Epoch Biosciences), which is incorporated here entirely by reference. cPent-G, cPent-AP and Pr-AP were shown to reduce immunostimulatory effects when incorporated in siRNA (Peacock H. et al.).
In an embodiment, an oligonucleotide of the invention comprises an abasic site or an abasic monomer. Within the context of the invention, such monomer may be called an abasic site or an abasic monomer. An abasic monomer or abasic site is a nucleotide residue or building block that lacks a nucleobase by comparison to a corresponding nucleotide residue comprising a nucleobase. Within the invention, an abasic monomer is thus a building block part of an oligonucleotide but lacking a nucleobase. Such abasic monomer may be present or linked or attached or conjugated to a free terminus of an oligonucleotide.
In a more preferred embodiment, an oligonucleotide of the invention comprises 1-10 or more abasic monomers. Therefore, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more abasic monomers may be present in an oligonucleotide of the invention.
An abasic monomer may be of any type known and conceivable by the skilled person, non-limiting examples of which are depicted below:
Herein, R1 and R2 are independently H, an oligonucleotide or other abasic site(s), provided that not both R1 and R2 are H and R1 and R2 are not both an oligonucleotide. An abasic monomer(s) can be attached to either or both termini of the oligonucleotide as specified before. It should be noted that an oligonucleotide attached to one or two an abasic site(s) or abasic monomer(s) may comprise less than 12 nucleotides. In this respect, the oligonucleotide according to the invention may comprise at least 12 nucleotides, optionally including one or more abasic sites or abasic monomers at one or both termini.
In the sequence listing, an oligonucleotide of the invention comprising an abasic monomer may be represented by its nucleotide or base sequence; the abasic monomer not being represented since it may be considered as linked or attached or conjugated to a free terminus of an oligonucleotide. This is the case for base sequences SEQ ID NO: 107 and 108. In table 2, the full sequence of preferred oligonucleotides comprising SEQ ID NO:107 or 108 is provided: such oligonucleotide comprises SEQ ID NO: 107 or 108 and 4 abasic monomers at the 3′ terminus of the corresponding SEQ ID NO: 107 or 108. SEQ ID NO: 220 and 221 correspond to SEQ ID NO: 107 and 108 further comprising 4 additional abasic monomers at the 3′ terminus of the oligonucleotide.
When an abasic monomer is present within a base sequence of an oligonucleotide, said abasic monomer is identified in the sequence listing as part of the sequence of said oligonucleotide as in SEQ ID NO:210 and 213.
In tables 1 and 2, an abasic monomer is identified using the letter Q.
Depending on its length an oligonucleotide of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base modifications. It is also encompassed by the invention to introduce more than one distinct base modification in said oligonucleotide.
A “sugar modification” indicates the presence of a modified version of the ribosyl moiety as naturally occurring in RNA (i.e. the furanosyl moiety), such as bicyclic sugars, tetrahydropyrans, morpholinos, 2′-modified sugars, 4′-modified sugars, 5′-modified sugars, and 4′-substituted sugars. Examples of suitable sugar modifications include, but are not limited to, 2′-O-modified RNA nucleotide residues, such as 2′-O-alkyl or 2′-O-(substituted)alkyl e.g. 2′-O-methyl, 2′-O-(2-cyanoethyl), 2′-O-(2-methoxyl)ethyl (2′-MOE), 2′-O-(2-thiomethyl)ethyl, 2′-O-butyryl, 2′-O-propargyl, 2′-O-allyl, 2′-O-(2-amino)propyl, 2′-O-(2-(dimethylamino)propyl), 2′-O-(2-amino)ethyl, 2′-O-(2-(dimethylamino)ethyl); 2′-deoxy (DNA); 2′-O-(haloalkoxy)methyl (Arai K. et al.) e.g. 2′-O-(2-chloroethoxyl)methyl (MCEM), 2′-O-(2,2-dichloroethoxy)methyl (DCEM); 2′-O-alkoxycarbonyl e.g. 2′-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2′-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2′-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2′-halo e.g. 2′-F, FANA (2′-F arabinosyl nucleic acid); carbasugar and azasugar modifications; 3′-O-alkyl e.g. 3′-O-methyl, 3′-O-butyryl, 3′-O-propargyl, 5′-alkyl e.g. 5′-methyl; and their derivatives. Another sugar modification includes “bridged” or “bicylic” nucleic acid (BNA), e.g. locked nucleic acid (LNA), xcylo-LNA, α-L-LNA, β-D-LNA, cEt (2′-O,4′-C constrained ethyl) LNA, cMOEt (2′-O,4′-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), BNANC[N-Me] (as described in Chem. Commun 2007, 3765, which is incorporated in its entirety by reference); tricyclo DNA (tcDNA); unlocked nucleic acid (UNA); 5′-methyl substituted BNAs (as described in U.S. patent application Ser. No. 13/530,218, which is incorporated in its entirety by reference); cyclohexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (as e.g. in PMO, PMOPlus, PMO-X) and their derivatives, preferably locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2′-O,4′-C constrained ethyl) LNA, cMOEt (2′-O,4′-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo DNA (tcDNA); cyclohexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (as e.g. in PMO, PMOPlus, PMO-X) and their derivatives. A preferred tcDNA is tc-PS-DNA (tricyclo DNA comprising phosphorothioate internucleoside linkage). Depending on its length, an oligonucleotide of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 sugar modifications. It is also encompassed by the invention to introduce more than one distinct sugar modification in said oligonucleotide. In an embodiment, an oligonucleotide as defined herein comprises or consists of an LNA or a derivative thereof. BNA derivatives are for example described in WO 2011/097641, which is incorporated in its entirety by reference. In a more preferred embodiment, an oligonucleotide of the invention is fully 2′-O-methyl modified. Examples of PMO-X are described in WO2011150408, which is incorporated here in its entirety by reference.
In a preferred embodiment, the oligonucleotide according to the invention comprises, apart from the mandatory 2′-O-methyl sugar modification, at least one other sugar modification selected from 2′-O-methyl, 2′-O-(2-methoxyl)ethyl, morpholino, a bridged nucleotide or BNA, or the oligonucleotide comprises both bridged nucleotides and 2′-deoxy modified nucleotides (BNA/DNA mixmers). More preferably, the oligonucleotide according to the invention is modified over its full length with a sugar modification selected from 2′-O-methyl, 2′-O-(2-methoxyl)ethyl, morpholino, bridged nucleic acid (BNA) or BNA/DNA mixmer.
In a more preferred embodiment, the oligonucleotide according to the invention comprises is fully 2′-O-methyl modified, preferably fully 2′-O-methyl phosphorothioate modified.
A “backbone modification” indicates the presence of a modified version of the ribosyl moiety (“sugar modification”), as indicated above, and/or the presence of a modified version of the phosphodiester as naturally occurring in RNA (“internucleoside linkage modification”). Examples of internucleoside linkage modifications, which are compatible with the present invention, are phosphorothioate (PS), chirally pure phosphorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, and their derivatives. Another modification includes phosphoramidite, phosphoramidate, N3′→P5′ phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives. Depending on its length, an oligonucleotide of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 backbone modifications. It is also encompassed by the invention to introduce more than one distinct backbone modification in said oligonucleotide.
An oligonucleotide of the invention comprises at least one phosphorothioate modification. In a more preferred embodiment, an oligonucleotide of the invention is fully phosphorothioate modified.
Other chemical modifications of an oligonucleotide of the invention include peptide-base nucleic acid (PNA), boron-cluster modified PNA, pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- or glycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA), acyclic threoninol-based nucleic acid (aTNA), morpholino-based oligonucleotide (PMO, PPMO, PMO-X), cationic morpholino-based oligomers (PMOPlus), oligonucleotides with integrated bases and backbones (ONIBs), pyrrolidine-amide oligonucleotides (POMs); and their derivatives.
In another embodiment, an oligonucleotide comprises a peptide nucleic acid and/or a morpholino phosphorodiamidate or a derivative thereof.
Thus, the preferred oligonucleotide according to one aspect of the invention comprises:
(a) at least one base modification selected from 5-methylpyrimidine and 2,6-diaminopurine; and/or
(b) at least one sugar modification, which is 2′-O-methyl, and/or
(c) at least one backbone modification, which is phosphorothioate.
Thus, a preferred oligonucleotide according to this aspect of the invention comprises a base modification (a) and no sugar modification (b) and no backbone modification (c). Another preferred oligonucleotide according to this aspect of the invention comprises a sugar modification (b) and no base modification (a) and no backbone modification (c). Another preferred oligonucleotide according to this aspect of the invention comprises a backbone modification (c) and no base modification (a) and no sugar modification (b). Also oligonucleotides having none of the above-mentioned modifications are understood to be covered by the present invention, as well as oligonucleotides comprising two, i.e. (a) and (b), (a) and (c) and/or (b) and (c), or all three of the modifications (a), (b) and (c), as defined above. In another preferred embodiment, any of the oligonucleotides as described in the previous paragraph may comprise:
(a) at least one (additional) base modification selected from 2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine; and/or
(b) at least one (additional) sugar modification selected from 2′-O-methyl, 2′-O-(2-methoxyl)ethyl, 2′-deoxy (DNA), morpholino, a bridged nucleotide or BNA, or the oligonucleotide comprises both bridged nucleotides and 2′-deoxy modified nucleotides (BNA/DNA mixmers); and/or
(c) at least one (additional) backbone modification selected from (another) phosphorothioate or phosphordiamidate.
In another preferred embodiment, the oligonucleotide according to the invention is modified over its entire length with one or more of the same modification, selected from (a) one of the base modifications; and/or (b) one of the sugar modifications; and/or (c) one of the backbone modifications.
With the advent of nucleic acid mimicking technology, it has become possible to generate molecules that have a similar, preferably the same hybridization characteristics in kind not necessarily in amount as nucleic acid itself. Such functional equivalents are of course also suitable for use in the invention.
The skilled person will understand that not each sugar, base, and/or backbone may be modified the same way. Several distinct modified sugars, bases and/or backbones may be combined into one single oligonucleotide of the invention.
A person skilled in the art will also recognize that there are many synthetic derivatives of oligonucleotides.
Preferably, said oligonucleotide comprises RNA, as RNA/RNA duplexes are very stable. It is preferred that an RNA oligonucleotide comprises a modification providing the RNA with an additional property, for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, increased activity, reduced toxicity, increased intracellular transport, tissue-specificity, etc. In addition, the mRNA complexed with the oligonucleotide of the invention is preferably not susceptible to RNaseH cleavage. Preferred modifications have been identified above. Oligonucleotides containing at least in part naturally occurring DNA nucleotides are useful for inducing degradation of DNA-RNA hybrid molecules in the cell by RNase H activity (EC.3.1.26.4).
Naturally occurring RNA ribonucleotides or RNA-like synthetic ribonucleotides comprising oligonucleotides are encompassed herein to form double stranded RNA-RNA hybrids that act as enzyme-dependent antisense through the RNA interference or silencing (RNAi/siRNA) pathways, involving target RNA recognition through sense-antisense strand pairing followed by target RNA degradation by the RNA-induced silencing complex (RISC).
Alternatively or in addition, an oligonucleotide can interfere with the processing or expression of precursor RNA or messenger RNA (steric blocking, RNaseH independent processes) in particular but not limited to RNA splicing and exon skipping, by binding to a target sequence of RNA transcript and getting in the way of processes such as translation or blocking of splice donor or splice acceptor sites. Moreover, the oligonucleotide may inhibit the binding of proteins, nuclear factors and others by steric hindrance and/or interfere with the authentic spatial folding of the target RNA and/or bind itself to proteins that originally bind to the target RNA and/or have other effects on the target RNA, thereby contributing to the destabilization of the target RNA, preferably pre-mRNA, and/or to the decrease in amount of diseased or toxic transcript and/or protein in diseases like HD as identified later herein.
As herein defined, an oligonucleotide may comprise nucleotides with (RNaseH resistant) chemical substitutions at least one of its 5′ or 3′ ends, to provide intracellular stability, and comprises less than 9, more preferably less than 6 consecutive (RNaseH-sensitive) deoxyribose nucleotides in the rest of its sequence. The rest of the sequence is preferably the center of the sequence. Such oligonucleotide is called a gapmer. Gapmers have been extensively described in WO 2007/089611. Gapmers are designed to enable the recruitment and/or activation of RNaseH. Without wishing to be bound by theory, it is believed that RNaseH is recruited and/or activated via binding to the central region of the gapmer made of deoxyriboses. An oligonucleotide of the invention which is preferably substantially independent of or independent of RNaseH is designed in order to have a central region which is substantially not able or is not able to recruit and/or activate RNaseH. In a preferred embodiment, the rest of the sequence of said oligonucleotide, more preferably its central part comprises less than 9, 8, 7, 6, 5, 4, 3, 2, 1, or no deoxyribose. Accordingly, this oligonucleotide of the invention is preferably partly to fully replaced as earlier defined herein. “Partly replaced” means that the oligonucleotide comprises at least some of nucleotides that have been replaced, preferably at least 50% of its nucleotides have been replaced, or at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% have been replaced. 100% replacement of nucleotides corresponds to “fully replaced”.
Accordingly, the invention provides an oligonucleotide comprising a 2′-O-methyl phosphorothioate RNA residue or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylpyrimidine and/or a 2,6-diaminopurine base. Most preferably, this oligonucleotide consists of 2′-O-methyl RNA residues connected through a phosphorothioate backbone and all of its cytosines and/or all of its uracils and/or all of its adenines, independently, have been replaced by 5-methylcytosine, 5-methyluracil and/or 2,6-diaminopurine, respectively. Thus, an oligonucleotide of the invention may have:
At least one and preferably all cytosines replaced with 5-methylcytosines,
At least one and preferably all cytosines replaced with 5-methylcytosines and at least one and preferably all uracils replaced with 5-methyluracils,
At least one and preferably all cytosines replaced with 5-methylcytosines and at least one and preferably all adenines replaced with 2,6-diaminopurines,
At least one and preferably all cytosines replaced with 5-methylcytosines and at least one and preferably all uracils replaced with 5-methyluracils and at least one and preferably all adenines replaced with 2,6-diaminopurines,
At least one and preferably all uracils replaced with 5-methyluracils,
At least one and preferably all uracils replaced with 5-methyluracils and at least one and preferably all adenines replaced with 2,6-diaminopurines, or
At least one and preferably all adenines replaced with 2,6-diaminopurines.
An oligonucleotide of the invention is for use as a medicament for preventing delaying and/or treating a human cis-element repeat instability associated genetic disorders preferably as exemplified herein. A human cis-element repeat instability associated genetic disorders as identified herein is preferably a neuromuscular disorder. Preferably said oligonucleotide is for use in therapeutic RNA modulation. Therefore, the oligonucleotide according to the invention may be described as an antisense oligonucleotide (AON). An antisense oligonucleotide is an oligonucleotide which binds (or is able to bind), targets, hybridizes to (or is able to hybridize to) and/or is reverse complementary to a specific sequence of a transcript of a gene which is known to be associated with or involved in a human cis-element repeat instability associated genetic neuromuscular disorder.
According to the invention, an antisense oligonucleotide comprising or consisting of 2′-O-methyl RNA nucleotide residues, having a backbone wherein at least one phosphate moiety is replaced by a phosphorothioate moiety, and further comprising at least one of a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a repetitive element in a RNA transcript having as repetitive nucleotide unit a repetitive nucleotide unit, which is selected from the (CAG)n, (GCG)n, (CGG)n, (GAA)n, (GCC)n, (CCG)n, (AUUCU)n, (GGGGCC)n or (CCUG)n. Said oligonucleotide is preferably a single stranded oligonucleotide.
Although it is to be understood that an oligonucleotide of the invention binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a repetitive element present in a RNA transcript as identified above, it can not be ruled out that such oligonucleotide may also interfere with or bind (or is able to bind) or hybridize to (or is able to hybridize) a corresponding DNA, this RNA transcript is derived from.
A repeat or repetitive element or repetitive sequence or repetitive stretch is herein defined as a repetition of at least 3, 4, 5, 10, 100, 1000 or more, of a repetitive unit or repetitive nucleotide unit or repeat nucleotide unit (as (CAG)n, (GCG)n, (CGG)n, (GAA)n, (GCC)n, (CCG)n, (AUUCU)n, (GGGGCC)n or (CCUG)n), comprising a trinucleotide repetitive unit, or alternatively a 4, 5 or 6 nucleotide repetitive unit, in a transcribed gene sequence in the genome of a subject, including a human subject. Accordingly, n is an integer and may be at least 3, 4, 5, 10, 100, 1000 or more. The invention is not limited to exemplified repetitive nucleotide units. Other repetitive nucleotide unit could be found on the following site http://neuromuscular.wustl.edu.mother/dnarep.htm. In the majority of patients, a “pure” repeat or repetitive element or repetitive sequence or repetitive stretch as identified above (as (CAG)n, (GCG)n, (CGG)n, (GAA)n, (GCC)n, (CCG)n, (AUUCU)n, (GGGGCC)n or (CCUG)n) is present in a transcribed gene sequence in the genome of said patient. However, it is also encompassed by the invention, that in some patients, said repeat or repetitive element or repetitive sequence or repetitive stretch as identified above is not qualified as “pure” or is qualified as a “variant” when for example said repeat or repetitive element or repetitive sequence or repetitive stretch as identified above is interspersed with at least 1, 2, or 3 nucleotide(s) that do not fit the nucleotide(s) of said repeat or repetitive element or repetitive sequence or repetitive stretch (Braida C., et al).
An oligonucleotide according to the invention therefore may not need to be 100% reverse complementary to a targeted repeat. Usually an oligonucleotide of the invention may be at least 90%, 95%, 97%, 99% or 100% reverse complementary to a targeted repeat.
In an embodiment, an antisense oligonucleotide comprises or consists of 2′-O-methyl phosphorothioate RNA, comprises a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (CAG)n tract in a transcript and is particularly useful for the treatment, delay, amelioration and/or prevention of the human genetic diseases Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA) and/or dentatorubropallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in the transcripts of the HTT (SEQ ID NO: 80), ATXN1 (SEQ ID NO:81), ATXN2 (SEQ ID NO: 82) ATXN3 (SEQ ID NO: 83), CACNA1A (SEQ ID NO:84), ATXN7 (SEQ ID NO: 85), PPP2R2B (SEQ ID NO: 86), TBP (SEQ ID NO: 87), AR (SEQ ID NO: 88) or ATN1 (SEQ ID NO: 89) genes. Preferably, these genes are from human origin. In this embodiment, an oligonucleotide comprises or consists of 2′-O-methyl phosphorothioate RNA, comprises a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (CAG)n repeat as identified above and has as repetitive nucleotide unit (CUG)m. The m in (CUG)m is preferably an integer which is 4, 5, 6, 7, 8, 9, 10, 11, 12. In a preferred embodiment, m is 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12.
It is to be noted that for ALS and FTD, it is known that at least two distinct repeats in at least two distinct transcripts may be involved or may be responsible or linked with the disease. One has been identified in the previous paragraph (i.e. (CAG)n in a ATXN2 transcript). Another one is being identified later as a (GGGGCC)n repeat or tract in a C9ORF72 transcript. It means that for each of these two diseases, one may envisage to use either one of these two distinct oligonucleotides of the invention to specifically induce the specific degradation of the corresponding (toxic) expanded repeat transcripts.
Throughout the application, an oligonucleotide defined as being reverse complementary to, binding (being able to bind), hybridizing (being able to hybridize) or targeting a repeat as identified above and has or comprises a repetitive nucleotide unit may have any length comprised from 12 to 36 nucleotides. If we take the example of CUG as repetitive nucleotide unit comprised within said oligonucleotide, any oligonucleotide comprising UGC or GCU as repetitive nucleotide unit is also encompassed by the present invention. Depending on the length of said oligonucleotide (for example from 12 to 36 nucleotides), the given repetitive nucleotide unit may not be complete at the 5′ and/or at the 3′ side of said oligonucleotide. Each of said oligonucleotide is encompassed within the scope of said invention.
Alternatively, if we still take as an example the oligonucleotide having CUG as repetitive nucleotide unit, it may be represented by H-(P)p-(CUG)m-(Q)q-H, wherein m is an integer as defined above. Each occurrence of P and Q is, individually, an abasic monomer as defined above or a nucleotide, such as A, C, G, U or an analogue or equivalent thereof and p and q are each individually an integer, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher up to 100. Thus, p and q are each individually an integer from 0 to 100, preferably an integer from 0 to 20, more preferably an integer from 0 to 10, more preferably from 0 to 6, even more preferably from 0 to 3. Thus, when p is 0, P is absent and when q is 0, Q is absent. The skilled person will appreciate that an oligonucleotide will always start with and end with a hydrogen atom (H), regardless of the amount and nature of the nucleotides present in the oligonucleotide.
It will be appreciated that herein (CUG)m may be replaced by any repeating nucleotide unit within the context of the invention. Thus, a preferred oligonucleotide according to the invention may be represented by H-(P)p-(R)r-(Q)q-H, wherein (R)r is a repeating nucleotide unit within the context of the invention and P, Q, p and q are as defined above.
In the context of the present invention, an “analogue” or an “equivalent” of a nucleotide is to be understood as a nucleotide which comprises at least one modification with respect to the nucleotides naturally occurring in RNA, such as A, C, G and U. Such a modification may be a internucleoside linkage modification and/or a sugar modification and/or a base modification, as explained and exemplified above. Again taking the oligonucleotide having CUG as repetitive nucleotide unit, it is to be understood that the repeating sequence may start with either a C, a U or a G. Thus, in a preferred embodiment, p is not 0, and (P)p is represented by (P′)p′UG or (P′)p″G, wherein each occurrence of P′ is, individually, an abasic site or a nucleotide, such as A, C, G, U or an analogue or equivalent thereof, and p′ is p−2 and p″ is p−1. Such oligonucleotides may be represented as:
H-(P′)p′UG-(CUG)m-(Q)q-H or
H-(P′)p″G-(CUG)m-(Q)q-H.
In an equally preferred embodiment, q is not 0, and (Q)q is represented by CU(Q′)q′ or C(Q′)q″ and each occurrence of Q′ is, individually, an abasic site or a nucleotide, such as A, C, G, U or an analogue or equivalent thereof, and q′ is q−2 and q″ is q−1. Such oligonucleotides may be represented as:
H-(P)p-(CUG)m-CU(Q′)q′-H or
H-(P)p-(CUG)m-C(Q′)q″-H.
In another preferred embodiment, both p and q are not 0, and both (P)p and (Q)q are represented by (P′)p′UG or (P′)p″G and CU(Q′)q′ or C(Q′)q″ respectively, wherein P′, Q′, p′, p″, q′ and q″ are as defined above. Such oligonucleotides may be represented as:
H-(P′)p′UG-(CUG)m-CU(Q′)q′-H,
H-(P′)p″G-(CUG)m-CU(Q′)q′-H,
H-(P′)p′UG-(CUG)m-C(Q′)q″-H, or
H-(P′)p″G-(CUG)m-C(Q′)q″-H.
It is to be understood that p′, p″, q′ and q″ may not be negative integers. Thus, when (P)p is represented by (P′)p′UG or (P′)p″G, p is at least 1 or at least 2 respectively, and when (Q)q is represented by CU(Q′)q′ or C(Q′)q″, q is at least 1 or at least 2 respectively.
It is to be understood that all said here regarding the CUG repeat unit can be extended to any repeat unit within the context of the invention.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (CAG)n repeat comprises or consists of a repetitive nucleotide unit (XYG)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil. m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XYG)m, wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil, and m is an integer from 4 to 12 (SEQ ID NO:2 to 12).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XYG)m, wherein each X is 5-methylcytosine, and/or each Y is 5-methyluracil, and m is an integer from 4 to 12 (SEQ ID NO:2 to 12).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XYG)5, (XYG)6 or (XYG)7, (XYG)8, or (XYG)9 wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil. More preferred is an oligonucleotide comprising or consisting of (XYG)7, wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil (SEQ ID NO:7).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XYG)7, wherein each X is 5-methylcytosine and each Y is a uracil (SEQ ID NO: 2), or each X is a cytosine and each Y is 5-methyluracil (SEQ ID NO:3). An even more preferred oligonucleotide comprises SEQ ID NO:2 or 3 and has a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XYG)m have been identified in table 2 as SEQ ID NO:90-118.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 90-106 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 90-106 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 90-106 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 107 or 108 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 107 or 108 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 107 or 108 and has a length of 21 nucleotides.
Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 107 or 108, has a length of 21 nucleotides and additionally comprises 4 abasic monomers at one of its termini, preferably at the 3′ terminus. Said most preferred oligonucleotide is represented by a base sequence consisting of SEQ ID NO: 220 or 221.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 109 or 110 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 109 or 110 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 109 or 110 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 111 or 112 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 111 or 112 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 111 or 112 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 113 or 114 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 113 or 114 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 113 or 114 and has a length of 30 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 115 or 116 and has a length from 33-36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 115 or 116 and has a length from 33-36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 115 or 116 and has a length of 33 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 117 or 118 and has a length of 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 117 or 118 and has a length of 36 nucleotides.
In another embodiment, an antisense oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA, and comprising a 5-methylcytosine is represented by a nucleotide sequence comprising or consisting of a sequence that binds to (or is able to bind to), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (GCG)n repeat in a transcript and is particularly useful for the treatment, delay, amelioration and/or prevention of the human genetic diseases: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyly, oculopharyngeal muscular dystrophy and/or holoprosencephaly, which are caused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM/PABP2, TCFBR1 or ZIC2 genes. Preferably, these genes are from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (GCG)n repeat comprises or consists of a repetitive nucleotide unit (XGX)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine, such that at least one X is 5-methylcytosine. m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XGX)m, wherein at least one X is 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 13 to 21). An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XGX)m, wherein each X is 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 13 to 21).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XGX)7 (SEQ ID NO: 16), wherein at least one X is 5-methylcytosine.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XGX)7 (SEQ ID NO: 16), wherein each X is 5-methylcytosine.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XGX)m have been identified in table 2 as SEQ ID NO:119-132.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 119 or 120 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 119 or 120 and has a length from 16-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 119 or 120 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 121 or 122 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 90-106 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 121 or 122 and has a length of 15 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 123 or 124 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 123 or 124 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 123 or 124 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 125 or 126 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 125 or 126 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 125 or 126 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 127 or 128 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 127 or 128 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 127 or 128 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 129 or 130 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 129 or 130 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 129 or 130 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 131 or 132 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 131 or 132 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 131 or 132 and has a length of 30 nucleotides.
In another embodiment, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), targets, hybridizes (or is able to hybridize) and/or is reverse complementary to a (CGG)n repeat in a transcript and is particularly useful for the treatment, delay, amelioration and/or prevention of human fragile X syndromes, caused by repeat expansion in the FMR1 gene. Preferably, these genes are from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or is able to bind), hybridizing (or is able to hybridize) or targeting a (CGG)n repeat comprises or consists of a repetitive nucleotide unit (XXG)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine, such that at least one X is 5-methylcytosine.
m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XXG), wherein each X is C or 5-methylcytosine, such that at least one X is 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 22 to 30).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XXG)m, wherein each X is 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 22 to 30).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XXG)7 (SEQ ID NO: 25), wherein each X is C or 5-methylcytosine, such that at least one X is 5-methylcytosine.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XXG)7 (SEQ ID NO: 25), wherein each X is 5-methylcytosine.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XXG)m have been identified in table 2 as SEQ ID NO: 133-146.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 133 or 134 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 133 or 134 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 133 or 134 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 135 or 136 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 135 or 136 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 135 or 136 and has a length of 15 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 137 or 138 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 137 or 138 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 137 or 138 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 139 or 140 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 139 or 140 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 139 or 140 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 141 or 142 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 141 or 142 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 141 or 142 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 143 or 144 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 143 or 144 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 143 or 144 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 145 or 146 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 145 or 146 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 145 or 146 and has a length of 30 nucleotides.
In another embodiment, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine and/or a 5-methyluracil, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), targets, hybridizes (or is able to hybridize) and/or is reverse complementary to a (GAA)n repeat in a transcript and is particularly useful for the treatment, delay and/or prevention of the human genetic disorder Friedreich's ataxia.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (GAA)n repeat comprises or consists of a repetitive nucleotide unit (YYX)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil.
m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (YYX)m, wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil, and m is an integer from 4 to 12 (SEQ ID NO: 31 to 39).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (YYX)m, wherein each X is 5-methylcytosine, and/or each Y is 5-methyluracil, and m is an integer from 4 to 12 (SEQ ID NO: 31 to 39).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (YYX)7 (SEQ ID NO: 34), wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil such that at least one X is 5-methylcytosine and/or at least one Y is 5-methyluracil.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (YYX)7 (SEQ ID NO: 34), wherein each X is 5-methylcytosine, and/or each Y is 5-methyluracil.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XYG)m have been identified in table 2 as SEQ ID NO: 147-167.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 147 or 148 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 147 or 148 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 147 or 148 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 149 or 150 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 149 or 150 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 149 or 150 and has a length of 15 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 151 or 152 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 151 or 152 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 151 or 152 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 153-157 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 153-157 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 153-157 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 158 or 159 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 158 or 159 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 158 or 159 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 160 or 161 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 160 or 161 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 160 or 161 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 162 or 163 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 162 or 163 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 162 or 163 and has a length of 30 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 164 or 165 and has a length from 33-36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 164 or 165 and has a length from 33-36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has a base sequence that consists of one of the base sequences SEQ ID NO: 164 or 165 and has a length of 33 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 166 or 167 and has a length of 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and has a base sequence that consists one of the base sequences SEQ ID NO: 166 or 167 and has a length of 36 nucleotides.
In another embodiment, an antisense oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds to (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (CCG)n or (GCC)n repeat in a transcript and is particularly useful for the treatment, delay, amelioration and/or prevention of the human genetic disorder fragile XE mental retardation, caused by repeat expansion in the FMR2 gene. Preferably, these genes are from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (CCG)n repeat comprises or consists of a repetitive nucleotide unit (XGG)m or (GGX)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine. m is an integer. In the context of this embodiment, m may be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value for m is 7.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XGG)m or (GGX)m, wherein each X is C or 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 49 to 57) or (SEQ ID NO: 40 to 48).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XGG)m or (GGX)m, wherein each X is 5-methylcytosine, and m is an integer from 4 to 12 (SEQ ID NO: 49 to 57) or (SEQ ID NO: 40 to 48).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XGG)7 (SEQ ID NO: 52) or (GGX)7 (SEQ ID NO: 43), wherein each X is C or 5-methylcytosine.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XGG)7 (SEQ ID NO: 52) or (GGX)7 (SEQ ID NO: 43), wherein each X is 5-methylcytosine.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (GGX)m have been identified in table 2 as SEQ ID NO: 168-177.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 168 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequences SEQ ID NO: 168 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequences SEQ ID NO: 168 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 169 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 169 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 169 and has a length of 15 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 170 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 170 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 170 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 171-174 has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 171-174 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 171-174 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 175 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 175 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 175 and has a length of 24 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 176 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 176 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 176 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 177 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 177 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 177 and has a length of 30 nucleotides.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XGG)m have been identified in table 2 as SEQ ID NO: 178-184.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 178 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 178 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 178 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 179 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 179 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 179 and has a length of 15 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequences SEQ ID NO: 180 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 180 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 180 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 181 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 181 and has a length from 21-36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 181 and has a length of 21 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 182 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 182 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 182 and has a length of 24 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 183 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 183 and has a length from 27-36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 183 and has a length of 27 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 184 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 184 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 184 and has a length of 30 nucleotides.
In another embodiment, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine and/or a 2,6-diaminopurine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (CCUG)n repeat in a transcript and is particularly useful for the treatment, delay and/or prevention of the human genetic disorder myotonic dystrophy type 2 (DM2), caused by repeat expansions in the DM2/ZNF9 gene. Preferably, these genes are from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (CCUG)n repeat comprises or consists of a repetitive nucleotide unit (XZGG)m and has a length comprised from 12 to 36 nucleotides and wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine such that at least one X is 5-methylcytosine and/or at least one Z is 2,6-diaminopurine.
m is an integer. In the context of this embodiment, m may be 3, 4, 5, 6, 7, 8, 9. A preferred value for m is 5.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XZGG)m, wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine such that at least one X is 5methyl-cytosine and/or at least one A is 2,6-diaminopurine, and m is an integer from 3 to 9 (SEQ ID NO: 63 to 69).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XZGG)m, wherein each X is 5-methylcytosine, and/or each Z is 2,6-diaminopurine, and m is an integer from 3 to 9 (SEQ ID NO: 63 to 69).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (XZGG)5 (SEQ ID NO: 65), wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine such that at least one X is 5-methylcytosine and/or at least one Z is 2,6-diaminopurine.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (XZGG)5 (SEQ ID NO: 65), wherein each X is 5-methyl-cytosine, and/or each Z is 2,6-diaminopurine.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (XZGG)m have been identified in table 2 as SEQ ID NO: 193-208.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 193 or 194 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 193 or 194 and has a length from 12-36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 193 or 194 and has a length of 12 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 195 or 196 and has a length from 16-36 nucleotides, more preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 195 or 196 and has a length from 16-36 nucleotides, more preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 195 or 196 and has a length of 16 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 197-200 and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 197-200 and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 197-200 and has a length of 20 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 201 or 202 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 201 or 202 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 201 or 202 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 203 or 204 and has a length from 28-36 nucleotides, more preferably 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 203 or 204 and has a length from 28-36 nucleotides, more preferably 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 203 or 204 and has a length of 28 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 205 or 206 and has a length from 32-36 nucleotides, more preferably 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 205 or 206 and has a length from 32-36 nucleotides, more preferably 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 205 or 206 and has a length of 32 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 207 or 208 and has a length of 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and has its base sequence that consists of one of the base sequences SEQ ID NO: 207 or 208 and has a length of 36 nucleotides.
In another embodiment, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methyluracil and/or a 2,6-diaminopurine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (AUUCU)n repeat in an intron and is particularly useful for the treatment, delay, amelioration and/or prevention of the human genetic disorder spinocerebellar ataxia type 10 (SCA10). Preferably, this gene is from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (AUUCU)n repeat comprises or consists of a repetitive nucleotide unit (ZGZZY)m and has a length comprised from 12 to 36 nucleotides and wherein each Y is U or 5-methyluracil, and each Z is A or 2,6-diaminopurine such that at least one Y is 5-methyluracil and/or at least one Z is 2,6-diaminopurine. m is an integer. In the context of this embodiment, m may be 3, 4, 5, 6, 7. A preferred value for m is 4.
A more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (ZGZZY)m, wherein each Y is U or 5-methyluracil, and each Z is A or 2,6-diaminopurine such that at least one Y is 5-methyluracil and/or at least one Z is 2,6-diaminopurine, and m is an integer from 3 to 7 (SEQ ID NO: 58 to 62).
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (ZGZZY)m, wherein each Y is 5-methyluracil, and/or each Z is 2,6-diaminopurine, and m is an integer from 3 to 7 (SEQ ID NO: 58 to 62).
An even more preferred oligonucleotide therefore comprises or consists of a repetitive nucleotide unit (ZGZZY)4 (SEQ ID NO: 59), wherein each Y is C or 5-methyluracil, and each Z is A or 2,6-diaminopurine such that at least one Y is 5-methyluracil and/or at least one Z is 2,6-diaminopurine.
An even more preferred oligonucleotide comprises or consists of a repetitive nucleotide unit (ZGZZY)4 (SEQ ID NO: 59), wherein each Y is 5-methyluracil, and/or each Z is 2,6-diaminopurine.
Most preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (ZGZZY)m have been identified in table 2 as SEQ ID NO:185-192.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 185 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 185 and has a length from 15-36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 185 and has a length of 15 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 186-189 and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 186-189 and has a length from 20-36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 186-189 and has a length of 20 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 190 and has a length from 25-36 nucleotides, more preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 190 and has a length from 25-36 nucleotides, more preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 190 and has a length of 25 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 191 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 191 and has a length from 30-36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 191 and has a length of 30 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 192 and has a length from 35-36 nucleotides, more preferably 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 192 and has a length from 35-36 nucleotides, more preferably 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 192 and has a length of 35 nucleotides.
In another embodiment, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA and comprising a 5-methylcytosine and/or a abasic monomer, and/or a inosine, is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a (GGGGCC)n repeat present in a C9ORF72 human transcript and is particularly useful for the treatment, delay, amelioration and/or prevention of the human genetic disorder amylotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). Preferably, this gene is from human origin.
In a preferred embodiment, an oligonucleotide defined as being reverse complementary to, binding (or being able to bind), hybridizing (or being able to hybridize) or targeting a (GGGGCC)n repeat comprises or consists of a repetitive nucleotide unit (GGXUXX)m, (GGXQXX)m, (GGXIXX)m, or (GGCCUC)m, and has a length comprised from 17 to 36 nucleotides and wherein each X is C or 5-methylcytosine such that at least one X is 5-methylcytosine, wherein each Q is an abasic monomer, wherein each I is an inosine, and wherein m is an integer. In the context of this embodiment, m may be 3, 4, 5, 6, 7. A preferred value for m is 3 or 4.
More preferably, said oligonucleotide comprises or consists of a repetitive nucleotide unit SEQ ID NO: 216-219 as defined in table 1. Even more preferred oligonucleotides sequences comprising or consisting of a repetitive nucleotide unit (GGXUXX)m, (GGXQXX), (GGXIXX), or (GGCCUC)m, have been identified in table 2 as SEQ ID NO:209-215.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 209 or 211 and has a length from 17-36 nucleotides, more preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 209 or 211 and has a length from 17-36 nucleotides, more preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 209 or 211 and has a length of 17 or 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 210 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 210 and has a length from 18-36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequence SEQ ID NO: 210 and has a length of 18 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises one of base sequences SEQ ID NO: 212 or 215 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises one of the base sequences SEQ ID NO: 212 or 215 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of one of the base sequences SEQ ID NO: 212 or 215 and has a length of 24 nucleotides. A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 213 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 213 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequences SEQ ID NO: 213 and has a length of 24 nucleotides.
A preferred oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA comprises base sequence SEQ ID NO: 214 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. An even more preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA and comprises base sequence SEQ ID NO: 214 and has a length from 24-36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotide consists of 2′-O-methyl phosphorothioate RNA, has its base sequence that consists of base sequences SEQ ID NO: 214 and has a length of 24 nucleotides.
In an embodiment, an oligonucleotide preferably comprises or consists of 2′-O-methyl phosphorothioate RNA, comprises a 5-methylcytosine and/or a 5-methyluracil and/or a 2,6-diaminopurine base, is represented by a nucleotide sequence comprising or consisting of at least 12 to 36 consecutive nucleotides, said oligonucleotide targeting, hybridizing (or is able to hybridize), binding (or is able to bind) and/or being reverse complementary to a repeat as earlier defined herein More preferably, said nucleotide sequence comprising or consisting of at least 12 to 36 nucleotides, even more preferably 15 to 24, and most preferably 20 or 21 nucleotides. The length of said oligonucleotide may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. Said oligonucleotide may be reverse complementary to and/or capable of hybridizing to and/or capable of targeting and/or capable of binding to a repeat in a coding region of a transcript, preferably a polyglutamine (CAG)n coding tract. Said oligonucleotide may also be reverse complementary to and/or capable of hybridizing to and/or capable of targeting and/or capable of binding to a non-coding region for instance 5′ or 3′ untranslated regions, or intronic sequences present in precursor RNA molecules.
In the context of the invention, the expression “capable of” may be replaced with “is able to”.
In a second aspect, the present invention relates to an oligonucleotide, which comprises one or more abasic sites, as defined further below, at one or both termini. Preferably 1 to 10, more preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 and most preferably 4 abasic sites are present at a single terminus or at both termini of the oligonucleotide. One or more abasic sites may be present and both free termini of the oligonucleotide (5′ and 3′), or at only one. The oligonucleotide according to this aspect of the invention preferably is represented by a nucleotide or a base sequence comprising or consisting of a sequence that binds (or is able to bind), hybridizes (or is able to hybridize), targets and/or is reverse complementary to a repetitive element in a RNA transcript selected from the (CAG)n, (GCG)n, (CGG)n, (GAA)n, (GCC)n, (CCG)n, (AUUCU)n, (GGGGCC)n or (CCUG)n, as indicated above. Said oligonucleotide is preferably a single stranded oligonucleotide, and may further optionally comprise any of the modifications as discussed herein, such as one or more base modifications, sugar modifications and/or backbone modifications, such as 5-methyl-C, 5-methyl-U, 2,6-diaminopurine, 2′-O-methyl, phosphorothioate, and combinations thereof. It is to be understood that in this aspect of the invention, these modification are not compulsory.
The oligonucleotide according to this aspect of the invention, comprising one or more abasic sites at one or both termini has an improved parameter over the oligonucleotides without such abasic sites. In this context, parameters may include: binding affinity and/or kinetics, silencing activity, allelic selectivity, biostability, (intra-tissue) distribution, cellular uptake and/or trafficking, and/or immunogenicity of said oligonucleotide, as explained earlier herein in connection with the improved parameter of an oligonucleotide of the invention of the first aspect. Each of the assays and definitions provided herein in connection with the improvement of a parameter of an oligonucleotide of the first aspect also hold for an oligonucleotide of the second aspect.
Below, an oligonucleotide comprising or consisting of 2′-O-methyl phosphorothioate RNA, comprising a 5-methylcytosine and/or a 5-methyluracil base and being represented by a nucleotide or a base sequence comprising (CUG)m and thus binding to (or being able to bind to), hybridizing (or being able to hybridize), targeting and/or being reverse complementary to (CAG)n is taken as an example to further illustrate the invention. Similar parameters defined in the context of such oligonucleotide could be defined by the skilled person for other oligonucleotides falling under the scope of the invention and binding to (or being able to bind to), hybridizing (or being able to hydridize), targeting and/or being reverse complementary to other repeats as identified herein. Other or similar symptoms may be identified by the skilled person concerning other diseases as identified herein.
In a preferred embodiment, in the context of the invention, an oligonucleotide as designed herein is able to delay and/or cure and/or treat and/or prevent and/or ameliorate a human genetic disorder as Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA) and/or dentatorubropallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in the transcripts of a HTT (SEQ ID NO: 80), ATXN1 (SEQ ID NO: 81), ATXN2 (SEQ ID NO: 82) ATXN3 (SEQ ID NO: 83), CACNA1A (SEQ ID NO: 84), ATXN7 (SEQ ID NO: 85), PPP2R2B (SEQ ID NO: 86), TBP (SEQ ID NO: 87), AR (SEQ ID NO: 88), ATN1 (SEQ ID NO: 89) genes when this oligonucleotide is able to reduce or decrease the amount of (toxic) transcript of a diseased allele of a HTT, ATXN1, ATXN2 ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 gene in a cell of a patient, in a tissue of a patient and/or in a patient. In an embodiment, said HTT, ATXN1, ATXN2 ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes are human genes.
In the case of HD, an expanded CAG repeat region is present in exon 1 of the HTT gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 38 to 180 repetitive units comprising a CAG trinucleotide, in a transcribed sequence of the HTT gene
In the case of SCA1, an expanded CAG repeat region is present in exon 8 of the ATXN1 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 41 to 83 repetitive units comprising a CAG trinucleotide, in a transcribed sequence of the ATXN1 gene.
In the case of SCA2, an expanded CAG repeat region is present in exon 1 of the ATXN2 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 32 to 200 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the ATXN2 gene.
In the case of SCA3, an expanded CAG repeat region is present in exon 8 of the ATXN3 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 52 to 86 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the ATXN3 gene.
In the case of SCA6, an expanded CAG repeat region is present in exon 47 of the CACNA1A gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 20 to 33 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the CACNA1A gene.
In the case of SCAT, an expanded CAG repeat region is present in exon 3 of the ATXN7 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 36 to at least 460 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the ATXN7 gene.
In the case of SCA12, an expanded CAG repeat region may be present in the 5′ untranslated region (UTR), in an intron or within an open reading frame of the PPP2R2B gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 66 to 78 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the PPP2R2B gene.
In the case of SCA17, an expanded CAG repeat region is present in exon 3 of the TBP gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 45 to 66 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the TBP gene.
In the case of ALS or FTD, an expanded CAG repeat region is present in exon 1 of the ATXN2 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 27 to 33 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the ATXN2 gene.
In the case of ALS or FTD, an expanded GGGGCC repeat region is present in the first intron of the C9ORF72 gene in the genome of a patient. An expanded GGGGCC repeat region may be defined herein as comprising a consecutive repetition of >30 repetitive units comprising a GGGGCC hexanucleotide in a transcribed sequence of the C9ORF72 gene.
In the case of SBMA, an expanded CAG repeat region is present in exon 1 of the AR gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 40 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the AR gene.
In the case of DRPLA, an expanded CAG repeat region is present in exon 5 of the ATN1 gene in the genome of a patient. An expanded CAG repeat region may be defined herein as comprising a consecutive repetition of 49 to 88 repetitive units comprising a CAG trinucleotide in a transcribed sequence of the ATN1 gene.
Throughout the invention, the term CAG repeat may be replaced by (CAG)n, and vice versa, wherein n is an integer that may be 6 to 29 when the repeat is present in exon 1 of the HTT transcript of a healthy individual, 6 to 39 when the repeat is present in exon 8 of the ATXN1 gene of a healthy individual, less than 31 when the repeat is present in exon 1 of the ATXN2 gene of a healthy individual, 12 to 40 when the repeat is present in exon 8 of the ATXN3 gene of a healthy individual, less than 18 when the repeat is present in exon 47 of the CACNA1A gene of a healthy individual, 4 to 17 when the repeat is present in exon 3 of the ATXN7 gene of a healthy individual, 7 to 28 when the repeat is present in the 5′UTR of the PPP2R2B gene of a healthy individual, 25 to 42 when the repeat is present in exon 3 of the TBP gene of a healthy individual, 13 to 31 when the repeat is present in exon 1 of the AR gene of a healthy individual, 12 to 40 when the repeat is present in exon 8 of the ATXN3 gene of a healthy individual, or 6 to 35 when the repeat is present in exon 5 of the ATN1 gene of a healthy individual.
It preferably means that an oligonucleotide of the invention reduces a detectable amount of disease-associated or disease-causing or mutant transcript containing an extending or unstable number of CAG repeats in a cell of said patient, in a tissue of said patient and/or in a patient. Alternatively or in combination with previous sentence, said oligonucleotide may reduce the translation of said mutant transcript and thus the amount of mutant (toxic) protein. The reduction or decrease of the amount of expanded CAG repeat transcripts may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% by comparison to the amount of expanded CAG repeat transcripts before the treatment. Another parameter may be the decrease in (CAG)n transcript or of the quantity of said mutant transcript. This may be of at least. 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% by comparison to the quantity of said transcript detected at the onset of the treatment
The reduction or decrease may be assessed by Northern Blotting or Q-RT-PCR, preferably as carried out in the experimental part. An oligonucleotide of the invention may first be tested in the cellular system as described in Example 1 in the experimental part.
Alternatively or in combination with previous preferred embodiment, in the context of the invention, an oligonucleotide as designed herein is able to delay and/or cure and/or treat and/or prevent and/or ameliorate a human genetic disorder as Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA) and/or dentatorubropallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in the transcripts of the HTT, ATXN1, ATXN2 ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes when this oligonucleotide is able to alleviate one or more symptom(s) and/or characteristic(s) and/or to improve a parameter linked with or associated with Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA) and/or dentatorubropallidoluysian atrophy (DRPLA) in an individual. An oligonucleotide as defined herein is able to improve one parameter or reduce a symptom or characteristic if after at least one week, one month, six month, one year or more of treatment using a dose of said oligonucleotide of the invention as identified herein said parameter is said to have been improved or said symptom or characteristic is said to have been reduced.
Improvement in this context may mean that said parameter had been significantly changed towards a value of said parameter for a healthy person and/or towards a value of said parameter that corresponds to the value of said parameter in the same individual at the onset of the treatment.
Reduction or alleviation in this context may mean that said symptom or characteristic had been significantly changed towards the absence of said symptom or characteristic which is characteristic for a healthy person and/or towards a change of said symptom or characteristic that corresponds to the state of the same individual at the onset of the treatment.
In this context, symptoms for Huntington's Disease are choreiform movements, progressive dementia and psychiatric manifestations (depression, psychosis, etc.). Choreiform movements consist of involuntary, rapid, irregular, jerky motor actions including facial twitching or writhing and twitching of distal extremities, and later more generalized forms that may impair gait (Ropper and Brown, 2005). Each of these symptoms may be assessed by the physician using known and described methods. A preferred method is monitoring of total functional capacity (TFC), a validated scale or symptom progression regarding the three main symptomatic areas of HD, measured by validated rating scales. These areas are specifically progression of motor signs, progression of neuropsychiatric symptoms and progression of cognitive decline. Another preferred scale therefore is the Unified HD Rating Scale (UHDRS; Huntington Study Group (Kieburtz K. et al. 1996; 11:136-142).
Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, or 17, X-linked spinal and bulbar muscular atrophy (SBMA) and dentatorubropallidoluysian atrophy (DRPLA) are all caused by CAG triplet repeat expansions in the coding region of the gene. Although the disease causing proteins in these diseases are different, in each case the resulting expanded stretch of glutamines results in a toxic-gain-of function of the protein and this leads to neurodegeneration. Protein aggregates are found in the nucleus and cytoplasm of cells, indicating that protein misfolding is a common feature of these disorders. A common preferred parameter is therefore (mutant) protein levels which can be determined by western blot analysis (Evers et al.), or the presence of protein aggregates in the nucleus and/or cytoplasm which can be monitored by in situ hybridization. An improvement of a HD parameter may be the decrease in the detection of the quantity or amount of protein aggregate. Such decrease may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% by comparison to the quantity or amount of protein aggregate before the onset of the treatment.
In the context of HD, various other proteins have been found to co-localize with htt aggregates, i.e. TATA box binding protein (TBP), CREB binding protein (CBP) and several molecular chaperones (Huang et al.; Muchowski et al.; Roon-Mom et al.; Steffan et al.). Also many affected cellular processes have been identified in HD, such as transcriptional de-regulation, mitochondrial dysfunction, and impaired vesicle transport, which may provide alternative parameters for HD (Bauer et al., 2009; Ross et al.). An improvement of each of these possible alternative HD parameters (i.e. TATA box binding protein (TBP), CREB binding protein (CBP) and several molecular chaperones) may be defined as for the improvement of protein aggregate as defined above.
Composition
In a second aspect, there is provided a composition comprising an oligonucleotide as described in the previous section entitled “Oligonucleotide”. This composition preferably comprises or consists of or essentially consists of an oligonucleotide as described above.
As explained in the first aspect of the invention for ALS and FTD, it is known that at least two distinct repeats in at least two distinct transcripts may be involved in, responsible for, or linked with the disease. All preferred features relating to each of these oligonucleotides have been disclosed in the section entitled “oligonucleotide”.
In a preferred embodiment, said composition is for use as a medicament. Said composition is therefore a pharmaceutical composition. A pharmaceutical composition usually comprises a pharmaceutically accepted carrier, diluent and/or excipient. In a preferred embodiment, a composition of the current invention comprises a compound as defined herein and optionally further comprises a pharmaceutically acceptable formulation, filler, preservative, solubilizer, carrier, diluent, excipient, salt, adjuvant and/or solvent. Such pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, salt, adjuvant, solvent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. The compound as described in the invention possesses at least one ionizable group. An ionizable group may be a base or acid, and may be charged or neutral. An ionizable group may be present as ion pair with an appropriate counterion that carries opposite charge(s). Examples of cationic counterions are sodium, potassium, cesium, Tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium, and tetraalkylammonium. Examples of anionic counterions are chloride, bromide, iodide, lactate, mesylate, acetate, trifluoroacetate, dichloroacetate, and citrate. Examples of counterions have been described [e.g. Kumar L. et al, 2008, which is incorporated here in its entirety by reference].
A pharmaceutical composition may be further formulated to further aid in enhancing the stability, solubility, absorption, bioavailability, pharmacokinetics and cellular uptake of said compound, in particular formulations comprising excipients capable of forming complexes, nanoparticles, microparticles, nanotubes, nanogels, hydrogels, poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes, and/or liposomes. Examples of nanoparticles include polymeric nanoparticles, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, protein nanoparticles and peptide nanoparticles.
A preferred composition comprises at least one excipient that may further aid in enhancing the targeting and/or delivery of said composition and/or said oligonucleotide to and/or into muscle and/or brain tissue and/or to a neuronal tissue and/or a cell. A cell may be a muscular or a neuronal cell.
Many of these excipients are known in the art (e.g. see Bruno, 2011) and may be categorized as a first type of excipient. Examples of first type of excipients include polymers (e.g. polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivatives, butylcyanoacrylate (PBCA), hexylcyanoacrylate (PHCA), poly(lactic-co-glycolic acid) (PLGA), polyamines (e.g. spermine, spermidine, putrescine, cadaverine), chitosan, poly(amido amines) (PAMAM), poly(ester amine), polyvinyl ether, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) cyclodextrins, hyaluronic acid, colominic acid, and derivatives thereof), dendrimers (e.g. poly(amidoamine)), lipids {e.g. 1,2-dioleoyl-3-dimethylammonium propane (DODAP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholine derivatives [e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)], lyso-phosphatidylcholine derivaties [e.g. 1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)], sphingomyeline, 2-{3-[bis-(3-amino-propyl)-amino]-propylamino}-N-ditetracedyl carbamoyl methylacetamide (RPR209120), phosphoglycerol derivatives [e.g. 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na), phosphaticid acid derivatives [1,2-distearoyl-sn-glycero-3-phosphaticid acid, sodium salt (DSPA), phosphatidylethanolamine derivatives [e.g. dioleoyl-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE),], N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), N-[1-(2,3-dioleylo xy)propyl]-N,N,N-trimethylammonium (DOTMA), 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER), (1,2-dimyristyolxypropyl-3-dimethylhydroxy ethyl ammonium (DMRIE), (N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN), dimethyldioctadecylammonium bromide (DDAB), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), (b-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-olelyl-amide trihydrochloride (AtuFECT01), N,N-dimethyl-3-aminopropane derivatives [e.g. 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA), 1,2-dilinoleyloxy-N,N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), phosphatidylserine derivatives [1,2-dioleyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS)], cholesterol}proteins (e.g. albumin, gelatins, atellocollagen), and peptides (e.g. protamine, PepFects, NickFects, polyarginine, polylysine, CADY, MPG).
Another preferred composition may comprise at least one excipient categorized as a second type of excipient. A second type of excipient may comprise or contain a conjugate group as described herein to enhance targeting and/or delivery of the composition and/or of the oligonucleotide of the invention to a tissue and/or cell and/or into a tissue and/or cell, as for example muscle or neuronal tissue or cell. Both types of excipients may be combined together into one single composition as identified herein.
The skilled person may select, combine and/or adapt one or more of the above or other alternative excipients and delivery systems to formulate and deliver a compound for use in the present invention.
Such a pharmaceutical composition of the invention may be administered in an effective concentration at set times to an animal, preferably a mammal More preferred mammal is a human being. An oligonucleotide or a composition as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing a disease or condition as identified herein, and may be administered directly in vivo, ex vivo or in vitro. Administration may be via systemic and/or parenteral routes, for example intravenous, subcutaneous, intraventricular, intrathecal, intramuscular, intranasal, enteral, intravitreal, intracerebral, epidural or oral route.
Preferably, such a pharmaceutical composition of the invention may be encapsulated in the form of an emulsion, suspension, pill, tablet, capsule or soft-gel for oral delivery, or in the form of aerosol or dry powder for delivery to the respiratory tract and lungs.
In an embodiment an oligonucleotide of the invention may be used together with another compound already known to be used for the treatment of said disease. Such other compounds may be used for slowing down progression of disease, for reducing abnormal behaviors or movements, for reducing muscle tissue inflammation, for improving muscle fiber and/or neuronal function, integrity and/or survival and/or improve, increase or restore cardiac function. Examples are, but not limited to, a steroid, preferably a (gluco)corticosteroid, an ACE inhibitor (preferably perindopril), an angiotensin II type 1 receptor blocker (preferably losartan), a tumor necrosis factor-alpha (TNFα) inhibitor, a TGFβ inhibitor (preferably decorin), human recombinant biglycan, a source of mIGF-1, a myostatin inhibitor, mannose-6-phosphate, dantrolene, halofuginone, an antioxidant, an ion channel inhibitor, a protease inhibitor, a phosphodiesterase inhibitor (preferably a PDES inhibitor, such as sildenafil or tadalafil, and/or PDE10A inhibitors and/or MP-10), L-arginine, dopamine blockers, amantadine, tetrabenazine, co-enzyme Q10, antidepressants, anti-psychotics, anti-epileptics, mood-stabilizers in general, omega-3-fatty acids, creatine monohydrate, KMO inhibitors (Kynurenine mono oxigenase) such as CHDI246, or HDAC4 inhibitors such as PBT2. Such combined use may be a sequential use: each component is administered in a distinct composition. Alternatively each compound may be used together in a single composition.
Use
In a further aspect, there is provided the use of a composition or an oligonucleotide as described in the previous sections for use as a medicament or part of therapy, or applications in which said oligonucleotide exerts its activity intracellularly.
Preferably, an oligonucleotide or composition of the invention is for use as a medicament or part of a therapy for preventing, delaying, curing, ameliorating and/or treating a human cis-element repeat instability associated genetic disorder. A human cis-element repeat instability associated genetic disorder is preferably a neuromuscular genetic disorder, more preferably as identified earlier herein.
Method
In a further aspect, there is provided a method for preventing, treating, curing, ameliorating and/or delaying a condition or disease as defined in the previous section in an individual, in a cell, tissue or organ of said individual. The method comprising administering an oligonucleotide or a composition of the invention to said individual or a subject in the need thereof.
The method according to the invention wherein an oligonucleotide or a composition as defined herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by any of the herein defined diseases or at risk of developing said disease, and may be administered in vivo, ex vivo or in vitro. An individual or a subject in need is preferably a mammal, more preferably a human being.
In a further aspect, there is provided a method for diagnosis wherein the oligonucleotide of the invention is provided with a radioactive label or fluorescent label. In this method, an oligonucleotide of the invention may be used as an in situ probe to detect foci (RNA/protein aggregates resulting from the repeat expansion) in a sample from a subject. Said sample comprises cells from said subject.
In an embodiment, in a method of the invention, a concentration of an oligonucleotide or composition is ranged from 0.01 nM to 1 μM. More preferably, the concentration used is from 0.05 to 500 nM, or from 0.1 to 500 nM, or from 0.02 to 500 nM, or from 0.05 to 500 nM, even more preferably from 1 to 200 nM.
Dose ranges of an oligonucleotide or composition according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. An oligonucleotide as defined herein may be used at a dose which is ranged from 0.01 to 200 mg/kg or 0.05 to 100 mg/kg or 0.1 to 50 mg/kg or 0.1 to 20 mg/kg, preferably from 0.5 to 10 mg/kg.
Dose ranges of an oligonucleotide or composition according to the invention may also be used at a dose which is
Ranged from 100 to 300 μg/week, 8 to 12 injections in total or
Ranged from 150 to 250 μg/week, 9 to 11 injections in total or
200 μg/week, 11 injections in total or
Ranged from 10 to 350 μg/day during two weeks or
Ranged from 50 to 250 μg/day during two weeks or
Ranged from 100 to 200 μg/day during two weeks or
Ranged from 20 to 80 μg/day during two weeks or
Ranged from 200 to 320 μg/day during two weeks or
320 μg/day, during two weeks or
30 μg/day, during two weeks.
The ranges of concentration or dose of oligonucleotide or composition as given above are preferred concentrations or doses for in vitro or ex vivo uses. The skilled person will understand that depending on the identity of the oligonucleotide used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of oligonucleotide used may further vary and may need to be optimised any further.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb “to comprise” is synonymous with the verb “to have” unless otherwise indicated. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that an oligonucleotide or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
DEFINITIONS
Throughout the application, the word “binds”, “targets”, “hybridizes” could be used interchangeably when used in the context of an antisense oligonucleotide which is reverse complementary to a part of a pre-mRNA as identified herein. In the context of the invention, “hybridizes” or “binds” is used under physiological conditions in a cell, preferably a human cell unless otherwise indicated.
As used herein, “hybridization” refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymine, 5-methyluracil and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl-cytosine. Hybridization can occur under varying circumstances. In particular, hybridization of an oligonucleotide of the invention with a targeted pre-mRNA can occur under varying circumstances. Similarly, binding of an oligonucleotide of the invention to a targeted pre-mRNA can occur under varying circumstances. Preferably, said hybridization or said binding is assessed under physiological conditions in a cell, more preferably in a human cell. An oligonucleotide of the invention is preferably said to be able to bind to, or capable of binding to, or able to hybridize with, or capable of hybridizing with, when said binding or hybridization occurs under physiological conditions in a cell, preferably a human cell.
As used herein, “nucleotide” refers to a nucleoside further comprising a modified or unmodified phosphate linking group or a non-phosphate internucleoside linkage.
As used herein, “nucleotide analogue” or “nucleotide equivalent” refers to a nucleotide, which comprises at least one modification with respect to the nucleotides naturally occurring in RNA, such as A, C, G and U. Such a modification may be an internucleoside linkage modification and/or a sugar modification and/or a base modification.
As used herein, “monomer” refers to a precursor in the synthesis of an oligomeric or polymeric compound. Also the monomeric unit or residue within such an oligomeric or polymeric compound is encompassed in the term “monomer”. Thus, “monomer” and “nucleotide residue” may be used interchangeably throughout the description. Within the context of the present invention, a monomer is preferably a nucleotide. Preferred monomers to be incorporated in the oligonucleotides according to the invention are nucleotides comprising a 2′-O-methyl substituent, a phosphorothioate internucleoside linkage and a 5-methylpyrimidine and/or a 2,6-diaminopurine nucleobase.
As used herein, “nucleobase” refers to the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or may be modified and therefore include, but are not limited to adenine, cytosine, guanine, uracil, thymine and analogues thereof such as 5-methyl-cytosine. In certain embodiments, a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.
As used herein, “Tm” means melting temperature which is the temperature at which the two strands of a duplex nucleic acid separate. Tm is often used as a measure of duplex stability or the binding affinity of an antisense compound toward a complementary RNA molecule.
As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a pentose sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′-substituents, such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), or O—CH2—C(═O)—N(Rm)(Rn), wherein each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. 2′-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used herein, “2′-O-Me”, “2′-OMe” or “2′-OCH3” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH3 group at the 2′ position of the sugar ring.
As used herein, “MOE” or “2′-MOE” or “2′-OCH2CH2OCH3” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH2CH2OCH3 group at the 2′ position of the sugar ring.
As used herein, the term “adenine analogue” means a chemically-modified purine nucleobase that, when incorporated into an oligomer, is capable of forming a base pair with either a thymine or uracil of a complementary strand of RNA or DNA. Preferably, such base pair is a Watson-Crick base pair, but analogues and slight deviations thereof are also considered allowable within the context of the present invention.
As used herein, the term “uracil analogue” means a chemically-modified pyrimidine nucleobase that, when incorporated into an oligomer, is capable of forming a base pair with either a adenine of a complementary strand of RNA or DNA. Preferably, such base pair is a Watson-Crick base pair, but analogues and slight deviations thereof are also considered allowable within the context of the present invention.
As used herein, the term “thymine analogue” means a chemically-modified pyrimidine nucleobase that, when incorporated into an oligomer, is capable of forming a base pair with an adenine of a complementary strand of RNA or DNA. Preferably, such base pair is a Watson-Crick base pair, but analogues and slight deviations thereof are also considered allowable within the context of the present invention.
As used herein, the term “cytosine analogue” means a chemically-modified pyrimidine nucleobase that, when incorporated into an oligomer, is capable of forming a base pair with a guanine of a complementary strand of RNA or DNA. For example, cytosine analogue can be a 5-methylcytosine. Preferably, such base pair is a Watson-Crick base pair, but analogues and slight deviations thereof are also considered allowable within the context of the present invention.
As used herein, the term “guanine analogue” means a chemically-modified purine nucleobase that, when incorporated into an oligomer, is capable of forming a base pair with a cytosine of a complementary strand of RNA or DNA. Preferably, such base pair is a Watson-Crick base pair, but analogues and slight deviations thereof are also considered allowable within the context of the present invention.
As used herein, the term “guanosine” refers to a nucleoside or sugar-modified nucleoside comprising a guanine or guanine analog nucleobase.
As used herein, the term “uridine” refers to a nucleoside or sugar-modified nucleoside comprising a uracil or uracil analog nucleobase.
As used herein, the term “thymidine” refers to a nucleoside or sugar-modified nucleoside comprising a thymine or thymine analog nucleobase.
As used herein, the term “cytidine” refers to a nucleoside or sugar-modified nucleoside comprising a cytosine or cytosine analog nucleobase.
As used herein, the term “adenosine” refers to a nucleoside or sugar-modified nucleoside comprising an adenine or adenine analog nucleobase.
As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
As used herein, “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides. An internucleoside linkage may be a naturally occurring internucleoside linkage, i.e. a 3′ to 5′ phosphodiester linkage, or a modified internucleoside linkage.
As used herein, “modified internucleoside linkage” refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.
As used herein, “backbone” refers to the chain of alternating sugar moieties and internucleoside linkages, as it occurs in an oligonucleotide. The oligonucleotide of the invention comprises at least one phosphorodithioate internucleoside linkage, but it has to be understood that more backbone modifications, such as sugar modifications and/or internucleoside linkage modifications may be present in the backbone.
As used herein, “oligomeric compound” refers to a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound is an oligonucleotide. In certain embodiments, an oligomeric compound is a single-stranded oligonucleotide. In certain embodiments, an oligomeric compound is a double-stranded duplex comprising two oligonucleotides. In certain embodiments, an oligomeric compound is a single-stranded or double-stranded oligonucleotide comprising one or more conjugate groups and/or terminal groups.
As used herein, “conjugate” refers to an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to the parent compound such as an oligomeric compound. In certain embodiments, conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. In certain embodiments, conjugates are terminal groups. In certain embodiments, conjugates are attached to a 3′ or 5′ terminal nucleoside or to an internal nucleoside of an oligonucleotide.
As used herein, “conjugate linking group” refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linking groups or bifunctional linking moieties such as those known in the art are amenable to the present invention.
As used herein, “antisense compound” refers to an oligomeric compound, at least a portion of which is at least partially complementary to, or at least partially directed to, a target nucleic acid to which it hybridizes and modulates the activity, processing or expression of said target nucleic acid.
As used herein, “expression” refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.
As used herein, “antisense oligonucleotide” refers to an antisense compound that is an oligonucleotide.
As used herein, “antisense activity” refers to any detectable and/or measurable activity attributable to the hybridization of an anti sense compound to its target nucleic acid. In certain embodiments, such activity may be an increase or decrease in an amount of a nucleic acid or protein. In certain embodiments, such activity may be a change in the ratio of splice variants of a nucleic acid or protein. Detection and/or measuring of antisense activity may be direct or indirect. In certain embodiments, antisense activity is assessed by observing a phenotypic change in a cell or animal.
As used herein, “target nucleic acid” refers to any nucleic acid molecule the expression, amount, or activity of which is capable of being modulated by an antisense compound. In certain embodiments, the target nucleic acid is DNA or RNA. In certain embodiments, the target RNA is miRNA, mRNA, pre-mRNA, non-coding RNA, or natural antisense transcripts. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state,
As used herein, “target mRNA” refers to a pre-selected RNA molecule that encodes a protein.
As used herein, “targeting” or “targeted to” refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently reverse complementary to the target nucleic acid to allow hybridization under physiological conditions. In this context “sufficiently reverse complementary” may be at least 90%, 95%, 97%, 99% or 100% reverse complementary with said targeted nucleic acid molecule.
As used herein, “target site” refers to a region of a target nucleic acid that is bound by an antisense compound. In certain embodiments, a target site is at least partially within the 3′ untranslated region of an RNA molecule. In certain embodiments, a target site is at least partially within the 5′ untranslated region of an RNA molecule. In certain embodiments, a target site is at least partially within the coding region of an RNA molecule. In certain embodiments, a target site is at least partially within an exon of an RNA molecule. In certain embodiments, a target site is at least partially within an intron of an RNA molecule. In certain embodiments, a target site is at least partially within a miRNA target site of an RNA molecule. In certain embodiments, a target site is at least partially within a repeat region of an RNA molecule.
As used herein, “target protein” refers to a protein, the expression of which is modulated by an antisense compound. In certain embodiments, a target protein is encoded by a target nucleic acid. In certain embodiments, expression of a target protein is otherwise influenced by a target nucleic acid.
As used herein, “complementarity” in reference to nucleobases refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
As used herein, “non-complementary” in reference to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
As used herein, “complementary” in reference to linked nucleosides, oligonucleotides, or nucleic acids, refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity. In certain embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). Preferably the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid or reverse complementarity to a target nucleic acid.
As used herein, “modulation” refers to a perturbation of amount or quality of a function or activity when compared to the function or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include perturbing splice site selection of pre-mRNA processing, resulting in a change in the amount of a particular splice-variant present compared to conditions that were not perturbed. As a further example, modulation includes perturbing translation of a protein.
As used herein, “motif” refers to a pattern of modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
As used herein, “the same modifications” refer to modifications relative to naturally occurring molecules that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same modification,” even though the DNA nucleoside is unmodified.
As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” refers to the modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
As used herein, “pharmaceutically acceptable salts” refers to salts of active compounds that retain the desired biological activity of the active compound and do not impart undesired toxicological effects thereto.
As used herein, the term “independently” means that each occurrence of a repetitive variable within a claimed oligonucleotide is selected independent of one another. For example, each repetitive variable can be selected so that (i) each of the repetitive variables are the same, (ii) two or more are the same, or (iii) each of the repetitive variables can be different.
General Chemistry Definitions
As used herein, “alkyl” refers to a saturated straight or branched hydrocarbon substituent or radical, typically containing up to twenty four carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to 24 carbon atoms, more typically from 1 to 12 carbon atoms (C1-C12 alkyl) with from 1 to 6 carbon atoms (C1-C6 alkyl) being more preferred. The term “lower alkyl” as used herein includes from 1 to 6 carbon atoms (C1-C6 alkyl). Alkyl groups as used herein may optionally contain one or more further substituents.
As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain radical or substituent, typically containing up to twenty four carbon atoms, and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadienyl and the like. Alkenyl groups typically include from 2 to 24 carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally contain one or more further substituents.
As used herein, “alkynyl” refers to a straight or branched hydrocarbon radical or substituent, typically containing up to twenty four carbon atoms, and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to 24 carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally contain one or more further substituents.
As used herein, “aminoalkyl” refers to an amino substituted alkyl radical or substituent. This term is meant to include C1-C12 alkyl groups having an amino substituent at any position and wherein the aminoalkyl group is attached to the parent molecule via its alkyl moiety. The alkyl and/or amino portions of the aminoalkyl group may optionally be further substituted with further substituents.
As used herein, “aliphatic” refers to a straight or branched hydrocarbon radical or substituent, typically containing up to twenty four carbon atoms, wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to 24 carbon atoms, more typically from 1 to 12 carbon atoms with from 1 to 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally contain further substituents.
As used herein, “alicyclic” or “alicyclyl” refers to a cyclic radical or substituent, wherein the ring system is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclic moieties include rings having from 5 to 9 carbon atoms in the ring. Alicyclic groups as used herein may optionally contain further substituents.
As used herein, “alkoxy” refers to a radical or substituent comprising an alkyl group and an oxygen atom, wherein the alkoxy group is attached to a parent molecule via its oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally contain further substituents.
As used herein, “halo”, “halide” and “halogen” refer to an atom, radical or substituent selected from fluorine, chlorine, bromine and iodine.
As used herein, “aryl” and “aromatic” refer to a radical or substituent comprising a mono- or polycyclic carbocyclic ring system having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from 5 to 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally contain further substituents.
As used herein, “aralkyl” and “arylalkyl” refer to a radical or substituent comprising an alkyl group and an aryl group, wherein the aralkyl or arylalkyl group is attached to a parent molecule via its alkyl moiety. Examples include, but are not limited to, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally contain further substituents attached to the alkyl, the aryl or both moieties that form the radical or substituent.
As used herein, “heterocyclyl” refers to a radical or substituent comprising a mono- or polycyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclyl is also meant to include fused ring system moieties wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic group typically includes at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic groups include[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as used herein may optionally contain further substituents.
As used herein, “heteroaryl” and “heteroaromatic” refer to a radical or substituent comprising a mono- or polycyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatom. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. Heteroaryl radicals or substituents can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. Heteroaryl groups as used herein may optionally contain further substituents.
As used herein, “heteroarylalkyl” refers to a radical or substituent comprising a heteroaryl group as previously defined and an alkyl moiety, wherein the heteroarylalkyl group is attached to a parent molecule via its alkyl moiety. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkyl groups as used herein may optionally contain further substituents on one or both of the heteroaryl or alkyl portions.
As used herein, “mono or polycyclic” refers to any ring systems, such as a single ring or a polycyclic system having rings that are fused or linked, and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and polycyclic structures can contain rings that have a uniform or varying degree of saturation, including fully saturated, partially saturated or fully unsaturated rings. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms. Heterocyclic and all-carbon rings can be present in a mixed motif, such as for example benzimidazole wherein one ring of the fused ring system has only carbon ring atoms and the other ring has two nitrogen atoms. The mono or polycyclic structures can be further substituted with substituents such as for example phthalimide which has two oxo groups (═O) attached to one of the rings. In another aspect, mono or polycyclic structures can be attached to a parent molecule directly through a ring atom, through a substituent or a bifunctional linking moiety.
As used herein, “acyl” refers to a radical or substituent comprising a carbonyl moiety (C═O or —C(O)—) and a further substituent X, wherein the acyl group is attached to a parent molecule via its carbonyl moiety. As such, an acyl group is formally obtained by removal of a hydroxyl group from an organic acid and has the general formula —C(O)—X, wherein X is typically aliphatic, alicyclic or aromatic. The term “acyl” is also meant to include heteroacyl radicals or substituents with general formula —Y(O)n—X, wherein X is as defined above and Y(O)n is typically sulfonyl, sulfinyl or phosphate. Examples of acyl groups include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally contain further substituents.
As used herein, “substituent” and “substituent group” include groups that are typically added to other substituents or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be attached to one available site or to many available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound. Herein, “hydrocarbyl” refers to any group comprising C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more heteroatoms selected from N, O and S and can be further substituted with one or more substituents.
Unless otherwise indicated, the term “substituted” or “optionally substituted” refers to the (optional) presence of any of the following substituents: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (—O—Raa), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (—NRbbRcc), imino (═NRbb), amido (—C(O)NRbbRcc or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)NRbbRcc or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)NRbbRcc), thioureido (—N(Rbb)C(S)NRbbRcc), guanidinyl (—N(Rbb)C(═NRbb)NRbbRcc), amidinyl (—C(═NRbb)NRbbRcc or —N(Rbb)C(NRbb)Raa), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb), sulfonamidyl (—S(O)2NRbbRcc or —N(Rbb)S(O)2Rbb) and conjugate groups. Herein, each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent, preferably but without limitation chosen from the group consisting of H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
In this context, “recursive substituent” means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target and practical properties such as ease of synthesis.
Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the invention, the total number will be determined as set forth above.
As used herein, a zero (0) in a range indicating number of a particular unit means that the unit may be absent. For example, an oligomeric compound comprising 0-2 regions of a particular motif means that the oligomeric compound may comprise one or two such regions having the particular motif, or the oligomeric compound may not have any regions having the particular motif. In instances where an internal portion of a molecule is absent, the portions flanking the absent portion are bound directly to one another. Likewise, the term “none” as used herein, indicates that a certain feature is not present.
As used herein, “analogue” or “derivative” means either a compound or moiety similar in structure but different in respect to elemental composition from the parent compound regardless of how the compound is made. For example, an analogue or derivative compound does not need to be made from the parent compound as a chemical starting material.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
LEGENDS TO THE FIGURE
FIGS. 1A-1B. In vitro activity assay for (XYG)7 in which X=5-methylcytosine and Y═U (PS659 SEQ ID NO:90; derived from SEQ ID NO:2) and (XYG)7 in which X═C and Y is 5-methyluracil (PS661 SEQ ID NO: 97; derived from SEQ ID NO:3). PS659 (1A) and PS661 (1B) were transfected into HD fibroblasts (GM04022) at increasing concentrations (0.5-200 nM). Efficacy and selectivity was determined with RT-PCR and lab-on-a-chip analysis. Silencing of the expanded ((CAG)44) and healthy ((CAG)18) HTT transcripts were compared to the relative HTT transcript levels in mock samples. For all AONs n=2 except for mock (n=3).
FIGS. 2A-2D. In vivo efficacy of PS659 ((XYG)7 in which X=5-methylcytosine and Y═U; SEQ ID NO:2) in a transgenic HD rat model. Transgenic HD rats ((CAG)51 repeat) received 15 times an intraventricular injection with PS659 (SEQ ID NO:90 derived from SEQ ID NO: 2), during 18 weeks at a final dose of 200 μg per injection, control HD rats received vehicle only. Rats were sacrificed one week after the final injection. From all rats tissue was isolated and HTT levels were determined with Q-RT-PCR analysis. Reduced levels of HTT transcript were found in (2A) cortex, (2B) hippocampus, (2C) olfactory bulb and (2D) thalamus after PS659 treatment compared to control.
TABLE 1
General structures of AONs. X = C or 5-methylcytosine Y = U or 5-
methyluracil, Z = A or 2,6-diaminopurine, I = inosine,
and Q = abasic monomer.
Target Repeat
AON Sequence (5′→3′)
SEQ ID NO
(CAG)n
(XYG)7 (PS57)
1
X = C, Y = U
(XYG)7 (PS659)
2
X = 5-methylcytosine,Y = U
(XYG)7 (PS661)
3
X = C, Y = 5-methyluracil
(XYG)4
4
(XYG)5
5
(XYG)6
6
(XYG)7
7
(XYG)8
8
(XYG)9
9
(XYG)10
10
(XYG)11
11
(XYG)12
12
(GCG)n
(XGX)4
13
(XGX)5
14
(XGX)6
15
(XGX)7
16
(XGX)8
17
(XGX)9
18
(XGX)10
19
(XGX)11
20
(XGX)12
21
(CGG)n
(XXG)4
22
(XXG)5
23
(XXG)6
24
(XXG)7
25
(XXG)8
26
(XXG)9
27
(XXG)10
28
(XXG)11
29
(XXG)12
30
(GAA)n
(YYX)4
31
(YYX)5
32
(YYX)6
33
(YYX)7
34
(YYX)8
35
(YYX)9
36
(YYX)10
37
(YYX)11
38
(YYX)12
39
(GCC)n
(GGX)4
40
(GGX)5
41
(GGX)6
42
(GGX)7
43
(GGX)8
44
(GGX)9
45
(GGX)10
46
(GGX)11
47
(GGX)12
48
(CCG)n
(XGG)4
49
(XGG)5
50
(XGG)6
51
(XGG)7
52
(XGG)8
53
(XGG)9
54
(XGG)10
55
(XGG)11
56
(XGG)12
57
(AUUCU)n
(ZGZZY)3
58
(ZGZZY)4
59
(ZGZZY)5
60
(ZGZZY)6
61
(ZGZZY)7
62
(CCUG)n
(XZGG)3
63
(XZGG)4
64
(XZGG)5
65
(XZGG)6
66
(XZGG)7
67
(XZGG)8
68
(XZGG)9
69
(GGGGCC)n
(GGXUXX)3
216
(GGXUXX)4
217
(GGXIXX)4
218
(GGXQXX)4
219
Note:
All AONs with SEQ ID NO: 4-69, or 216-219 comprise at least one base modification selected from 5-methylcytosine, 5-methyluracil, and 2,6-diaminopurine.
TABLE 2
General structures of AONs. All AONs are 2′-O-methyl phosphorothioate
AONs wherein C is 5-methylcytosine, U is 5-methyluracil, A is 2,6-
diaminopurine, I is inosine and Q is an abasic monomer.
Target
AON
SEQ
Repeat
ID
AON Sequence (5′→3′)
ID NO
(CAG)n
PS659
CUG CUG CUG CUG CUG CUG CUG
90
CUG CUG CUG CUG CUG CUG CUG
91
CUG CUG CUG CUG CUG CUG CUG
92
CUG CUG CUG CUG CUG CUG CUG
93
CUG CUG CUG CUG CUG CUG CUG
94
CUG CUG CUG CUG CUG CUG CUG
95
CUG CUG CUG CUG CUG CUG CUG
96
PS661
CUG CUG CUG CUG CUG CUG CUG
97
CUG CUG CUG CUG CUG CUG CUG
98
CUG CUG CUG CUG CUG CUG CUG
99
CUG CUG CUG CUG CUG CUG CUG
100
CUG CUG CUG CUG CUG CUG CUG
101
CUG CUG CUG CUG CUG CUG CUG
102
CUG CUG CUG CUG CUG CUG CUG
103
PS660
CUG CUG CUG CUG CUG CUG CUG
104
CUG CUG CUG CUG CUG CUG CUG
105
CUG CUG CUG CUG CUG CUG CUG
106
PS684
CUG CUG CUG CUG CUG CUG CUG
107
CUG CUG CUG CUG CUG CUG CUG QQQQ
220
CUG CUG CUG CUG CUG CUG CUG
108
CUG CUG CUG CUG CUG CUG CUG QQQQ
221
CUG CUG CUG CUG CUG CUG CUG CUG
109
CUG CUG CUG CUG CUG CUG CUG CUG
110
CUG CUG CUG CUG CUG CUG CUG CUG CUG
111
CUG CUG CUG CUG CUG CUG CUG CUG CUG
112
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
113
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
114
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
115
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
116
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
117
CUG
CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG
118
CUG
(GCG)n
CGC CGC CGC CGC
119
CGC CGC CGC CGC
120
CGC CGC CGC CGC CGC
121
CGC CGC CGC CGC CGC
122
CGC CGC CGC CGC CGC CGC
123
CGC CGC CGC CGC CGC CGC
124
CGC CGC CGC CGC CGC CGC CGC
125
CGC CGC CGC CGC CGC CGC CGC
126
CGC CGC CGC CGC CGC CGC CGC CGC
127
CGC CGC CGC CGC CGC CGC CGC CGC
128
CGC CGC CGC CGC CGC CGC CGC CGC CGC
129
CGC CGC CGC CGC CGC CGC CGC CGC CGC
130
CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC
131
CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC
132
(CGG)n
CCG CCG CCG CCG
133
CCG CCG CCG CCG
134
CCG CCG CCG CCG CCG
135
CCG CCG CCG CCG CCG
136
CCG CCG CCG CCG CCG CCG
137
CCG CCG CCG CCG CCG CCG
138
CCG CCG CCG CCG CCG CCG CCG
139
CCG CCG CCG CCG CCG CCG CCG
140
CCG CCG CCG CCG CCG CCG CCG CCG
141
CCG CCG CCG CCG CCG CCG CCG CCG
142
CCG CCG CCG CCG CCG CCG CCG CCG CCG
143
CCG CCG CCG CCG CCG CCG CCG CCG CCG
144
CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG
145
CCG CCG CCG CCG CCG CCG CCG CCG CCG CCG
146
(GAA)n
UUC UUC UUC UUC
147
UUC UUC UUC UUC
148
UUC UUC UUC UUC UUC
149
UUC UUC UUC UUC UUC
150
UUC UUC UUC UUC UUC UUC
151
UUC UUC UUC UUC UUC UUC
152
UUC UUC UUC UUC UUC UUC UUC
153
UUC UUC UUC UUC UUC UUC UUC
154
UUC UUC UUC UUC UUC UUC UUC
155
UUC UUC UUC UUC UUC UUC UUC
156
UUC UUC UUC UUC UUC UUC UUC
157
UUC UUC UUC UUC UUC UUC UUC UUC
158
UUC UUC UUC UUC UUC UUC UUC UUC
159
UUC UUC UUC UUC UUC UUC UUC UUC UUC
160
UUC UUC UUC UUC UUC UUC UUC UUC UUC
161
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
162
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
163
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
164
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
165
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
166
UUC
UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC UUC
167
UUC
(GCC)n
GGC GGC GGC GGC
168
GGC GGC GGC GGC GGC
169
GGC GGC GGC GGC GGC GGC
170
GGC GGC GGC GGC GGC GGC GGC
171
GGC GGC GGC GGC GGC GGC GGC
172
GGC GGC GGC GGC GGC GGC GGC
173
GGC GGC GGC GGC GGC GGC GGC
174
GGC GGC GGC GGC GGC GGC GGC GGC
175
GGC GGC GGC GGC GGC GGC GGC GGC GGC
176
GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC
177
(CCG)n
CGG CGG CGG CGG
178
CGG CGG CGG CGG CGG
179
CGG CGG CGG CGG CGG CGG
180
CGG CGG CGG CGG CGG CGG CGG
181
CGG CGG CGG CGG CGG CGG CGG CGG
182
CGG CGG CGG CGG CGG CGG CGG CGG CGG
183
CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG
184
(AUUCU)n
AGAAU AGAAU AGAAU
185
AGAAU AGAAU AGAAU AGAAU
186
AGAAU AGAAU AGAAU AGAAU
187
AGAAU AGAAU AGAAU AGAAU
188
AGAAU AGAAU AGAAU AGAAU
189
AGAAU AGAAU AGAAU AGAAU AGAAU
190
AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU
191
AGAAU AGAAU AGAAU AGAAU AGAAU AGAAU
192
AGAAU
(CCUG)n
CAGG CAGG CAGG
193
CAGG CAGG CAGG
194
CAGG CAGG CAGG CAGG
195
CAGG CAGG CAGG CAGG
196
CAGG CAGG CAGG CAGG CAGG
197
CAGG CAGG CAGG CAGG CAGG
198
CAGG CAGG CAGG CAGG CAGG
199
CAGG CAGG CAGG CAGG CAGG
200
CAGG CAGG CAGG CAGG CAGG CAGG
201
CAGG CAGG CAGG CAGG CAGG CAGG
202
CAGG CAGG CAGG CAGG CAGG CAGG CAGG
203
CAGG CAGG CAGG CAGG CAGG CAGG CAGG
204
CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG
205
CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG
206
CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG
207
CAGG
CAGG CAGG CAGG CAGG CAGG CAGG CAGG CAGG
208
CAGG
(GGGGCC)n
PS1252
GGCUCC GGCUCC GGCUC
209
GGCQCC GGCQCC GGCQCC
210
GGCUCC GGCUCC GGCUCC
211
GGCUCC GGCUCC GGCUCC GGCUCC
212
GGCQCC GGCQCC GGCQCC GGCQCC
213
GGCICC GGCICC GGCICC GGCICC
214
GGCCUC GGCCUC GGCCUC GGCCUC
215
EXAMPLES
Example 1
Introduction
The particular characteristics of a chosen antisense oligonucleotide (AON) chemistry may at least in part enhance binding affinity and stability, enhance activity, improve safety, and/or reduce cost of goods by reducing length or improving synthesis and/or purification procedures. This example describes the comparative analysis of the activity of AONs designed to target the expanded (CAG)n repeat in HTT transcripts in HD fibroblasts in vitro, and includes AONs with either 5-methylcytosines (XYG)7, wherein X is 5-methylcytosine and Y═U being also identified as SEQ ID NO:90 (and derived from SEQ ID NO:2), or 5-methyluracils (XYG)7, wherein X═C and Y=5-methyluracil being also identified as SEQ ID NO: 97 (and derived from SEQ ID NO:3).
Materials and Methods
Cell Culture.
Patient derived HD fibroblasts (GM04022) (purchased from Coriell Cell Repositories, Camden, USA) were cultured at 37° C. and 5% CO2 in Minimal Essential Medium (MEM) (Gibco Invitrogen, Carlsbad, USA) with 15% heat inactivated Fetal Bovine Serum (FBS) (Clontech, Palo Alto USA), 1% Glutamax (Gibco) and 100 Um′ penicillin/streptomycin (P/S) (Gibco).
Oligonucleotides.
The AONs were fully 2′-O-methyl phosphorothioate modified: PS659; (XYG)7, wherein X is 5-methylcytosine and Y═U being also identified as SEQ ID NO: 90 (and derived from SEQ ID NO:2), and PS661; (XYG)7, wherein X═C and Y=5-methyluracil being also identified as SEQ ID NO:97 (and derived from SEQ ID NO:3).
Transfection.
Cells were transfected with AONs complexed with PEI (2 μL per μg AON, in 0.15 M NaCl). AON-PEI complex was added in MEM medium with 5% FBS to cells to a final AON concentration varying from 0.5-200 nM. Fresh medium was supplemented after four hours and after 24 hours RNA was isolated.
RNA Isolation.
RNA from cultured cells was isolated using the Aurum Total RNA Mini Kit (Bio-Rad, Hercules, Calif.) according to the manufacturer's protocol.
RT-PCR and Lab-on-a-Chip Analysis.
Approximately 200 ng RNA was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 μL. PCR was performed with primers for HTT (across the CAG repeat) and β-actin. The PCR program started with a 4 min initial denaturation at 95° C., followed by 35 cycles of 30 sec denaturation at 94° C., 30 sec annealing at 60° C., 45 sec elongation at 72° C., after which a final elongation step was performed at 72° C. for 7 min Lab-on-a-Chip was performed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), using the Agilent DNA 1000 Kit. Expression levels were normalized for β-actin levels and relative to transcript levels without transfection. The following primers were used:
HTT forward;
(SEQ ID NO: 70)
5′-ATGGCGACCCTGGAAAAGCTGAT-3′
HTT reverse:
(SEQ ID NO: 71)
5′-TGAGGCAGCAGCGGCTG-3′
β-actin forward;
(SEQ ID NO: 72)
5′-GGACTTCGAGCAAGAGATGG-3′
β-actin reverse;
(SEQ ID NO: 73)
5′-AGCACTGTGTTGGCGTACAG-3′
Results
Both PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) and PS661 (SEQ ID NO: 97 derived from SEQ ID NO:3) were highly effective and reduced the HTT transcripts in HD fibroblasts in a dose-dependent manner (FIG. 1a, b). Both AONs also showed a preference for the allele with the expanded CAG repeats. PS659 (SEQ ID NO: 90 derived from SEQ ID NO:2) was more effective and more allele-specific at lower concentrations (strongest effect at 5 nM) (1a) than PS661 (SEQ ID NO: 97 derived from SEQ ID NO:3) (strongest effect at 20 nM) (1b).
Example 2
Introduction
PS659 (XYG)7, wherein X is 5-methylcytosine and Y═U also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2), was selected from in vitro studies as most efficient and safe candidate. This example describes its activity in a transgenic HD rat model after a series of direct intraventricular injections.
Materials and Methods
Animals.
Transgenic HD rats carry a truncated Huntington cDNA fragment with 51 CAG repeats under the control of the native rat Huntington promoter. The expressed gene product is about 75 kDa, corresponding to 22% of the full-length Huntington (cDNA position 324-2321, amino acid position 1-709/825, corresponding to exon 1-16), under the control of 886 bp of the rat Huntington promoter (von Horsten S. et al.). All animal experiments were approved by the Institutional Animal Care and Use Committees of the Maastricht University, Maastricht.
Oligonucleotides.
PS659 (XYG)7, wherein X is 5-methylcytosine and Y═U also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2), is a fully 2′-O-methyl phosphorothioate modified AON.
In Vivo Treatment.
Transgenic HD rats received 15 times an intraventricular injection at a final dose of 200 ng PS659 also identified as SEQ ID NO: 90 (derived from SEQ ID NO:2) during 18 weeks. Control HD rats received vehicle only. Rats were sacrificed one week after the final injection.
RNA Isolation.
RNA from brain tissue was isolated using RNA-Bee reagent (Tel Test, Inc). In brief, tissue samples were homogenized in MagNA Lyser green bead tubes (Roche) by adding RNA-Bee (50 mg tissue/mL RNA-Bee) and homogenizing using a MagNA Lyser instrument (Roche). Lysate was transferred to a new tube, chloroform (SIGMA) was added (0.2 mL per mL RNA-Bee), mixed, incubated on ice for 5 minutes and centrifuged at 13,000 rpm for 15 minutes at 4° C. The upper aqueous phase was collected and an equal volume isopropanol (SIGMA) was added, followed by a 1 hour incubation period at 4° C. and centrifugation (13,000 rpm, 15 min, 4° C.). The RNA precipitate was washed with 70% (v/v) ethanol (BioSolve), air dried and dissolved in MilliQ.
Quantitative RT-PCR Analysis.
Approximately 200 ng was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 μL 3 μL of 1/40 cDNA dilution preparation was subsequently used in a quantitative PCR analysis according to standard procedures in presence of iQ™ SYBR® Green Supermix (Bio-Rad). Quantitative PCR primers were designed based on NCBI database sequence information. Product identity was confirmed by DNA sequencing. The signal for Rab2 and YWHAZ was used for normalization. The following primers were used:
Rat Htt-F;
(SEQ ID NO: 74)
5′-CGCCGCCTCCTCAGCTTC-3′
Rat Htt-R;
(SEQ ID NO: 75)
5′-GAGAGTTCCTTCTTTGGTCGGTGC-3′
Rab2-F;
(SEQ ID NO: 76)
5′-TGGGAAACAGATAAAACTCCAGA-3′
Rab2-R;
(SEQ ID NO: 77)
5′-AATATGACCTTGTGATAGAACGAAAG-3′
YWHAZ-F;
(SEQ ID NO: 78)
5′-AAATGAGCTGGTGCAGAAGG-3′
YWHAZ-R;
(SEQ ID NO: 79)
5′-GGCTGCCATGTCATCGTAT-3′
Results
PS659 (also identified as SEQ ID NO: 90 or derived from SEQ ID NO: 2) reduced transgenic Htt transcript levels in cortex (FIG. 2a), hippocampus (FIG. 2b), olfactory bulb (FIG. 2c) as well as in thalamus (FIG. 3d) when compared to saline treated rats. These results demonstrate that PS659 (also identified as SEQ ID NO: 90 or derived from SEQ ID NO; 2) is effective in vivo after direct intraventricular injection.
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14522002
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Jun 23rd, 2020 12:00AM
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Dec 27th, 2017 12:00AM
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https://www.uspto.gov?id=US10689646-20200623
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Treatment of genetic disorders associated with DNA repeat instability
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The current invention provides for methods and medicaments that apply oligonucleotide molecules complementary only to a repetitive sequence in a human gene transcript, for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders. The invention also pertains to modified oligonucleotides which can be applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.
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10689646
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1. A method for treating myotonic dystrophy type 1 in a subject, comprising administering to the subject an oligonucleotide comprising 10 to 50 nucleotides that is complementary to a repetitive nucleotide unit sequence of a pre-mRNA transcript, wherein the repetitive nucleotide unit is a CUG repetitive nucleotide unit.
2. The method according to claim 1, wherein said repeats are present in a coding sequence of the gene transcript.
3. The method according to claim 1, wherein said repeats are present in a non-coding sequence of the gene transcript.
4. The method according to claim 1, wherein the oligonucleotide comprises a ribonucleotide and/or a deoxyribonucleotide.
5. The method according to claim 4, wherein the oligonucleotide comprises a 2′-O-substituted phosphorothioate nucleotide.
6. The method of claim 5, wherein said oligonucleotide comprises a 2′-O-methyl phosphorothioate nucleotide or a 2′-O-meth[y]oxy ethyl phosphorothioate nucleotide.
7. The method according to claim 1, wherein the oligonucleotide is administered in a nucleic acid vector.
8. The method according to claim 1, wherein the oligonucleotide is in a pharmaceutical composition comprising an excipient and/or a targeting ligand.
9. A method for reducing the number of repeat-containing gene transcripts in a cell comprising providing to said cell an oligonucleotide comprising 10 to 50 nucleotides that is complementary to a repetitive nucleotide unit of a pre-mRNA transcript, wherein said repetitive nucleotide unit is a CUG repetitive nucleotide unit; and wherein the oligonucleotide has a phosphorothioate-containing backbone.
10. The method according to claim 1, wherein the oligonucleotide has a length selected from the group consisting of about 12 to about 30 nucleotides, about 12 to about 45 nucleotides and about 12 to about 25 nucleotides.
11. The method of claim 1, wherein the oligonucleotide has a phosphorothioate-containing backbone.
12. The method according to claim 1, where the oligonucleotide comprises a morpholino phosphorodiamidate oligonucleotide, a phosphorothioate oligonucleotide, a locked nucleic acid (LNA), a peptide nucleic acid (PNA) and/or an ethylene-bridged nucleic acid.
13. The method of claim 1, wherein the oligonucleotide binds only to the sequence of consecutive trinucleotide repeats.
14. The method of claim 1, wherein the oligonucleotide reduces the amount of an aberrant protein.
15. The method of claim 12, wherein the oligonucleotide is a 2′-O-methyl phosphorothioate.
16. The method of claim 15, wherein the oligonucleotide comprises the base sequence of: SEQ ID NO: 6.
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PRIORITY
This application is a U.S. divisional application of U.S. patent application Ser. No. 14/809,483, filed Jul. 27, 2015, which is a divisional application of U.S. patent application Ser. No. 12/377,160, filed Feb. 24, 2010, which is a U.S. National Stage Entry of PCT/NL07/050399, filed on Aug. 10, 2007, which claims priority to EP 06118809.0, filed Aug. 11, 2006 and EP 06119247.2, filed Aug. 21, 2006, the disclosure of each of which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The current invention relates to the field of medicine, in particular to the treatment of genetic disorders associated with genes that have unstable repeats in their coding or non-coding sequences, most in particular unstable repeats in the human Huntington disease causing HD gene or the myotonic dystrophy type 1 causing DMPK gene.
BACKGROUND OF THE INVENTION
Instability of gene-specific microsatellite and minisatellite repetitive sequences, leading to increase in length of the repetitive sequences in the satellite, is associated with about 35 human genetic disorders. Instability of trinucleotide repeats is for instance found in genes causing X-linked spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy type 1 (DM1), fragile X syndrome (FRAX genes A, E, F), Huntington's disease (HD) and several spinocerebellar ataxias (SCA gene family). Unstable repeats are found in coding regions of genes, such as the Huntington's disease gene, whereby the phenotype of the disorder is brought about by alteration of protein function and/or protein folding. Unstable repeat units are also found in untranslated regions, such as in myotonic dystrophy type 1 (DM1) in the 3′ UTR or in intronic sequences such as in myotonic dystrophy type 2 (DM2). The normal number of repeats is around 5 to 37 for DMPK, but increases to premutation and full disease state two to ten fold or more, to 50, 100 and sometimes 1000 or more repeat units. For DM2/ZNF9 increases to 10,000 or more repeats have been reported. (Cleary and Pearson, Cytogenet. Genome Res. 100: 25-55, 2003).
The causative gene for Huntington's disease, HD, is located on chromosome 4. Huntington's disease is inherited in an autosomal dominant fashion. When the gene has more than 35 CAG trinucleotide repeats coding for a polyglutamine stretch, the number of repeats can expand in successive generations. Because of the progressive increase in length of the repeats, the disease tends to increase in severity and presents at an earlier age in successive generations, a process called anticipation. The product of the HD gene is the 348 kDa cytoplasmic protein huntingtin. Huntingtin has a characteristic sequence of fewer than 40 glutamine amino acid residues in the normal form; the mutated huntingtin causing the disease has more than 40 residues. The continuous expression of mutant huntingtin molecules in neuronal cells results in the formation of large protein deposits which eventually give rise to cell death, especially in the frontal lobes and the basal ganglia (mainly in the caudate nucleus). The severity of the disease is generally proportional to the number of extra residues.
DM1 is the most common muscular dystrophy in adults and is an inherited, progressive, degenerative, multisystemic disorder of predominantly skeletal muscle, heart and brain. DM1 is caused by expansion of an unstable trinucleotide (CTG)n repeat in the 3′ untranslated region of the DMPK gene (myotonic dystrophy protein kinase) on human chromosome 19q (Brook et al, Cell, 1992). Type 2 myotonic dystrophy (DM2) is caused by a CCTG expansion in intron 1 of the ZNF9 gene, (Liguori et al, Science 2001). In the case of myotonic dystrophy type 1, the nuclear-cytoplasmic export of DMPK transcripts is blocked by the increased length of the repeats, which form hairpin-like secondary structures that accumulate in nuclear foci. DMPK transcripts bearing a long (CUG)n tract can form hairpin-like structures that bind proteins of the muscleblind family and subsequently aggregate in ribonuclear foci in the nucleus. These nuclear inclusions are thought to sequester muscleblind proteins, and potentially other factors, which then become limiting to the cell. In DM2, accumulation of ZNF9 RNA carrying the (CCUG)n expanded repeat form similar foci. Since muscleblind proteins are splicing factors, their depletion results in a dramatic rearrangement in splicing of other transcripts. Transcripts of many genes consequently become aberrantly spliced, for instance by inclusion of fetal exons, or exclusion of exons, resulting in non-functional proteins and impaired cell function.
The observations and new insights above have led to the understanding that unstable repeat diseases, such as myotonic dystrophy type 1, Huntington's disease and others can be treated by removing, either fully or at least in part, the aberrant transcript that causes the disease. For DM1, the aberrant transcript that accumulates in the nucleus could be down regulated or fully removed. Even relatively small reductions of the aberrant transcript could release substantial and possibly sufficient amounts of sequestered cellular factors and thereby help to restore normal RNA processing and cellular metabolism for DM (Kanadia et al., PNAS 2006). In the case of HD, a reduction in the accumulation of huntingtin protein deposits in the cells of an HD patient can ameliorate the symptoms of the disease.
A few attempts have been made to design methods of treatment and medicaments for unstable repeat disease myotonic dystrophy type 1 using antisense nucleic acids, RNA interference or ribozymes. (i) Langlois et al. (Molecular Therapy, Vol. 7 No. 5, 2003) designed a ribozyme capable of cleaving DMPK mRNA. The hammerhead ribozyme is provided with a stretch RNA complementary to the 3′ UTR of DMPK just before the CUG repeat. In vivo, vector transcribed ribozyme was capable of cleaving and diminishing in transfected cells both the expanded CUG repeat containing mRNA as well as the normal mRNA species with 63 and 50% respectively. Hence, also the normal transcript is gravely affected by this approach and the affected mRNA species with expanded repeats are not specifically targeted.
(ii) Another approach was taken by Langlois et al., (Journal Biological Chemistry, vol 280, no. 17, 2005) using RNA interference. A lentivirus-delivered short-hairpin RNA (shRNA) was introduced in DM1 myoblasts and demonstrated to down regulate nuclear retained mutant DMPK mRNAs. Four shRNA molecules were tested, two were complementary against coding regions of DMPK, one against a unique sequence in the 3′ UTR and one negative control with an irrelevant sequence. The first two shRNAs were capable of down regulating the mutant DMPK transcript with the amplified repeat to about 50%, but even more effective in down regulating the cytoplasmic wildtype transcript to about 30% or less. Equivalent synthetic siRNA delivered by cationic lipids was ineffective. The shRNA directed at the 3′ UTR sequence proved to be ineffective for both transcripts. Hence, also this approach is not targeted selectively to the expanded repeat mRNA species.
(iii) A third approach by Furling et al. (Gene Therapy, Vol. 10, p 795-802, 2003) used a recombinant retrovirus expressing a 149-bp long antisense RNA to inhibit DMPK mRNA levels in human DM1 myoblasts. A retrovirus was designed to provide DM1 cells with the 149 bp long antisense RNA complementary to a 39 bp-long (CUG)13 repeat and a 110 bp region following the repeat to increase specificity. This method yielded a decrease in mutated (repeat expanded) DMPK transcript of 80%, compared to a 50% reduction in the wild type DMPK transcript and restoration of differentiation and functional characteristics in infected DM1 myoblasts. Hence, also this approach is not targeted selectively to the expanded repeat mRNA species, it depends on a very long antisense RNA and can only be used in combination with recombinant viral delivery techniques.
DETAILED DESCRIPTION OF THE INVENTION
The methods and techniques described above provide nucleid acid based methods that cause non-selective breakdown of both the affected repeat expanded allele and unaffected (normal) allele for genetic diseases that are associated with repeat instability and/or expansion. Moreover, the art employs sequences specific for the gene associated with the disease and does not provide a method that is applicable to several genetic disorders associated with repeat expansion. Finally, the art only teaches methods that involve use of recombinant DNA vector delivery systems, which need to be adapted for each oligonucleotide and target cell and which still need to be further optimised.
The current invention provides a solution for these problems by using a short single stranded nucleic acid molecule that comprises or consists of a sequence, which is complementary to the expanded repeat region only, i.e. it does not rely on hybridisation to unique sequences in exons or introns of the repeat containing gene. Furthermore, it is not essential that the employed nucleic acid (oligonucleotide) reduces transcripts by the RNAse H mediated breakdown mechanism.
Without wishing to be bound by theory, the current invention may cause a decrease in transcript levels by alterations in posttranscriptional processing and/or splicing of the premature RNA. A decrease in transcript levels via alternative splicing and/or postranscriptional processing is thought to result in transcripts lacking the overly expanded or instable (tri)nucleotide repeat, but still possessing functional activities. The reduction of aberrant transcripts by altered RNA processing and/or splicing may prevent accumulation and/or translation of aberrant, repeat expanded transcripts in cells.
Without wishing to be bound by theory the method of the current invention is also thought to provide specificity for the affected transcript with the expanded repeat because the kinetics for hybridisation to the expanded repeat are more favourable. The likelihood that a repeat specific complementary nucleic acid oligonucleotide molecule will hybridise to a complementary stretch in an RNA or DNA molecule increases with the size of the repetitive stretch. RNA molecules and in particular RNA molecules comprising repetitive sequences are normally internally paired, forming a secondary structure comprising open loops and closed hairpin parts. Only the open parts are relatively accessible for complementary nucleic acids. The short repeat stretches of a wild type transcript not associated with disease is often only 5 to about 20-40 repeats and due to the secondary structure relatively inaccessible for base pairing with a complementary nucleic acid. In contrast, the repeat units of the expanded repeat and disease associated allele is normally at least 2 fold expanded but usually even more, 3, 5, 10 fold, up to 100 or even more than 1000 fold expansion for some unstable repeat disorders. This expansion increases the likelihood that part of the repeat is, at least temporarily, in an open loop structure and thereby more accessible to base pairing with a complementary nucleic acid molecule, relative to the wild type allele. So despite the fact that the oligonucleotide is complementary to a repeat sequence present in both wildtype and repeat-expanded transcripts and could theoretically hybridise to both transcripts, the current invention teaches that oligonucleotides complementary to the repetitive tracts preferably hybridise to the disease-associated or disease-causing transcripts and leave the function of normal transcripts relatively unaffected. This selectivity is beneficial for treating diseases associated with repeat instability irrespective of the mechanism of reduction of the aberrant transcript.
The invention thus provides a method for the treatment of unstable cis-element DNA repeat associated genetic disorders, by providing nucleic acid molecules that are complementary to and/or capable of hybridising to the repetitive sequences only. This method thereby preferentially targets the expanded repeat transcripts and leaves the transcripts of the normal, wild type allele relatively unaffected. This is advantageous since the normal allele can thereby provide for the normal function of the gene, which is at least desirable and, depending on the particular gene with unstable DNA repeats, may in many cases be essential for the cell and/or individual to be treated.
Furthermore, this approach is not limited to a particular unstable DNA repeat associated genetic disorder, but may be applied to any of the known unstable DNA repeat diseases, such as, but not limited to: coding regions repeat diseases having a polyglutamine (CAG) repeat: Huntington's disease, Haw River syndrome, Kennedy's disease/spinobulbar muscular atrophy, spino-cerebellar ataxia, or diseases having polyalanine (GCG) repeats such as: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis/ptosis/epicanthus invensus syndrome, hand-foot-genital syndrome, synpolydactyly, oculopharyngeal muscular dystrophy, holoprosencephaly. Diseases with repeats in non-coding regions of genes to be treated according to the invention comprise the trinucleotide repeat disorders (mostly CTG and/or CAG and/or CCTG repeats): myotonic dystrophy type 1, myotonic dystrophy type 2, Friedreich's ataxia (mainly GAA), spino-cerebellar ataxia, autism. Furthermore, the method of the invention can be applied to fragile site associated repeat disorder comprising various fragile X-syndromes, Jacobsen syndrome and other unstable repetitive element disorders such as myoclonus epilepsy, facioscapulohumeral dystrophy and certain forms of diabetes mellitus type 2.
Another advantage of the current invention is that the oligonucleotides specific for a repeat region may be administered directly to cells and it does not rely on vector-based delivery systems. The techniques described in the prior art, for instance those mentioned above for treatment of DM1 and removal of DMPK transcripts from cells, require the use of vector based delivery systems to administer sufficient levels of oligonucleotides to the cell. The use of plasmid or viral vectors is yet less desirable for therapeutic purposes because of current strict safety regulations for therapeutic recombinant DNA vectors, the production of sufficient recombinant vectors for broad clinical application and the limited control and reversibility of an exaggerated (or non-specific) response after application. Nevertheless, optimisation in future is likely in these areas and viral delivery of plasmids could yield an advantageous long lasting effect. The current inventors have surprisingly found that oligonucleotides that comprise or consist of a sequence that is complementary to repetitive sequences of expanded repeat transcripts, due to the expansion of their molecular target for hybridisation, have a much increased affinity and/or avidity for their target in comparison to oligonucleotides that are specific for unique sequences in a transcript. Because of this high affinity and avidity for the repeat expanded target transcript, lower amounts of oligonucleotide suffice to yield sufficient inhibition and/or reduction of the repeat expanded allele by RNase H degradation, RNA interference degradation or altered post-transcriptional processing (comprising but not limited to splicing or exon skipping) activities. The oligonucleotides of the current invention which are complementary to repetitive sequences only, may be produced synthetically and are potent enough to be effective when delivered directly to cells using commonly applied techniques for direct delivery of oligonucleotides to cells and/or tissues. Recombinant vector delivery systems may, when desired, be circumvented by using the method and the oligonucleotide molecules of the current invention.
In a first aspect, the current invention discloses and teaches the use of an oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders.
In a second aspect, the invention also pertains to an oligonucleotide which can be used in the first aspect of the invention and/or applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.
An oligonucleotide of the invention may comprise a sequence that is complementary only to a repetitive sequence as defined below. Preferably, the repetitive sequence is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or more. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary only to a repetitive sequence as defined below. For example, an oligonucleotide may comprise a sequence that is complementary only to a repetitive sequence as defined below and a targeting part, which is later on called a targeting ligand.
A repeat or repetitive element or repetitive sequence or repetitive stretch is herein defined as a repetition of at least 3, 4, 5, 10, 100, 1000 or more, of a repetitive unit or repetitive nucleotide unit or repeat nucleotide unit comprising a trinucleotide repetitive unit, or alternatively a 4, 5 or 6 nucleotide repetitive unit, in a transcribed gene sequence in the genome of a subject, including a human subject.
An oligonucleotide may be single stranded or double stranded. Double stranded means that the oligonucleotide is an heterodimer made of two complementary strands, such as in a siRNA. In a preferred embodiment, an oligonucleotide is single stranded. A single stranded oligonucleotide has several advantages compared to a double stranded siRNA oligonucleotide: (i) its synthesis is expected to be easier than two complementary siRNA strands; (ii) there is a wider range of chemical modifications possible to optimise more effective uptake in cells, a better (physiological) stability and to decrease potential generic adverse effects; and (iii) siRNAs have a higher potential for non-specific effects and exaggerated pharmacology (e.g. less control possible of effectiveness and selectivity by treatment schedule or dose) and (iv) siRNAs are less likely to act in the nucleus and cannot be directed against introns. Therefore, in a preferred embodiment of the first aspect, the invention relates to the use of a single stranded oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans.
The oligonucleotide(s) preferably comprise at least 10 to about 50 consecutive nucleotides complementary to a repetitive element, more preferably 12 to 45 nucleotides, even more preferably 12 to 30, and most preferably 12 to 25 nucleotides complementary to a repetitive stretch, preferably having a trinucleotide repeat unit or a tetranucleotide repeat unit. The oligonucleotide may be complementary to and/or capable of hybridizing to a repetitive stretch in a coding region of a transcript, preferably a polyglutamine (CAG) or a polyalanine (GCG) coding tract. The oligonucleotide may also be complementary to and/or capable of hybridizing to a non-coding region for instance 5′ or 3′ untranslated regions, or intronic sequences present in precursor RNA molecules.
In a preferred embodiment the oligonucleotide to be used in the method of the invention comprises or consists of a sequence that is complementary to a repetitive element having as repetitive nucleotide unit a repetitive nucleotide unit selected from the group consisting of (CAG)n (SEQ ID NO:18), (GCG)n (SEQ ID NO:19), (CUG)n (SEQ ID NO:20), (CGG)n (SEQ ID NO:21) (GAA)n (SEQ ID NO:35), (GCC)n (SEQ ID NO:36) and (CCUG)n (SEQ ID NO:22) and said oligonucleotide being a single or double stranded oligonucleotide. Preferably, the oligonucleotide is double stranded.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a polyglutamine (CAG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders Huntington's disease, several forms of spino-cerebellar ataxia or Haw River syndrome, X-linked spinal and bulbar muscular atrophy and/or dentatorubral-pallidoluysian atrophy caused by repeat expansions in the HD, HDL2/JPH3, SBMA/AR, SCA1/ATX1, SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCAT, SCA17, AR or DRPLA human genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a polyalanine (GCG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyly, oculopharyngeal muscular dystrophy and/or holoprosencephaly, which are caused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM/PABP2, TCFBR1 or ZIC2 human genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CUG)n repeat in a transcript and is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 1, spino-cerebrellar ataxia 8 and/or Huntington's disease-like 2 caused by repeat expansions in the DM1/DMPK, SCA8 or JPH3 genes respectively. Preferably, these genes are from human origin.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCUG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 2, caused by repeat expansions in the DM2/ZNF9 gene.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CGG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of human fragile X syndromes, caused by repeat expansion in the FRAXA/FMR1, FRAXE/FMR2 and FRAXF/FAM11A genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Jacobsen syndrome, caused by repeat expansion in the FRA11B/CBL2 gene.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (GAA)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Friedreich's ataxia.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (ATTCT)n repeat in an intron is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Spinocerebellar ataxia type 10 (SCA10).
The repeat-complementary oligonucleotide to be used in the method of the invention may comprise or consist of RNA, DNA, Locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino phosphorodiamidates (PMO), ethylene bridged nucleic acid (ENA) or mixtures/hybrids thereof that comprise combinations of naturally occurring DNA and RNA nucleotides and synthetic, modified nucleotides. In such oligonucleotides, the phosphodiester backbone chemistry may further be replaced by other modifications, such as phosphorothioates or methylphosphonates. Other oligonucleotide modifications exist and new ones are likely to be developed and used in practice. However, all such oligonucleotides have the character of an oligomer with the ability of sequence specific binding to RNA. Therefore in a preferred embodiment, the oligonucleotide comprises or consists of RNA nucleotides, DNA nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, morpholino phosphorodiamidates, ethylene-bridged nucleic acid (ENA) nucleotides or mixtures thereof with or without phosphorothioate containing backbones.
Oligonucleotides containing at least in part naturally occurring DNA nucleotides are useful for inducing degradation of DNA-RNA hybrid molecules in the cell by RNase H activity (EC.3.1.26.4).
Naturally occurring RNA ribonucleotides or RNA-like synthetic ribonucleotides comprising oligonucleotides may be applied in the method of the invention to form double stranded RNA-RNA hybrids that act as enzyme-dependent antisense through the RNA interference or silencing (RNAi/siRNA) pathways, involving target RNA recognition through sense-antisense strand pairing followed by target RNA degradation by the RNA-induced silencing complex (RISC).
Alternatively or in addition, steric blocking antisense oligonucleotides (RNase-H independent antisense) interfere with gene expression or other precursor RNA or messenger RNA-dependent cellular processes, in particular but not limited to RNA splicing and exon skipping, by binding to a target sequence of RNA transcript and getting in the way of processes such as translation or blocking of splice donor or splice acceptor sites. Alteration of splicing and exon skipping techniques using modified antisense oligonucleotides are well documented, known to the skilled artisan and may for instance be found in U.S. Pat. No. 6,210,892, WO9426887, WO04/083446 and WO02/24906. Moreover, steric hindrance may inhibit the binding of proteins, nuclear factors and others and thereby contribute to the decrease in nuclear accumulation or ribonuclear foci in diseases like DM1.
The oligonucleotides of the invention, which may comprise synthetic or modified nucleotides, complementary to (expanded) repetitive sequences are useful for the method of the invention for reducing or inactivating repeat containing transcripts via the siRNA/RNA interference or silencing pathway.
Single or double stranded oligonucleotides to be used in the method of the invention may comprise or consist of DNA nucleotides, RNA nucleotides, 2′-O substituted ribonucleotides, including alkyl and methoxy ethyl substitutions, peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino (PMO) antisense oligonucleotides and ethylene-bridged nucleotides (ENA) and combinations thereof, optionally chimeras with RNAse H dependent antisense. Integration of locked nucleic acids in the oligonucleotide changes the conformation of the helix after base pairing and increases the stability of the duplex. Integration of LNA bases into the oligonucleotide sequence can therefore be used to increase the ability of complementary oligonucleotides of the invention to be active in vitro and in vivo to increase RNA degradation inhibit accumulation of transcripts or increase exon skipping capabilities. Peptide nucleic acids (PNAs), an artificial DNA/RNA analog, in which the backbone is a pseudopeptide rather than a sugar, have the ability to form extremely stable complexes with complementary DNA oligomers, by increased binding and a higher melting temperature. Also PNAs are superior reagents in antisense and exon skipping applications of the invention. Most preferably, the oligonucleotides to be used in the method of this invention comprise, at least in part or fully, 2′-O-methoxy ethyl phosphorothioate RNA nucleotides or 2′-O-methyl phosphorothioate RNA nucleotides. Oligonucleotides comprising or consisting of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (CCG)n, (GAA)n, (GCC)n and (CCUG)n having a length of 10 to 50, more preferably 12 to 35, most preferably 12 to 25 nucleotides, and comprising 2′-O-methoxyethyl phosphorothioate RNA nucleotides, 2′-O-methyl phosphorothioate RNA nucleotides, LNA nucleotides or PMO nucleotides are most preferred for use in the invention for the diagnosis, treatment of prevention of cis-element repeat instability genetic disorders.
Accordingly, in a preferred embodiment, an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
a) comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides could be used in any combination and/or with DNA phosphorothioate or RNA nucleotides; and/or
b) being a single stranded oligonucleotide.
Accordingly, in another preferred embodiment, an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
c) comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides could be used in combination and/or with DNA phosphorothioate or RNA nucleotides; and/or
d) being a double stranded oligonucleotide.
In case, the invention relates to a double stranded oligonucleotide with two complementary strands, the antisense strand, complementary only to a repetitive sequence in a human gene transcript, this double stranded oligonucleotide is preferably not the siRNA with antisense RNA strand (CUG)7 and sense RNA strand (GCA)7 applied to cultured monkey fibroblast (COS-7) or human neuroblastoma (SH-SY5Y) cell lines with or without transfection with a human Huntington gene exon 1 fused to GFP and as depicted in Wanzhao Liu et al (Wanzhao Liu et al, (2003), Proc. Japan Acad, 79: 293-298). More preferably, the invention does neither relate to the double stranded oligonucleotide siRNA (with antisense strand (CUG)7 and sense strand (GCA)7) nor to its use for the manufacture of a medicament for the treatment or prevention of Huntington disease, even more preferably for the treatment or prevention of Huntington disease gene exon 1 containing construct.
Although use of a single oligonucleotide may be sufficient for reducing the amount of repeat expanded transcripts, such as nuclear accumulated DMPK or ZNF9 transcripts or segments thereof or sufficient reduction of accumulation of repeat expanded HD protein, it is also within the scope of the invention to combine 2, 3, 4, 5 or more oligonucleotides. The oligonucleotide comprising or consisting of a sequence that is complementary to a repetitive part of a transcript may be advantageously combined with oligonucleotides that comprise or consist of sequences that are complementary to and/or capable of hybridizing with unique sequences in a repeat containing transcript. The method of the invention and the medicaments of the invention comprising repeat specific oligonucleotides may also be combined with any other treatment or medicament for cis-element repeat instability genetic disorders. For diagnostic purposes the oligonucleotide used in the method of the invention may be provided with a radioactive label or fluorescent label allowing detection of transcripts in samples, in cells in situ in vivo, ex vivo or in vitro. For myotonic dystrophy, labelled oligonucleotides may be used for diagnostic purposes, for visualisation of nuclear aggregates of DMPK or ZNF9 RNA transcript molecules with associated proteins. Fluorescent labels may comprise Cy3, Cy5, FITC, TRITC, Rhodamine, GFP and the like. Radioactive labels may comprise 3H, 35S, 32/33P, 125I. Enzymes and/or immunogenic haptens such as digoxigenin, biotin and other molecular tags (HA, Myc, FLAG, VSV, lexA) may also be used. Accordingly, in a further aspect, the invention discloses an vitro or ex vivo detection and/or diagnostic method wherein a oligonucleotide as defined above is used.
The oligonucleotides for use according to the invention are suitable for direct administration to cells, tissues and/or organs in vivo of individuals affected by or at risk of developing a cis-element repeat instability disorder, and may be administered directly in vivo, ex vivo or in vitro. Alternatively, the oligonucleotides may be provided by a nucleic acid vector capable of conferring expression of the oligonucleotide in human cells, in order to allow a sustainable source of the oligonucleotides. Oligonucleotide molecules according to the invention may be provided to a cell, tissue, organ and/or subject to be treated in the form of an expression vector that is capable of conferring expression of the oligonucleotide in human cells. The vector is preferably introduced in the cell by a gene delivery vehicle. Preferred vehicles for delivery are viral vectors such as retroviral vectors, adeno-associated virus vectors (AAV), adenoviral vectors, Semliki Forest virus vectors (SFV), EBV vectors and the like. Also plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of oligonucleotides. Preferred for the current invention are those vectors wherein transcription is driven from polIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts.
In a preferred embodiment, a concentration of oligonucleotide, which is ranged between about 0.1 nM and about 1 μM is used. More preferably, the concentration used is ranged between about 0.3 to about 400 nM, even more preferably between about 1 to about 200 nM. If several oligonucleotides are used, this concentration may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, the oligonucleotides to be used in the invention to prevent, treat or diagnose cis-element repeat instability disorders are synthetically produced and administered directly to cells, tissues, organs and/or patients in formulated form in pharmaceutically acceptable compositions. The delivery of the pharmaceutical compositions to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body. An intrathecal or intraventricular administration (in the cerebrospinal fluid) is preferably realized by introducing a diffusion pump into the body of a subject. Several diffusion pumps are known to the skilled person.
Pharmaceutical compositions that are to be used to target the oligonucleotide molecules comprising or consisting of a sequence that is complementary to repetitive sequences may comprise various excipients such as diluents, fillers, preservatives, solubilisers and the like, which may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
Particularly preferred for the method of the invention is the use of excipients that will aid in delivery of the oligonucleotides to the cells and into the cells, in particular excipients capable of forming complexes, vesicles and/or liposomes that deliver substances and/or oligonucleotide(s) complexed or trapped in the vesicles or liposomes through a cell membrane. Many of these substances are known in the art. Suitable substances comprise polyethylenimine (PEI), ExGen 500, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver oligonucleotides to cells. Lipofectin represents an example of liposomal transfection agents. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles. Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver oligonucleotides across cell membranes into cells. In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate oligonucleotides as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of the oligonucleotides. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver oligonucleotides for use in the current invention to deliver oligonucleotides for the treatment of cis-element repeat instability disorders in humans.
In addition, the oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes. Such targeting ligand would also encompass molecules facilitating the uptake of oligonucleotides into the brain through the blood brain barrier. Therefore, in a preferred embodiment, an oligonucleotide in a medicament is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to cells and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide of the invention and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to the cell and/or enhancing its intracellular delivery.
The invention also pertains to a method for the reduction of repeat containing gene transcripts in a cell comprising the administration of a single strand or double stranded oligonucleotide molecule, preferably comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothioate nucleotides or LNA nucleotides or PMO nucleotides, and having a length of 10 to 50 nucleotides that are complementary to the repetitive sequence only. The nucleotides could be used in combination and/or with DNA phosphorothioate nucleotides.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but combinations and/or items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
FIGURE LEGENDS
FIG. 1: Northern blot of RNA isolated from myotubes transfected with different oligonucleotides or mock control. The myotubes were derived from immorto mouse myoblast cell lines containing a transgenic human DMPK genes with (CTG)n repeat expansion length of approximately 500 next to its normal mouse DMPK gene without (CTG) repeat. The blot shows human DMPK mRNA (top), mouse DMPK (mDMPK) mRNA (middle) and mouse GAPDH mRNA (bottom).
FIG. 2: The human and mouse DMPK signals of FIG. 1 were quantified by phosphoimager analysis and normalized to the GAPDH signal. The results are expressed relative to the mock treatment (set to 100).
FIG. 3: Northern blot of total RNA isolated from murine myotubes containing a mouse-human chimaeric DMPK gene in which the 3′ part of the mDMPK gene was replaced by the cognate segment of the human DMPK gene including a (CTG)110-repeat. The blot was probed for DMPK mRNA (upper panel) and mouse GAPDH mRNA (bottom). Cells were transfected with antisense oligonucleotide PS58 or control.
FIG. 4 shows the response of DM500 myotubes treated with various concentrations of oligonucleotide PS58. The expression of hDMPK was quantified via Northern blot analysis followed by phosphoimager analysis. The signal was normalised to the GAPDH signal and expressed relative to the response after mock treatment.
FIG. 5 shows the Northern blot of total RNA of DM500 myotubes transfected with 200 nM PS58 at different time points: 2 h, 4 h, 8 h and 48 h before harvesting. Mock treatment was performed 48 h before harvesting. Northern blots show human and mouse DMPK and mouse GAPDH mRNA. These were quantified by phosphoimager and the normalized DMPK signal was expressed relative to mock treatment.
FIG. 6 shows the Northern blot of total RNA of DM500 myotubes harvested 2 d, 4 d, 6 d and 8 d after transfection with 200 nM PS58 or mock control. Northern blot analysis and quantification was performed as before.
FIG. 7 shows a Northern blot of total RNA from MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM) or mock control. The myoblasts were derived from fibroblasts obtained from a congenital myotonic dystrophy type I patient bearing a hDMPK allele with a triplet repeat expansion length of approximately 1500 and a hDMPK allele with normal length of 11 repeats. The Northern blot was hybridized with a human DMPK (hDMPK) probe and GAPDH mRNA probe. The human DMPK signals were normalized to the GAPDH signal and expressed relative to mock control.
FIG. 8 shows the RT-PCR analysis of DM500 myotubes transfected with 200 nM of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control. RT-PCR analysis was performed with primers specific for hDMPK mRNA and three other gene transcripts with a naturally occurring (CUG) repeat in mice: Ptbp1 mRNA with a (CUG)6, Syndecan3 mRNA with a (CUG)6 and Taxilinbeta mRNA with a (CUG)9. The intensity of the signals were normalized to the actin signal and expressed relative to mock control.
FIG. 9 shows FISH analysis of DM500 myoblasts transfected with 200 nM PS58 (B) or mock control (A). Fourty eight hours after the start of the treatment, the cells were washed and fixed and subsequently hybridized with fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3. The ribonuclear foci indicative of hDMPK (CUG)500 mRNA aggregation in the nucleus were visualized using a Bio-Rad MRC1024 confocal laser scanning microscope and LaserSharp2000 acquisition software.
FIG. 10 shows the relative cell count for the presence of ribonuclear foci in the nucleus of DM500 myoblasts transfected with PS58 or mock control from the experiment depicted in FIG. 9.
FIG. 11 shows the RT-PCR analysis of hDMPK mRNA in muscle of DM500 mice treated with PS58 or mock control. Shortly, PS58 (2 nmol) was injected in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h. After 15 days, M. plantaris and M. gastrocnemius were isolated and RT-PCR was performed on total RNA for hDMPK and mouse actin. The intensity of the hDMPK signal was normalized to the actin signal and the values expressed relative to mock control.
FIG. 12 shows a Northern blot analysis of DM500 myotubes treated with different oligonucleotides (200 nM) or mock control. PS58 (SEQ ID NO:6), PS146 (SEQ ID NO:16) and PS147 (SEQ ID NO:17 carried a full 2′O-methyl phosphorothiate backbone, but differed in length, (CAG)7, (CUG)10 and (CUG)5, respectively. PS142 has a complete phosphorothiate DNA backbone with a (CAG)7 sequence (SEQ ID NO:15). hDMPK and mDMPK signals were normalized to mouse GAPDH and expressed as percentage to mock control. Quantification is shown in the lower panel.
EXAMPLES
Example 1
Immortomyoblast cell lines were derived from DM500 or CTG110 mice using standard techniques known to the skilled person. DM500 mice were derived from mice obtained from de Gourdon group in Paris. CTG110 mice are described below and present at the group of Wieringa and Wansink in Nijmegen. Immortomyoblast cell lines DM500 or CTG110 with variable (CTG)n repeat length in the DMPK gene were grown subconfluent and maintained in a 5% CO2 atmosphere at 33° C. on 0.1% gelatin coated dishes. Myoblast cells were grown subconfluent in DMEM supplemented with 20% FCS, 50 μg/ml gentamycin and 20 units of γ-interferon/ml. Myotube formation was induced by growing myoblast cells on Matrigel (BD Biosciences) coated dishes and placing a confluent myoblast culture at 37° C. and in DMEM supplemented with 5% horse serum and 50 μg/ml gentamycin. After five days on this low serum media contracting myotubes arose in culture and were transfected with the desired oligonucleotides. For transfection NaCl (500 mM, filter sterile), oligonucleotide and transfection reagens PEI (ExGen 500, Fermentas) were added in this specific order and directly mixed. The oligonucleotide transfection solution contained a ratio of 5 μl ExGen500 per ug oligonucleotide which is according to the instructions (ExGen 500, Fermentas). After 15 minutes of incubation at room temperature the oligonucleotide transfection solution was added to the low serum medium with the cultured myotubes and gently mixed. The final oligonucleotide concentration was 200 nM. Mock control treatment is carried out with transfection solution without an oligonucleotide. After four hours of incubation at 37° C., fresh medium was added to the culture (resulting in a dilution of approximately 2.3×) and incubation was extended overnight at 37° C. The next day the medium containing the oligonucleotide was removed and fresh low serum medium was added to the myotubes which were kept in culture at 37° C. for another day. Fourty eight hours after the addition of oligonucleotide to the myotube culture (which is seven days after switching to low serum conditions to induced myotube formation), RNA was isolated with the “Total RNA mini kit” (Bio-Rad) and prepared for Northern blot and RT-PCR analysis. The Northern blot was hybridized with a radioactive human DMPK (hDMPK) probe and a mouse GAPDH probe. The probe used for DMPK is a human DMPK cDNA consisting of the DMPK open reading frame with full 3′ UTR and 11 CTGs.
The human and mouse DMPK signal were quantified by phosphoimager analysis and normalized to the GAPDH signal. Primers that were used for the RT-PCR for hDMPK mRNA were situated in the 3′ untranslated part with the sequence 5′-GGGGGATCACAGACCATT-3′ (SEQ ID NO:23) and 5′-TCAATGCATCCAAAACGTGGA-3′ (SEQ ID NO:24) and for murine actin the primers were as followed: Actin sense 5′-GCTAYGAGCTGCCTGACGG-3′ (SEQ ID NO:25) and Actin antisense 5′-GAGGCCAGGATGGAGCC-3′ (SEQ ID NO:26). PCR products were run on an agarose gel and the signal was quantified using Labworks 4.0 (UVP Biolmaging systems, Cambridge, United Kingdom). The intensity of each band was normalized to the intensity of the corresponding actin band and expressed relative to mock control.
Thirteen different oligonucleotides were tested (for an overview see Table 1) as described above on the immortomyoblast DM500 cell line containing transgenic human DMPK gene with (CTG)n repeat length of approximately 500 and a normal mouse DMPK gene without (CTG) repeat. FIG. 1 shows the Northern blot of the isolated RNA from the oligonucleotide transfected myotubes visualized with the hDMPK probe and a GAPDH probe for loading control. Quantification of the human DMPK (with CTG repeat) and murine DMPK (without CTG repeat) signal on the Northern blot is shown in FIG. 2. The signal was normalized to murine GAPDH and expressed relative to mock control.
Table 2 indicates the level of hDMPK mRNA reduction that is caused by a specific oligonucleotide. The minus (−) stands for no reduction and the number of positive signs (+) stands for the relative level of hDMPK mRNA break-down. Clearly, oligonucleotide PS58, specifically targeted to the repeat sequence, is much more potent in reducing or altering hDMPK transcripts than the other oligonucleotides complementary to unique sequences in the hDMPK transcripts.
FIG. 3 shows the effect of PS58 in murine immortomyotubes derived from CTG110 mice, a knock-in mouse containing a DMPK gene with the 3′ part of the human DMPK gene including a (CTG) repeat of approximately 110. Northern blot analysis showed that the DMPK transcript containing the (CTG)110 repeat was reduced by the treatment with oligonucleotide PS58 but not after mock treatment.
Example 2 (FIG. 4)
The DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1). In this example, the transfection was carried out with PS58 at different concentrations. Eighty four hours after start of treatment, the myotubes were harvested and Northern blot analysis was performed on isolated RNA as described above (see example 1).
FIG. 4 shows the quantification of the hDMPK mRNA signal preformed by phosphoimager analysis and normalized to the GAPDH signal at different concentrations. Under these conditions, a half maximal effect was observed at around 1 nM.
Example 3 (FIGS. 5 and 6)
The DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1). However, in this example the transfection with 200 nM PS58 was carried out at different time points. Usually DM500 myotubes were harvested seven days after switching to low serum conditions to induce myotube formation. The standard procedure (as in example 1 and 2) was to start treatment (transfection) 48 h (two days) before harvesting. Now, treatment with PS58 was started 2 h-48 h (FIG. 5) or 2 d-8 d (FIG. 6) before harvesting. Northern blot analysis and quantification was performed as before.
FIG. 5 shows that expanded hDMPK mRNA in DM500 myotubes was decreased rapidly within 2 h of treatment with oligonucleotide PS58 compared to mock control treatment.
FIG. 6 shows a persistent decrease in expanded hDMPK mRNA in DM500 myotubes for at least 8 days. Please note that in the case of the 8 d experiment, cells were transfected in the myoblast stage (approximately 60% confluent, 33° C., high serum) and that they have received fresh medium on various occasions until harvesting (including a change to low serum at 37° C., two days after transfection). Example 2 and 3 are indicative of a highly efficient inhibitory intervention by an oligonucleotide directed solely to the repeat expansion. The magnitude of this effect might be influenced by the relative low levels of hDMPK expression in these model cell systems, which normally is also seen in humans.
Example 4 (FIG. 7)
In this example, fibroblasts obtained from a human patient with congenital myotonic dystrophy type 1 (cDM1) were used. These patient cells carry one disease causing DMPK allele with a triplet repeat expansion length of 1500 and one normal DMPK allele with a repeat length of 11. The size of the (CTG)n expansion on both alleles was confirmed with PCR and Southern blotting.
The fibroblasts were grown subconfluent and maintained in a 5% CO2 atmosphere at 37° C. on 0.1% gelatin coated dishes. Fibroblasts were grown subconfluent in DMEM supplemented with 10% FCS and 50 μg/ml gentamycin. Myotube formation was induced by growing fibroblasts cells on Matrigel (BD Biosciences) coated dishes and infecting the cells at 75% confluency with MyoD-expressing adenovirus (Ad5Fib50MyoD, Crucell, Leiden) (MOI=100) in DMEM supplemented with 2% HS and 50 μg/ml gentamycin for 2 hours. After the incubation period MyoD adenovirus was removed and DMEM supplemented with 10% FCS and 50 μg/ml gentamycin was added. The cells were maintained in this medium in a 5% CO2 atmosphere at 37° C. until 100% confluency. At this point cells were placed in DMEM supplemented with 2% FCS and 50 μg/ml gentamycin. After five days on this low serum media cells were transfected with PS58 following the procedure according to the instructions (ExGen 500, Fermentas) and as described above. The final oligonucleotide concentration was 200 nM and 20 nM. Fourty eight hours after start of the treatment (which is seven days after switching to low serum conditions), RNA was isolated with the “Total RNA mini kit” (Bio-Rad) and prepared for Northern blot. The Northern blot was hybridized with a radioactive human DMPK (hDMPK) and mouse GAPDH mRNA probe. The human DMPK signals were quantified by phosphoimager analysis and normalized to the GAPDH signal and expressed relative to mock control.
FIG. 7 shows the Northern blot analysis of the MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM). The results demonstrate an effective complete inhibition of the disease-causing hDMPK (CUG)1500 RNA transcript, while the smaller normal hDMPK (CUG)11 RNA transcript is only moderately affected at the two concentrations. Thus, oligonucleotides directed to the repeat region exhibit selectivity towards the larger repeat size (or disease causing expansion).
Example 5 (FIG. 8)
In this example, the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, transfected and analysed as described before in example 1. The DM500 myotubes were treated 48 h before harvesting with 200 nM of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control. RT-PCR analysis was performed on hDMPK mRNA expressed in this murine cell line (for primers see example 1) and on three other gene transcripts with a naturally occurring (CUG) repeat in mice, Ptbp1 with a (CUG)6, Syndecan3 with a (CUG)6 and Taxilinbeta with a (CUG)9.
The PCR primers used were for Ptbp1: 5′-TCTGTCCCTAATGTCCATGG-3′ (SEQ ID NO:27) and 5′-GCCATCTGCACAAGTGCGT-3′ (SEQ ID NO:28); for Syndecan3: 5′-GCTGTTGCTGCCACCGCT-3′ (SEQ ID NO:29) and 5′-GGCGCCTCGGGAGTGCTA-3′ (SEQ ID NO:30); and for Taxilinbeta: 5′-CTCAGCCCTGCTGCCTGT-3′ (SEQ ID NO:31) and 5′-CAGACCCATACGTGCTTATG-3′ (SEQ ID NO:32). The PCR products were run on an agarose gel and signals were quantified using the Labworks 4.0 program (UVP Biolmaging systems, Cambridge, United Kingdom). The intensity of each signal was normalized to the corresponding actin signal and expressed relative to mock control.
FIG. 8 shows the RT-PCR results with a maximal inhibition of hDMPK mRNA expression by PS58. The other gene transcripts carrying a naturally occurring small (CUG) repeat were not or only marginally affected by the oligonucleotide PS58, specific to the (CUG) repeat, compared to oligonucleotide PS113, which has no complementary sequence to these gene transcripts.
This example confirms the selectivity of an oligonucleotide, directed solely to the repeat region, towards the long repeat size (or disease causing expansion) compared to naturally occurring shorter repeat sizes.
Example 6 (FIG. 9 en 10)
In this example, the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured and transfected with PS58 (200 nM). Here, FISH analysis was carried out on the cells. Fourty eight hours after the start of the treatment, the cells were fixed with 4% formaldehyde, 5 mM MgCl2 and 1×PBS for 30 minutes. Hybridization with fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3 was performed overnight at 37° C. in a humid chamber. After hybridization the material was washed and mounted in mowiol and allowed to dry overnight. Nuclear inclusions (ribonuclear foci) were visualized using a Bio-Rad MRC1024 confocal laser scanning microscope and LaserSharp2000 acquisition software. In total 50 cells were counted and scored for the presence of inclusions in the nuclei of these cells.
Literature indicates that DMPK mRNA containing a (CUG) expanded repeat accumulates and aggregates in the nucleus to form ribonuclear foci with regulatory nuclear proteins and transcription factors. Therefore, normal nuclear gene processing and cell function gets impaired.
FIG. 9 shows a mock treated cell containing ribonuclear inclusions in the nucleus, while these are no longer present in the cell nucleus after treatment with PS58. FIG. 10 shows that the percentage of nuclei containing ribonuclear foci seen under control conditions in DM500 myotubes is strongly decreased by the treatment with PS58. This result demonstrates that inhibition of hDMPK mRNA expression also inhibits the disease related triplet repeat (CUG) rich inclusions.
Example 7 (FIG. 11)
Here, the effect of PS58 was evaluated in vivo in DM500 mice containing hDMPK with a (CTG)n expansion of approximately 500 triplets. The DM500 mice were derived by somatic expansion from the DM300 mouse (e.g. see Gomes-Pereira M et al (2007) PLoS Genet. 2007 3(4): e52). A (CTG) triplet repeat expansion of approximately 500 was confirmed by southern blot and PCR analysis.
In short, PS58 was mixed with transfection agent ExGen 500 (Fermentas) according to the accompanying instructions for in vivo use. PS58 (2 nmol, in the transfection solution with Exgen 500) was injected (40μl) in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h. As a control, DM500 mice were treated similarly with the transfection solution without PS58. After 15 days, the mice were sacrificed, muscles were isolated and total RNA was isolated from the tissues (using Trizol, Invitrogen). RT-PCR analysis was performed to detect hDMPK mRNA in the muscle similar as described above. The intensity of each band was performed using the Labworks 4.0 program (UVP Biolmaging systems, Cambridge, United Kingdom) and normalized to the intensity of the corresponding actin band. Primer location is indicated in the figure.
FIG. 11 shows that in vivo treatment of DM500 mice with PS58 strongly reduced the presence of hDMPK mRNA containing a (CUG)n repeat expansion compared to mock treatment in the M. plantaris and M. gastrocnemius.
Example 8 (FIG. 12)
In this example, different oligonucleotides (in length and backbone chemistry) but all with a sequence directed solely to the (CTG)n repeat expansion were compared. DM500 myotubes were cultured, transfected and analysed as described above in example 1. Northern blots were quantified by phosphoimager analysis and DMPK signals were normalized to GAPDH.
Here, the DM500 myotubes were treated with the following oligonucleotides (200 nM), all with a complete phosphorothioate backbone (see Table 3).
FIG. 12 shows that treatment of the DM500 myotubes results in a complete reduction of (CUG)n expanded hDMPK mRNA for all oligonucleotides tested. Under the present conditions, the maximal effect obtainable is independent of oligonucleotide length, backbone modification or potential mechanism of inhibition by the employed single stranded oligonucleotides.
Example 9
Fibroblasts (GM 00305) from a male patient with Huntington's Disease were obtained from Coriell Cell Repository (Camden, N.J., US) and cultured according to the accompanying instructions and standard techniques known to the skilled person in the art. Huntington patients carry one healthy and one disease-causing allele of the Huntington gene resulting in the expression of both mRNAs with respectively a normal number and an expanded number of (CAG) repeats, respectively.
The fibroblasts were transfected with a 21-mer 2′O-methyl phosphorothioate RNA antisense oligonucleotide PS57 with a (CUG)7 sequence (SEQ ID NO:5), complementary to the (CAG) triplet repeat in Huntington mRNA. Transfection occurred at 100 or 200 nM in the presence of PEI as indicated by the manufacturer. Twenty four hours after transfection the cells were harvested and total RNA was isolated and analysed by RT-PCR. The Huntington transcript was determined using primers in downstream exon 64 (5′ GAAAG TCAGT CCGGG TAGAA CTTC 3′ (SEQ ID NO:33) and 5′ CAGAT ACCCG CTCCA TAGCA A 3′ (SEQ ID NO:34)).
This method detects both types of Huntington mRNAs, the normal and mutant transcript with the additional (CAG) expansion. GAPDH mRNA (housekeeping gene) was also determined. The signals were quantified and the total amount of Huntington mRNA was normalised to the amount of GAPDH mRNA in the same sample. The results are expressed relative to a control treated (without oligonucleotide) sample from fibroblasts (which was to 100%).
In the samples from fibroblasts transfected with either 100 or 200 nM of PS57, significantly lower levels of total Huntington mRNA levels were observed of approximately 53% and 66% compared to the levels in control-treated cells, respectively.
Thus, PS57, an oligonucleotide directed only to the (CAG) repeat, induces a decrease in Huntington mRNA levels and these results are consistent with a selective inhibition of mutant over normal Huntington mRNA.
TABLE 1
Overview oligonucleotides tested
Oligo
name
Modification
Sequence
Position
PS40
2′OMe RNA
GAGGGGCGUCCAGG
intron 14-
phosphorothioate/
GAUCCG
exon 15
FAM
(SEQ ID NO: 1)
PS41
2′OMe RNA
GCGUCCAGGGAUCC
intron 14-
phosphorothioate
GGACCG
exon 15
(SEQ ID NO: 2)
PS42
2′OMe RNA
CAGGGAUCCGGACC
intron 14-
phosphorothioate
GGAUAG
exon 15
(SEQ ID NO: 3)
PS56
DNA
CAGCAGCAGCAGCA
repeat in
GCAGCAG
exon 15
(SEQ ID NO: 4)
PS58
2′OMe RNA
CAGCAGCAGCAGCA
repeat in
phosphorothioate/
GCAGCAG
exon 15
FAM
(SEQ ID NO: 6)
P559
2′OMe RNA
UGAGUUGGCCGGCG
ESE exon
phosphorothioate
UGGGCC
15
(SEQ ID NO: 7)
PS60
2′OMe RNA
UUCUAGGGUUCAGG
ESE exon
phosphorothioate
GAGCGCGG
15
(SEQ ID NO: 8)
PS61
2′OMe RNA
ACUGGAGCUGGGCG
ESE exon
phosphorothioate
GAGACCC
15
(SEQ ID NO: 9)
PS62
2′OMe RNA
CUCCCCGGCCGCUA
ESE exon
phosphorothioate
GGGGGC
15
(SEQ ID NO: 10)
PS113
DNA
GAGCCGCCTCAGCC
Exon 1
phosphothioroate
GCACCTC
(SEQ ID NO: 11)
PS114
DNA
GAAGTCGGCCACGT
Exon 1
phosphothioroate
ACTTGTC
(SEQ ID NO: 12)
PS115
DNA
GGAGTCGAAGACAG
Exon 15
phosphothioroate
TTCTAGG
(SEQ ID NO: 13)
PS116
DNA
GGTACACAGGACTG
Exon 15
phosphothioroate
GAGCTGG
(SEQ ID NO: 14)
TABLE 2
Reduction of hDMPK mRNA after oligo transfection:
Oligo
Reduction hDMPK mRNA
SEQ ID No.'s
PS40
+
1
PS41
−
2
PS42
−
3
PS59
−
7
PS60
−
8
PS61
+/−
9
PS62
−
10
PS58
++++
6
PS56
−
4
PS113
−
11
PS114
−
12
PS115
+/−
13
PS116
+
14
(−) indicates no reduction, (+) indicates level of reduction in hDMPK mRNA.
TABLE 3
Oligonucleotides used in example 9
RNAse H
Substitution
breakdown
#
Length
(CAG)n
ribose
possible
PS58
21-mer
n = 7
2′O-Methyl
No
(SEQ ID NO: 6)
PS146
30-mer
n = 10
2′O-Methyl
No
(SEQ ID NO: 16)
PS147
15-mer
n = 5
2′O-Methyl
No
(SEQ ID NO: 17)
PS142
21-mer
n = 7
Deoxyribose
Yes
(SEQ ID NO: 15)
(DNA)
* all oligonucleotides full length phosphorothioate and substitution
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15855848
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Feb 13th, 2018 12:00AM
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Jul 27th, 2015 12:00AM
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https://www.uspto.gov?id=US09890379-20180213
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Treatment of genetic disorders associated with DNA repeat instability
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The current invention provides for methods and medicaments that apply oligonucleotide molecules complementary only to a repetitive sequence in a human gene transcript, for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders. The invention also pertains to modified oligonucleotides which can be applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.
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9890379
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1. A method of treating in a subject having a genetic disorder associated with human cis-element repeat instability comprising administering to the subject an oligonucleotide comprising 10 to 50 nucleotides that is complementary to a sequence of a pre-mRNA transcript, said cis-element repeat instability is characterized by CAG repeat instability, said sequence comprising (CAGCAGCAGCAG) SEQ ID NO: 18, said oligonucleotide comprises either ribonucleotides or deoxyribonucleotides, said oligonucleotide comprising a modification selected from the group consisting of: a morpholino phosphorodiamidate oligonucleotide, a phosphorothioate oligonucleotide, a locked nucleic acid (LNA), a peptide nucleic acid (PNA) and an ethylene-bridged nucleic acid, wherein said administered oligonucleotide does not induce RNaseH-mediated degradation of said pre-mRNA transcripts.
2. The method according to claim 1, wherein said sequence is present in a coding sequence of the gene pre-mRNA transcript.
3. The method according to claim 1, wherein said sequence is present in a non-coding sequence of the gene pre-mRNA transcript.
4. The method according to claim 1, wherein the disorder is Huntington's disease.
5. The method according to claim 1, wherein the oligonucleotide comprises a 2′-O-substituted phosphorothioate nucleotide.
6. The method of claim 5, wherein said oligonucleotide comprises a 2′-O-methyl phosphorothioate nucleotide or a 2′-O-methyoxy ethyl phosphorothioate nucleotide.
7. A method for reducing the number of CAG repeat-containing gene transcripts in a cell comprising providing to said cell an oligonucleotide comprising a 10 to 50 nucleotides that is complementary to a sequence of a pre-mRNA transcript, said sequence comprising (CAGCAGCAGCAG) SEQ ID NO: 18, said oligonucleotide comprises either ribonucleotides or deoxyribonucleotides, said oligonucleotide comprising a modification selected from the group consisting of: a morpholino phosphorodiamidate oligonucleotide, a phosphorothioate oligonucleotide, a locked nucleic acid (LNA), a peptide nucleic acid (PNA) and an ethylene-bridged nucleic acid, wherein said oligonucleotide does not induce RNaseH-mediated degradation of said pre-mRNA transcripts.
8. The method according to claim 1, wherein the oligonucleotide has a length selected from the group consisting of 12 to 30 nucleotides, 12 to 45 nucleotides and 12 to 25 nucleotides.
9. The method of claim 7, wherein the oligonucleotide has a length selected from the group consisting of 12 to 30 nucleotides, 12 to 45 nucleotides and 12 to 25 nucleotides.
10. The method of claim 1, wherein the oligonucleotide is fully complementary to said sequence.
11. The method of claim 1, wherein the oligonucleotide is a 2′-O-methyl phosphorothioate.
12. A method of treating a subject having a genetic disorder associated with human cis-element CAG repeat instability comprising administering to the subject an oligonucleotide comprising 10 to 50 nucleotides that is completely complementary to a sequence of a pre-mRNA transcript, wherein the oligonucleotide comprises a sequence selected from the group consisting of: SEQ ID NOs: 5 (cug cug cug cug cug cug cug) and 20 (cug cug cug cug).
13. The method of claim 12, wherein the oligonucleotide comprises a modification selected from the group consisting of: a morpholino phosphorodiamidate oligonucleotide, a phosphorothioate oligonucleotide, a locked nucleic acid (LNA), a peptide nucleic acid (PNA) and an ethylene-bridged nucleic acid.
14. The method of claim 1, wherein the oligonucleotide is a RNA phosphorothioate comprising the sequence of SEQ ID NO: 5 (cug cug cug cug cug cug cug) or 20 (cug cug cug cug).
15. The method of claim 1, wherein the oligonucleotide is a DNA phosphorothioate oligonucleotide.
16. The method of claim 12, wherein the oligonucleotide is RNA phosphorothioate consisting of the sequence of SEQ ID NO: 5 (cug cug cug cug cug cug cug) or 20 (cug cug cug cug).
17. The method of claim 12, wherein the oligonucleotide consists of the sequence of SEQ ID NOs: 5 (cug cug cug cug cug cug cug) or 20 (cug cug cug cug).
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FIELD OF THE INVENTION
The current invention relates to the field of medicine, in particular to the treatment of genetic disorders associated with genes that have unstable repeats in their coding or non-coding sequences, most in particular unstable repeats in the human Huntington disease causing HD gene or the myotonic dystrophy type 1 causing DMPK gene.
BACKGROUND OF THE INVENTION
Instability of gene-specific microsatellite and minisatellite repetitive sequences, leading to increase in length of the repetitive sequences in the satellite, is associated with about 35 human genetic disorders. Instability of trinucleotide repeats is for instance found in genes causing X-linked spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy type 1 (DM1), fragile X syndrome (FRAX genes A, E, F), Huntington's disease (HD) and several spinocerebellar ataxias (SCA gene family). Unstable repeats are found in coding regions of genes, such as the Huntington's disease gene, whereby the phenotype of the disorder is brought about by alteration of protein function and/or protein folding. Unstable repeat units are also found in untranslated regions, such as in myotonic dystrophy type 1 (DM1) in the 3′ UTR or in intronic sequences such as in myotonic dystrophy type 2 (DM2). The normal number of repeats is around 5 to 37 for DMPK, but increases to premutation and full disease state two to ten fold or more, to 50, 100 and sometimes 1000 or more repeat units. For DM2/ZNF9 increases to 10,000 or more repeats have been reported. (Cleary and Pearson, Cytogenet. Genome Res. 100: 25-55, 2003).
The causative gene for Huntington's disease, HD, is located on chromosome 4. Huntington's disease is inherited in an autosomal dominant fashion. When the gene has more than 35 CAG trinucleotide repeats coding for a polyglutamine stretch, the number of repeats can expand in successive generations. Because of the progressive increase in length of the repeats, the disease tends to increase in severity and presents at an earlier age in successive generations, a process called anticipation. The product of the HD gene is the 348 kDa cytoplasmic protein huntingtin. Huntingtin has a characteristic sequence of fewer than 40 glutamine amino acid residues in the normal form; the mutated huntingtin causing the disease has more than 40 residues. The continuous expression of mutant huntingtin molecules in neuronal cells results in the formation of large protein deposits which eventually give rise to cell death, especially in the frontal lobes and the basal ganglia (mainly in the caudate nucleus). The severity of the disease is generally proportional to the number of extra residues.
DM1 is the most common muscular dystrophy in adults and is an inherited, progressive, degenerative, multisystemic disorder of predominantly skeletal muscle, heart and brain. DM1 is caused by expansion of an unstable trinucleotide (CTG)n repeat in the 3′ untranslated region of the DMPK gene (myotonic dystrophy protein kinase) on human chromosome 19q (Brook et al, Cell, 1992). Type 2 myotonic dystrophy (DM2) is caused by a CCTG expansion in intron 1 of the ZNF9 gene, (Liguori et al, Science 2001). In the case of myotonic dystrophy type 1, the nuclear-cytoplasmic export of DMPK transcripts is blocked by the increased length of the repeats, which form hairpin-like secondary structures that accumulate in nuclear foci. DMPK transcripts bearing a long (CUG)n tract can form hairpin-like structures that bind proteins of the muscleblind family and subsequently aggregate in ribonuclear foci in the nucleus. These nuclear inclusions are thought to sequester muscleblind proteins, and potentially other factors, which then become limiting to the cell. In DM2, accumulation of ZNF9 RNA carrying the (CCUG)n expanded repeat form similar foci. Since muscleblind proteins are splicing factors, their depletion results in a dramatic rearrangement in splicing of other transcripts. Transcripts of many genes consequently become aberrantly spliced, for instance by inclusion of fetal exons, or exclusion of exons, resulting in non-functional proteins and impaired cell function.
The observations and new insights above have led to the understanding that unstable repeat diseases, such as myotonic dystrophy type 1, Huntington's disease and others can be treated by removing, either fully or at least in part, the aberrant transcript that causes the disease. For DM1, the aberrant transcript that accumulates in the nucleus could be down regulated or fully removed. Even relatively small reductions of the aberrant transcript could release substantial and possibly sufficient amounts of sequestered cellular factors and thereby help to restore normal RNA processing and cellular metabolism for DM (Kanadia et al., PNAS 2006). In the case of HD, a reduction in the accumulation of huntingtin protein deposits in the cells of an HD patient can ameliorate the symptoms of the disease.
A few attempts have been made to design methods of treatment and medicaments for unstable repeat disease myotonic dystrophy type 1 using antisense nucleic acids, RNA interference or ribozymes. (i) Langlois et al. (Molecular Therapy, Vol. 7 No. 5, 2003) designed a ribozyme capable of cleaving DMPK mRNA. The hammerhead ribozyme is provided with a stretch RNA complementary to the 3′ UTR of DMPK just before the CUG repeat. In vivo, vector transcribed ribozyme was capable of cleaving and diminishing in transfected cells both the expanded CUG repeat containing mRNA as well as the normal mRNA species with 63 and 50% respectively. Hence, also the normal transcript is gravely affected by this approach and the affected mRNA species with expanded repeats are not specifically targeted.
(ii) Another approach was taken by Langlois et al., (Journal Biological Chemistry, vol 280, no. 17, 2005) using RNA interference. A lentivirus-delivered short-hairpin RNA (shRNA) was introduced in DM1 myoblasts and demonstrated to down regulate nuclear retained mutant DMPK mRNAs. Four shRNA molecules were tested, two were complementary against coding regions of DMPK, one against a unique sequence in the 3′ UTR and one negative control with an irrelevant sequence. The first two shRNAs were capable of down regulating the mutant DMPK transcript with the amplified repeat to about 50%, but even more effective in down regulating the cytoplasmic wildtype transcript to about 30% or less. Equivalent synthetic siRNA delivered by cationic lipids was ineffective. The shRNA directed at the 3′ UTR sequence proved to be ineffective for both transcripts. Hence, also this approach is not targeted selectively to the expanded repeat mRNA species.
(iii) A third approach by Furling et al. (Gene Therapy, Vol. 10, p 795-802, 2003) used a recombinant retrovirus expressing a 149-bp long antisense RNA to inhibit DMPK mRNA levels in human DM1 myoblasts. A retrovirus was designed to provide DM1 cells with the 149 bp long antisense RNA complementary to a 39 bp-long (CUG)13 repeat and a 110 bp region following the repeat to increase specificity. This method yielded a decrease in mutated (repeat expanded) DMPK transcript of 80%, compared to a 50% reduction in the wild type DMPK transcript and restoration of differentiation and functional characteristics in infected DM1 myoblasts. Hence, also this approach is not targeted selectively to the expanded repeat mRNA species, it depends on a very long antisense RNA and can only be used in combination with recombinant viral delivery techniques.
DETAILED DESCRIPTION OF THE INVENTION
The methods and techniques described above provide nucleid acid based methods that cause non-selective breakdown of both the affected repeat expanded allele and unaffected (normal) allele for genetic diseases that are associated with repeat instability and/or expansion. Moreover, the art employs sequences specific for the gene associated with the disease and does not provide a method that is applicable to several genetic disorders associated with repeat expansion. Finally, the art only teaches methods that involve use of recombinant DNA vector delivery systems, which need to be adapted for each oligonucleotide and target cell and which still need to be further optimised.
The current invention provides a solution for these problems by using a short single stranded nucleic acid molecule that comprises or consists of a sequence, which is complementary to the expanded repeat region only, i.e. it does not rely on hybridisation to unique sequences in exons or introns of the repeat containing gene. Furthermore, it is not essential that the employed nucleic acid (oligonucleotide) reduces transcipts by the RNAse H mediated breakdown mechanism.
Without wishing to be bound by theory, the current invention may cause a decrease in transcript levels by alterations in posttranscriptional processing and/or splicing of the premature RNA. A decrease in transcript levels via alternative splicing and/or postranscriptional processing is thought to result in transcripts lacking the overly expanded or instable (tri)nucleotide repeat, but still possessing functional activities. The reduction of aberrant transcripts by altered RNA processing and/or splicing may prevent accumulation and/or translation of aberrant, repeat expanded transcripts in cells.
Without wishing to be bound by theory the method of the current invention is also thought to provide specificity for the affected transcript with the expanded repeat because the kinetics for hybridisation to the expanded repeat are more favourable. The likelihood that a repeat specific complementary nucleic acid oligonucleotide molecule will hybridise to a complementary stretch in an RNA or DNA molecule increases with the size of the repetitive stretch. RNA molecules and in particular RNA molecules comprising repetitive sequences are normally internally paired, forming a secondary structure comprising open loops and closed hairpin parts. Only the open parts are relatively accessible for complementary nucleic acids. The short repeat stretches of a wild type transcript not associated with disease is often only 5 to about 20-40 repeats and due to the secondary structure relatively inaccessible for base pairing with a complementary nucleic acid. In contrast, the repeat units of the expanded repeat and disease associated allele is normally at least 2 fold expanded but usually even more, 3, 5, 10 fold, up to 100 or even more than 1000 fold expansion for some unstable repeat disorders. This expansion increases the likelihood that part of the repeat is, at least temporarily, in an open loop structure and thereby more accessible to base pairing with a complementary nucleic acid molecule, relative to the wild type allele. So despite the fact that the oligonucleotide is complementary to a repeat sequence present in both wildtype and repeat-expanded transcripts and could theoretically hybridise to both transcripts, the current invention teaches that oligonucleotides complementary to the repetitive tracts preferably hybridise to the disease-associated or disease-causing transcripts and leave the function of normal transcripts relatively unaffected. This selectivity is beneficial for treating diseases associated with repeat instability irrespective of the mechanism of reduction of the aberrant transcript.
The invention thus provides a method for the treatment of unstable cis-element DNA repeat associated genetic disorders, by providing nucleic acid molecules that are complementary to and/or capable of hybridising to the repetitive sequences only. This method thereby preferentially targets the expanded repeat transcripts and leaves the transcripts of the normal, wild type allele relatively unaffected. This is advantageous since the normal allele can thereby provide for the normal function of the gene, which is at least desirable and, depending on the particular gene with unstable DNA repeats, may in many cases be essential for the cell and/or individual to be treated.
Furthermore, this approach is not limited to a particular unstable DNA repeat associated genetic disorder, but may be applied to any of the known unstable DNA repeat diseases, such as, but not limited to: coding regions repeat diseases having a polyglutamine (CAG) repeat: Huntington's disease, Haw River syndrome, Kennedy's disease/spinobulbar muscular atrophy, spino-cerebellar ataxia, or diseases having polyalanine (GCG) repeats such as: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis/ptosis/epicanthus invensus syndrome, hand-foot-genital syndrome, synpolydactyly, oculopharyngeal muscular dystrophy, holoprosencephaly. Diseases with repeats in non-coding regions of genes to be treated according to the invention comprise the trinucleotide repeat disorders (mostly CTG and/or CAG and/or CCTG repeats): myotonic dystrophy type 1, myotonic dystrophy type 2, Friedreich's ataxia (mainly GAA), spino-cerebellar ataxia, autism. Furthermore, the method of the invention can be applied to fragile site associated repeat disorder comprising various fragile X-syndromes, Jacobsen syndrome and other unstable repetitive element disorders such as myoclonus epilepsy, facioscapulohumeral dystrophy and certain forms of diabetes mellitus type 2.
Another advantage of the current invention is that the oligonucleotides specific for a repeat region may be administered directly to cells and it does not rely on vector-based delivery systems. The techniques described in the prior art, for instance those mentioned above for treatment of DM1 and removal of DMPK transcripts from cells, require the use of vector based delivery systems to administer sufficient levels of oligonucleotides to the cell. The use of plasmid or viral vectors is yet less desirable for therapeutic purposes because of current strict safety regulations for therapeutic recombinant DNA vectors, the production of sufficient recombinant vectors for broad clinical application and the limited control and reversibility of an exaggerated (or non-specific) response after application. Nevertheless, optimisation in future is likely in these areas and viral delivery of plasmids could yield an advantageous long lasting effect. The current inventors have surprisingly found that oligonucleotides that comprise or consist of a sequence that is complementary to repetitive sequences of expanded repeat transcripts, due to the expansion of their molecular target for hybridisation, have a much increased affinity and/or avidity for their target in comparison to oligonucleotides that are specific for unique sequences in a transcript. Because of this high affinity and avidity for the repeat expanded target transcript, lower amounts of oligonucleotide suffice to yield sufficient inhibition and/or reduction of the repeat expanded allele by RNase H degradation, RNA interference degradation or altered post-transcriptional processing (comprising but not limited to splicing or exon skipping) activities. The oligonucleotides of the current invention which are complementary to repetitive sequences only, may be produced synthetically and are potent enough to be effective when delivered directly to cells using commonly applied techniques for direct delivery of oligonucleotides to cells and/or tissues. Recombinant vector delivery systems may, when desired, be circumvented by using the method and the oligonucleotide molecules of the current invention.
In a first aspect, the current invention discloses and teaches the use of an oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders.
In a second aspect, the invention also pertains to an oligonucleotide which can be used in the first aspect of the invention and/or applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.
An oligonucleotide of the invention may comprise a sequence that is complementary only to a repetitive sequence as defined below. Preferably, the repetitive sequence is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or more. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary only to a repetitive sequence as defined below. For example, an oligonucleotide may comprise a sequence that is complementary only to a repetitive sequence as defined below and a targeting part, which is later on called a targeting ligand.
A repeat or repetitive element or repetitive sequence or repetitive stretch is herein defined as a repetition of at least 3, 4, 5, 10, 100, 1000 or more, of a repetitive unit or repetitive nucleotide unit or repeat nucleotide unit comprising a trinucleotide repetitive unit, or alternatively a 4, 5 or 6 nucleotide repetitive unit, in a transcribed gene sequence in the genome of a subject, including a human subject.
An oligonucleotide may be single stranded or double stranded. Double stranded means that the oligonucleotide is an heterodimer made of two complementary strands, such as in a siRNA. In a preferred embodiment, an oligonucleotide is single stranded. A single stranded oligonucleotide has several advantages compared to a double stranded siRNA oligonucleotide: (i) its synthesis is expected to be easier than two complementary siRNA strands; (ii) there is a wider range of chemical modifications possible to optimise more effective uptake in cells, a better (physiological) stability and to decrease potential generic adverse effects; and (iii) siRNAs have a higher potential for non-specific effects and exaggerated pharmacology (e.g. less control possible of effectiveness and selectivity by treatment schedule or dose) and (iv) siRNAs are less likely to act in the nucleus and cannot be directed against introns. Therefore, in a preferred embodiment of the first aspect, the invention relates to the use of a single stranded oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans.
The oligonucleotide(s) preferably comprise at least 10 to about 50 consecutive nucleotides complementary to a repetitive element, more preferably 12 to 45 nucleotides, even more preferably 12 to 30, and most preferably 12 to 25 nucleotides complementary to a repetitive stretch, preferably having a trinucleotide repeat unit or a tetranucleotide repeat unit. The oligonucleotide may be complementary to and/or capable of hybridizing to a repetitive stretch in a coding region of a transcript, preferably a polyglutamine (CAG) or a polyalanine (GCG) coding tract. The oligonucleotide may also be complementary to and/or capable of hybridizing to a non-coding region for instance 5′ or 3′ untranslated regions, or intronic sequences present in precursor RNA molecules.
In a preferred embodiment the oligonucleotide to be used in the method of the invention comprises or consists of a sequence that is complementary to a repetitive element having as repetitive nucleotide unit a repetitive nucleotide unit selected from the group consisting of (CAG)n (SEQ ID NO:18), (GCG)n (SEQ ID NO:19), (CUG)n (SEQ ID NO:20), (CGG)n (SEQ ID NO:21) (GAA)n (SEQ ID NO:35), (GCC)n (SEQ ID NO:36) and (CCUG)n (SEQ ID NO:22). and said oligonucleotide being a single or double stranded oligonucleotide. Preferably, the oligonucleotide is double stranded.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a polyglutamine (CAG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders Huntington's disease, several forms of spino-cerebellar ataxia or Haw River syndrome, X-linked spinal and bulbar muscular atrophy and/or dentatorubral-pallidoluysian atrophy caused by repeat expansions in the HD, HDL2/JPH3, SBMA/AR, SCA1/ATX1, SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCA7, SCA17, AR or DRPLA human genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a polyalanine (GCG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyly, oculopharyngeal muscular dystrophy and/or holoprosencephaly, which are caused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM/PABP2, TCFBR1 or ZIC2 human genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CUG)n repeat in a transcript and is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 1, spino-cerebrellar ataxia 8 and/or Huntington's disease-like 2 caused by repeat expansions in the DM1/DMPK, SCA8 or JPH3 genes respectively. Preferably, these genes are from human origin.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCUG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 2, caused by repeat expansions in the DM2/ZNF9 gene.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CGG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of human fragile X syndromes, caused by repeat expansion in the FRAXA/FMR1, FRAXE/FMR2 and FRAXF/FAM11A genes.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Jacobsen syndrome, caused by repeat expansion in the FRA11B/CBL2 gene.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (GAA)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Friedreich's ataxia.
The use of an oligonucleotide that comprises or consists of a sequence that is complementary to a (ATTCT)n repeat in an intron is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Spinocerebellar ataxia type 10 (SCA10).
The repeat-complementary oligonucleotide to be used in the method of the invention may comprise or consist of RNA, DNA, Locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino phosphorodiamidates (PMO), ethylene bridged nucleic acid (ENA) or mixtures/hybrids thereof that comprise combinations of naturally occurring DNA and RNA nucleotides and synthetic, modified nucleotides. In such oligonucleotides, the phosphodiester backbone chemistry may further be replaced by other modifications, such as phosphorothioates or methylphosphonates. Other oligonucleotide modifications exist and new ones are likely to be developed and used in practice. However, all such oligonucleotides have the character of an oligomer with the ability of sequence specific binding to RNA. Therefore in a preferred embodiment, the oligonucleotide comprises or consists of RNA nucleotides, DNA nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, morpholino phosphorodiamidates, ethylene-bridged nucleic acid (ENA) nucleotides or mixtures thereof with or without phosphorothioate containing backbones.
Oligonucleotides containing at least in part naturally occurring DNA nucleotides are useful for inducing degradation of DNA-RNA hybrid molecules in the cell by RNase H activity (EC.3.1.26.4).
Naturally occurring RNA ribonucleotides or RNA-like synthetic ribonucleotides comprising oligonucleotides may be applied in the method of the invention to form double stranded RNA-RNA hybrids that act as enzyme-dependent antisense through the RNA interference or silencing (RNAi/siRNA) pathways, involving target RNA recognition through sense-antisense strand pairing followed by target RNA degradation by the RNA-induced silencing complex (RISC).
Alternatively or in addition, steric blocking antisense oligonucleotides (RNase-H independent antisense) interfere with gene expression or other precursor RNA or messenger RNA-dependent cellular processes, in particular but not limited to RNA splicing and exon skipping, by binding to a target sequence of RNA transcript and getting in the way of processes such as translation or blocking of splice donor or splice acceptor sites. Alteration of splicing and exon skipping techniques using modified antisense oligonucleotides are well documented, known to the skilled artisan and may for instance be found in U.S. Pat. No. 6,210,892, WO9426887, WO04/083446 and WO02/24906. Moreover, steric hindrance may inhibit the binding of proteins, nuclear factors and others and thereby contribute to the decrease in nuclear accumulation or ribonuclear foci in diseases like DM1.
The oligonucleotides of the invention, which may comprise synthetic or modified nucleotides, complementary to (expanded) repetitive sequences are useful for the method of the invention for reducing or inactivating repeat containing transcripts via the siRNA/RNA interference or silencing pathway.
Single or double stranded oligonucleotides to be used in the method of the invention may comprise or consist of DNA nucleotides, RNA nucleotides, 2′-O substituted ribonucleotides, including alkyl and methoxy ethyl substitutions, peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino (PMO) antisense oligonucleotides and ethylene-bridged nucleotides (ENA) and combinations thereof, optionally chimeras with RNAse H dependent antisense. Integration of locked nucleic acids in the oligonucleotide changes the conformation of the helix after base pairing and increases the stability of the duplex. Integration of LNA bases into the oligonucleotide sequence can therefore be used to increase the ability of complementary oligonucleotides of the invention to be active in vitro and in vivo to increase RNA degradation inhibit accumulation of transcripts or increase exon skipping capabilities. Peptide nucleic acids (PNAs), an artificial DNA/RNA analog, in which the backbone is a pseudopeptide rather than a sugar, have the ability to form extremely stable complexes with complementary DNA oligomers, by increased binding and a higher melting temperature. Also PNAs are superior reagents in antisense and exon skipping applications of the invention. Most preferably, the oligonucleotides to be used in the method of this invention comprise, at least in part or fully, 2′-O-methoxy ethyl phosphorothioate RNA nucleotides or 2′-O-methyl phosphorothioate RNA nucleotides. Oligonucleotides comprising or consisting of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (CCG)n, (GAA)n, (GCC)n and (CCUG)n having a length of 10 to 50, more preferably 12 to 35, most preferably 12 to 25 nucleotides, and comprising 2′-O-methoxyethyl phosphorothioate RNA nucleotides, 2′-O-methyl phosphorothioate RNA nucleotides, LNA nucleotides or PMO nucleotides are most preferred for use in the invention for the diagnosis, treatment of prevention of cis-element repeat instability genetic disorders.
Accordingly, in a preferred embodiment, an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
a) comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides could be used in any combination and/or with DNA phosphorothioate or RNA nucleotides; and/or
b) being a single stranded oligonucleotide.
Accordingly, in another preferred embodiment, an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
c) comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides could be used in combination and/or with DNA phosphorothioate or RNA nucleotides; and/or
d) being a double stranded oligonucleotide.
In case, the invention relates to a double stranded oligonucleotide with two complementary strands, the antisense strand, complementary only to a repetitive sequence in a human gene transcript, this double stranded oligonucleotide is preferably not the siRNA with antisense RNA strand (CUG)7 and sense RNA strand (GCA)7 applied to cultured monkey fibroblast (COS-7) or human neuroblastoma (SH-SY5Y) cell lines with or without transfection with a human Huntington gene exon 1 fused to GFP and as depicted in Wanzhao Liu et al (Wanzhao Liu et al, (2003), Proc. Japan Acad, 79: 293-298). More preferably, the invention does neither relate to the double stranded oligonucleotide siRNA (with antisense strand (CUG)7 and sense strand (GCA)7) nor to its use for the manufacture of a medicament for the treatment or prevention of Huntington disease, even more preferably for the treatment or prevention of Huntington disease gene exon 1 containing construct.
Although use of a single oligonucleotide may be sufficient for reducing the amount of repeat expanded transcripts, such as nuclear accumulated DMPK or ZNF9 transcripts or segments thereof or sufficient reduction of accumulation of repeat expanded HD protein, it is also within the scope of the invention to combine 2, 3, 4, 5 or more oligonucleotides. The oligonucleotide comprising or consisting of a sequence that is complementary to a repetitive part of a transcript may be advantageously combined with oligonucleotides that comprise or consist of sequences that are complementary to and/or capable of hybridizing with unique sequences in a repeat containing transcript. The method of the invention and the medicaments of the invention comprising repeat specific oligonucleotides may also be combined with any other treatment or medicament for cis-element repeat instability genetic disorders. For diagnostic purposes the oligonucleotide used in the method of the invention may be provided with a radioactive label or fluorescent label allowing detection of transcripts in samples, in cells in situ in vivo, ex vivo or in vitro. For myotonic dystrophy, labelled oligonucleotides may be used for diagnostic purposes, for visualisation of nuclear aggregates of DMPK or ZNF9 RNA transcript molecules with associated proteins. Fluorescent labels may comprise Cy3, Cy5, FITC, TRITC, Rhodamine, GFP and the like. Radioactive labels may comprise 3H, 35S, 32/33P, 125I. Enzymes and/or immunogenic haptens such as digoxigenin, biotin and other molecular tags (HA, Myc, FLAG, VSV, lexA) may also be used. Accordingly, in a further aspect, the invention discloses an vitro or ex vivo detection and/or diagnostic method wherein a oligonucleotide as defined above is used.
The oligonucleotides for use according to the invention are suitable for direct administration to cells, tissues and/or organs in vivo of individuals affected by or at risk of developing a cis-element repeat instability disorder, and may be administered directly in vivo, ex vivo or in vitro. Alternatively, the oligonucleotides may be provided by a nucleic acid vector capable of conferring expression of the oligonucleotide in human cells, in order to allow a sustainable source of the oligonucleotides. Oligonucleotide molecules according to the invention may be provided to a cell, tissue, organ and/or subject to be treated in the form of an expression vector that is capable of conferring expression of the oligonucleotide in human cells. The vector is preferably introduced in the cell by a gene delivery vehicle. Preferred vehicles for delivery are viral vectors such as retroviral vectors, adeno-associated virus vectors (AAV), adenoviral vectors, Semliki Forest virus vectors (SFV), EBV vectors and the like. Also plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of oligonucleotides. Preferred for the current invention are those vectors wherein transcription is driven from polIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts.
In a preferred embodiment, a concentration of oligonucleotide, which is ranged between about 0.1 nM and about 1 μM is used. More preferably, the concentration used is ranged between about 0.3 to about 400 nM, even more preferably between about 1 to about 200 nM. If several oligonucleotides are used, this concentration may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, the oligonucleotides to be used in the invention to prevent, treat or diagnose cis-element repeat instability disorders are synthetically produced and administered directly to cells, tissues, organs and/or patients in formulated form in pharmaceutically acceptable compositions. The delivery of the pharmaceutical compositions to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body. An intrathecal or intraventricular administration (in the cerebrospinal fluid) is preferably realized by introducing a diffusion pump into the body of a subject. Several diffusion pumps are known to the skilled person.
Pharmaceutical compositions that are to be used to target the oligonucleotide molecules comprising or consisting of a sequence that is complementary to repetitive sequences may comprise various excipients such as diluents, fillers, preservatives, solubilisers and the like, which may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
Particularly preferred for the method of the invention is the use of excipients that will aid in delivery of the oligonucleotides to the cells and into the cells, in particular excipients capable of forming complexes, vesicles and/or liposomes that deliver substances and/or oligonucleotide(s) complexed or trapped in the vesicles or liposomes through a cell membrane. Many of these substances are known in the art. Suitable substances comprise polyethylenimine (PEI), ExGen 500, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver oligonucleotides to cells. Lipofectin represents an example of liposomal transfection agents. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles. Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver oligonucleotides across cell membranes into cells. In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate oligonucleotides as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of the oligonucleotides. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver oligonucleotides for use in the current invention to deliver oligonucleotides for the treatment of cis-element repeat instability disorders in humans.
In addition, the oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes. Such targeting ligand would also encompass molecules facilitating the uptake of oligonucleotides into the brain through the blood brain barrier. Therefore, in a preferred embodiment, an oligonucleotide in a medicament is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to cells and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide of the invention and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to the cell and/or enhancing its intracellular delivery.
The invention also pertains to a method for the reduction of repeat containing gene transcripts in a cell comprising the administration of a single strand or double stranded oligonucleotide molecule, preferably comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothioate nucleotides or LNA nucleotides or PMO nucleotides, and having a length of 10 to 50 nucleotides that are complementary to the repetitive sequence only. The nucleotides could be used in combination and/or with DNA phosphorothioate nucleotides.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but combinations and/or items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
FIGURE LEGENDS
FIG. 1: Northern blot of RNA isolated from myotubes transfected with different oligonucleotides or mock control. The myotubes were derived from immorto mouse myoblast cell lines containing a transgenic human DMPK genes with (CTG)n repeat expansion length of approximately 500 next to its normal mouse DMPK gene without (CTG) repeat. The blot shows human DMPK mRNA (top), mouse DMPK (mDMPK) mRNA (middle) and mouse GAPDH mRNA (bottom).
FIG. 2: The human and mouse DMPK signals of FIG. 1 were quantified by phosphoimager analysis and normalized to the GAPDH signal. The results are expressed relative to the mock treatment (set to 100).
FIG. 3: Northern blot of total RNA isolated from murine myotubes containing a mouse-human chimaeric DMPK gene in which the 3′ part of the mDMPK gene was replaced by the cognate segment of the human DMPK gene including a (CTG)110-repeat. The blot was probed for DMPK mRNA (upper panel) and mouse GAPDH mRNA (bottom). Cells were transfected with antisense oligonucleotide PS58 or control.
FIG. 4 shows the response of DM500 myotubes treated with various concentrations of oligonucleotide PS58. The expression of hDMPK was quantified via Northern blot analysis followed by phosphoimager analysis. The signal was normalised to the GAPDH signal and expressed relative to the response after mock treatment.
FIG. 5 shows the Northern blot of total RNA of DM500 myotubes transfected with 200 nM PS58 at different time points: 2 h, 4 h, 8 h and 48 h before harvesting. Mock treatment was performed 48 h before harvesting. Northern blots show human and mouse DMPK and mouse GAPDH mRNA. These were quantified by phosphoimager and the normalized DMPK signal was expressed relative to mock treatment.
FIG. 6 shows the Northern blot of total RNA of DM500 myotubes harvested 2 d, 4 d, 6 d and 8 d after transfection with 200 nM PS58 or mock control. Northern blot analysis and quantification was performed as before.
FIG. 7 shows a Northern blot of total RNA from MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM) or mock control. The myoblasts were derived from fibroblasts obtained from a congenital myotonic dystrophy type I patient bearing a hDMPK allele with a triplet repeat expansion length of approximately 1500 and a hDMPK allele with normal length of 11 repeats. The Northern blot was hybridized with a human DMPK (hDMPK) probe and GAPDH mRNA probe. The human DMPK signals were normalized to the GAPDH signal and expressed relative to mock control.
FIG. 8 shows the RT-PCR analysis of DM500 myotubes transfected with 200 nM of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control. RT-PCR analysis was performed with primers specific for hDMPK mRNA and three other gene transcripts with a naturally occurring (CUG) repeat in mice: Ptbp1 mRNA with a (CUG)6, Syndecan3 mRNA with a (CUG)6 and Taxilinbeta mRNA with a (CUG)9. The intensity of the signals were normalized to the actin signal and expressed relative to mock control.
FIG. 9 shows FISH analysis of DM500 myoblasts transfected with 200 nM PS58 (B) or mock control (A). Forty eight hours after the start of the treatment, the cells were washed and fixed and subsequently hybridized with fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3. The ribonuclear foci indicative of hDMPK (CUG)500 mRNA aggregation in the nucleus were visualized using a Bio-Rad MRC1024 confocal laser scanning microscope and LaserSharp2000 acquisition software.
FIG. 10 shows the relative cell count for the presence of ribonuclear foci in the nucleus of DM500 myoblasts transfected with PS58 or mock control from the experiment depicted in FIG. 9.
FIG. 11 shows the RT-PCR analysis of hDMPK mRNA in muscle of DM500 mice treated with PS58 or mock control. Shortly, PS58 (2 nmol) was injected in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h. After 15 days, M. plantaris and M. gastrocnemius were isolated and RT-PCR was performed on total RNA for hDMPK and mouse actin. The intensity of the hDMPK signal was normalized to the actin signal and the values expressed relative to mock control.
FIG. 12 shows a Northern blot analysis of DM500 myotubes treated with different oligonucleotides (200 nM) or mock control. PS58(SEQ ID NO:6), PS146 (SEQ ID NO:16) and PS147 (SEQ ID NO:17 carried a full 2′O-methyl phosphorothiate backbone, but differed in length, (CAG)7, (CUG)10 and (CUG)5, respectively. PS142 has a complete phosphorothiate DNA backbone with a (CAG)7 sequence (SEQ ID NO:15). hDMPK and mDMPK signals were normalized to mouse GAPDH and expressed as percentage to mock control. Quantification is shown in the lower panel.
EXAMPLES
Example 1
Immortomyoblast cell lines were derived from DM500 or CTG110 mice using standard techniques known to the skilled person. DM500 mice were derived from mice obtained from de Gourdon group in Paris. CTG110 mice are described below and present at the group of Wieringa and Wansink in Nijmegen Immortomyoblast cell lines DM500 or CTG110 with variable (CTG)n repeat length in the DMPK gene were grown subconfluent and maintained in a 5% CO2 atmosphere at 33° C. on 0.1% gelatin coated dishes. Myoblast cells were grown subconfluent in DMEM supplemented with 20% FCS, 50 μg/ml gentamycin and 20 units of γ-interferon/ml. Myotube formation was induced by growing myoblast cells on Matrigel (BD Biosciences) coated dishes and placing a confluent myoblast culture at 37° C. and in DMEM supplemented with 5% horse serum and 50 μg/ml gentamycin. After five days on this low serum media contracting myotubes arose in culture and were transfected with the desired oligonucleotides. For transfection NaCl (500 mM, filter sterile), oligonucleotide and transfection reagens PEI (ExGen 500, Fermentas) were added in this specific order and directly mixed. The oligonucleotide transfection solution contained a ratio of 5 μl ExGen500 per ug oligonucleotide which is according to the instructions (ExGen 500, Fermentas). After 15 minutes of incubation at room temperature the oligonucleotide transfection solution was added to the low serum medium with the cultured myotubes and gently mixed. The final oligonucleotide concentration was 200 nM. Mock control treatment is carried out with transfection solution without an oligonucleotide. After four hours of incubation at 37° C., fresh medium was added to the culture (resulting in a dilution of approximately 2.3×) and incubation was extended overnight at 37° C. The next day the medium containing the oligonucleotide was removed and fresh low serum medium was added to the myotubes which were kept in culture at 37° C. for another day. Forty eight hours after the addition of oligonucelotide to the myotube culture (which is seven days after switching to low serum conditions to induced myotube formation), RNA was isolated with the “Total RNA mini kit” (Bio-Rad) and prepared for Northern blot and RT-PCR analysis. The Northern blot was hybridized with a radioactive human DMPK (hDMPK) probe and a mouse GAPDH probe. The probe used for DMPK is a human DMPK cDNA consisting of the DMPK open reading frame with full 3′ UTR and 11 CTGs.
The human and mouse DMPK signal were quantified by phosphoimager analysis and normalized to the GAPDH signal. Primers that were used for the RT-PCR for hDMPK mRNA were situated in the 3′untranslated part with the sequence 5′-GGGGGATCACAGACCATT-3′ (SEQ ID NO:23) and 5′-TCAATGCATCCAAAACGTGGA-3′ (SEQ ID NO:24) and for murine actin the primers were as followed: Actin sense 5′-GCTAYGAGCTGCCTGACGG-3′ (SEQ ID NO:25) and Actin antisense 5′-GAGGCCAGGATGGAGCC-3′ (SEQ ID NO:26). PCR products were run on an agarose gel and the signal was quantified using Labworks 4.0 (UVP BioImaging systems, Cambridge, United Kingdom). The intensity of each band was normalized to the intensity of the corresponding actin band and expressed relative to mock control.
Thirteen different oligonucleotides were tested (for an overview see Table 1) as described above on the immortomyoblast DM500 cell line containing transgenic human DMPK gene with (CTG)n repeat length of approximately 500 and a normal mouse DMPK gene without (CTG) repeat. FIG. 1 shows the Northern blot of the isolated RNA from the oligonucleotide transfected myotubes visualized with the hDMPK probe and a GAPDH probe for loading control. Quantification of the human DMPK (with CTG repeat) and murine DMPK (without CTG repeat) signal on the Northern blot is shown in FIG. 2. The signal was normalized to murine GAPDH and expressed relative to mock control.
Table 2 indicates the level of hDMPK mRNA reduction that is caused by a specific oligonucleotide. The minus (−) stands for no reduction and the number of positive signs (+) stands for the relative level of hDMPK mRNA break-down. Clearly, oligonucleotide PS58, specifically targeted to the repeat sequence, is much more potent in reducing or altering hDMPK transcripts than the other oligonucleotides complementary to unique sequences in the hDMPK transcripts.
FIG. 3 shows the effect of PS58 in murine immortomyotubes derived from CTG110 mice, a knock-in mouse containing a DMPK gene with the 3′ part of the human DMPK gene including a (CTG) repeat of approximately 110. Northern blot analysis showed that the DMPK transcript containing the (CTG)110 repeat was reduced by the treatment with oligonucleotide PS58 but not after mock treatment.
Example 2
FIG. 4
The DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1). In this example, the transfection was carried out with PS58 at different concentrations. Eighty four hours after start of treatment, the myotubes were harvested and Northern blot analysis was performed on isolated RNA as described above (see example 1).
FIG. 4 shows the quantification of the hDMPK mRNA signal preformed by phosphoimager analysis and normalized to the GAPDH signal at different concentrations. Under these conditions, a half maximal effect was observed at around 1 nM.
Example 3
FIGS. 5 and 6
The DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1). However, in this example the transfection with 200 nM PS58 was carried out at different time points. Usually DM500 myotubes were harvested seven days after switching to low serum conditions to induce myotube formation. The standard procedure (as in example 1 and 2) was to start treatment (transfection) 48 h (two days) before harvesting. Now, treatment with PS58 was started 2 h-48 h (FIG. 5) or 2 d-8 d (FIG. 6) before harvesting. Northern blot analysis and quantification was performed as before.
FIG. 5 shows that expanded hDMPK mRNA in DM500 myotubes was decreased rapidly within 2 h of treatment with oligonucleotide PS58 compared to mock control treatment.
FIG. 6 shows a persistent decrease in expanded hDMPK mRNA in DM500 myotubes for at least 8 days. Please note that in the case of the 8 d experiment, cells were transfected in the myoblast stage (approximately 60% confluent, 33° C., high serum) and that they have received fresh medium on various occasions until harvesting (including a change to low serum at 37° C., two days after transfection). Example 2 and 3 are indicative of a highly efficient inhibitory intervention by an oligonucleotide directed solely to the repeat expansion. The magnitude of this effect might be influenced by the relative low levels of hDMPK expression in these model cell systems, which normally is also seen in humans.
Example 4
FIG. 7
In this example, fibroblasts obtained from a human patient with congenital myotonic dystrophy type 1 (cDM1) were used. These patient cells carry one disease causing DMPK allele with a triplet repeat expansion length of 1500 and one normal DMPK allele with a repeat length of 11. The size of the (CTG)n expansion on both alleles was confirmed with PCR and Southern blotting.
The fibroblasts were grown subconfluent and maintained in a 5% CO2 atmosphere at 37° C. on 0.1% gelatin coated dishes. Fibroblasts were grown subconfluent in DMEM supplemented with 10% FCS and 50 μg/ml gentamycin. Myotube formation was induced by growing fibroblasts cells on Matrigel (BD Biosciences) coated dishes and infecting the cells at 75% confluency with MyoD-expressing adenovirus (Ad5Fib50MyoD, Crucell, Leiden) (MOI=100) in DMEM supplemented with 2% HS and 50 μg/ml gentamycin for 2 hours. After the incubation period MyoD adenovirus was removed and DMEM supplemented with 10% FCS and 50 μg/ml gentamycin was added. The cells were maintained in this medium in a 5% CO2 atmosphere at 37° C. until 100% confluency. At this point cells were placed in DMEM supplemented with 2% FCS and 50 μg/ml gentamycin. After five days on this low serum media cells were transfected with PS58 following the procedure according to the instructions (ExGen 500, Fermentas) and as described above. The final oligonucleotide concentration was 200 nM and 20 nM. Forty eight hours after start of the treatment (which is seven days after switching to low serum conditions), RNA was isolated with the “Total RNA mini kit” (Bio-Rad) and prepared for Northern blot. The Northern blot was hybridized with a radioactive human DMPK (hDMPK) and mouse GAPDH mRNA probe. The human DMPK signals were quantified by phosphoimager analysis and normalized to the GAPDH signal and expressed relative to mock control.
FIG. 7 shows the Northern blot analysis of the MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM). The results demonstrate an effective complete inhibition of the disease-causing hDMPK (CUG)1500 RNA transcript, while the smaller normal hDMPK (CUG)11 RNA transcript is only moderately affected at the two concentrations. Thus, oligonucleotides directed to the repeat region exhibit selectivity towards the larger repeat size (or disease causing expansion).
Example 5
FIG. 8
In this example, the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, transfected and analysed as described before in example 1. The DM500 myotubes were treated 48 h before harvesting with 200 nM of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control. RT-PCR analysis was performed on hDMPK mRNA expressed in this murine cell line (for primers see example 1) and on three other gene transcripts with a naturally occurring (CUG) repeat in mice, Ptbp1 with a (CUG)6, Syndecan3 with a (CUG)6 and Taxilinbeta with a (CUG)9.
The PCR primers used were for Ptbp1: 5′-TCTGTCCCTAATGTCCATGG-3′ (SEQ ID NO:27) and 5′-GCCATCTGCACAAGTGCGT-3′ (SEQ ID NO:28) ; for Syndecan3: 5′-GCTGTTGCTGCCACCGCT-3′ (SEQ ID NO:29) and 5′-GGCGCCTCGGGAGTGCTA-3′ (SEQ ID NO:30) ; and for Taxilinbeta: 5′-CTCAGCCCTGCTGCCTGT-3′ (SEQ ID NO:31) and 5′-CAGACCCATACGTGCTTATG-3′ (SEQ ID NO:32) . The PCR products were run on an agarose gel and signals were quantified using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, United Kingdom). The intensity of each signal was normalized to the corresponding actin signal and expressed relative to mock control.
FIG. 8 shows the RT-PCR results with a maximal inhibition of hDMPK mRNA expression by PS58. The other gene transcripts carrying a naturally occurring small (CUG) repeat were not or only marginally affected by the oligonucleotide PS58, specific to the (CUG) repeat, compared to oligonucleotide PS113, which has no complementary sequence to these gene transcripts.
This example confirms the selectivity of an oligonucleotide, directed solely to the repeat region, towards the long repeat size (or disease causing expansion) compared to naturally occurring shorter repeat sizes.
Example 6
FIG. 9 en 10
In this example, the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured and transfected with PS58 (200 nM). Here, FISH analysis was carried out on the cells. Forty eight hours after the start of the treatment, the cells were fixed with 4% formaldehyde, 5 mM MgCl2 and 1×PBS for 30 minutes. Hybridization with fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3 was performed overnight at 37° C. in a humid chamber. After hybridization the material was washed and mounted in mowiol and allowed to dry overnight. Nuclear inclusions (ribonuclear foci) were visualized using a Bio-Rad MRC1024 confocal laser scanning microscope and LaserSharp2000 acquisition software. In total 50 cells were counted and scored for the presence of inclusions in the nuclei of these cells.
Literature indicates that DMPK mRNA containing a (CUG) expanded repeat accumulates and aggregates in the nucleus to form ribonuclear foci with regulatory nuclear proteins and transcription factors. Therefore, normal nuclear gene processing and cell function gets impaired.
FIG. 9 shows a mock treated cell containing ribonuclear inclusions in the nucleus, while these are no longer present in the cell nucleus after treatment with PS58. FIG. 10 shows that the percentage of nuclei containing ribonuclear foci seen under control conditions in DM500 myotubes is strongly decreased by the treatment with PS58. This result demonstrates that inhibition of hDMPK mRNA expression also inhibits the disease related triplet repeat (CUG) rich inclusions.
Example 7
FIG. 11
Here, the effect of PS58 was evaluated in vivo in DM500 mice containing hDMPK with a (CTG)n expansion of approximately 500 triplets. The DM500 mice were derived by somatic expansion from the DM300 mouse (e.g. see Gomes-Pereira M et al (2007) PLoS Genet. 2007 3(4): e52). A (CTG) triplet repeat expansion of approximately 500 was confirmed by southern blot and PCR analysis.
In short, PS58 was mixed with transfection agent ExGen 500 (Fermentas) according to the accompanying instructions for in vivo use. PS58 (2 nmol, in the transfection solution with Exgen 500) was injected (40 μl) in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h. As a control, DM500 mice were treated similarly with the transfection solution without PS58. After 15 days, the mice were sacrificed, muscles were isolated and total RNA was isolated from the tissues (using Trizol, Invitrogen). RT-PCR analysis was performed to detect hDMPK mRNA in the muscle similar as described above. The intensity of each band was performed using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, United Kingdom) and normalized to the intensity of the corresponding actin band. Primer location is indicated in the figure.
FIG. 11 shows that in vivo treatment of DM500 mice with PS58 strongly reduced the presence of hDMPK mRNA containing a (CUG)n repeat expansion compared to mock treatment in the M. plantaris and M. gastrocnemius.
Example 8
FIG. 12
In this example, different oligonucleotides (in length and backbone chemistry) but all with a sequence directed solely to the (CTG)n repeat expansion were compared. DM500 myotubes were cultured, transfected and analysed as described above in example 1. Northern blots were quantified by phosphoimager analysis and DMPK signals were normalized to GAPDH.
Here, the DM500 myotubes were treated with the following oligonucleotides (200 nM), all with a complete phosphorothioate backbone (see Table 3).
FIG. 12 shows that treatment of the DM500 myotubes results in a complete reduction of (CUG)n expanded hDMPK mRNA for all oligonucleotides tested. Under the present conditions, the maximal effect obtainable is independent of oligonucleotide length, backbone modification or potential mechanism of inhibition by the employed single stranded oligonucleotides.
Example 9
Fibroblasts (GM 00305) from a male patient with Huntington's Disease were obtained from Coriell Cell Repository (Camden, N.J., US) and cultured according to the accompanying instructions and standard techniques known to the skilled person in the art. Huntington patients carry one healthy and one disease-causing allele of the Huntington gene resulting in the expression of both mRNAs with respectively a normal number and an expanded number of (CAG) repeats, respectively.
The fibroblasts were transfected with a 21-mer 2′O-methyl phosphorothioate RNA antisense oligonucleotide PS57 with a (CUG)7 sequence (SEQ ID NO:5), complementary to the (CAG) triplet repeat in Huntington mRNA. Transfection occurred at 100 or 200 nM in the presence of PEI as indicated by the manufacturer. Twenty four hours after transfection the cells were harvested and total RNA was isolated and analysed by RT-PCR. The Huntington transcript was determined using primers in downstream exon 64 (5′ GAAAG TCAGT CCGGG TAGAA CTTC 3′ (SEQ ID NO:33) and 5′ CAGAT ACCCG CTCCA TAGCA A 3′ (SEQ ID NO:34)). This method detects both types of Huntington mRNAs, the normal and mutant transcript with the additional (CAG) expansion. GAPDH mRNA (housekeeping gene) was also determined. The signals were quantified and the total amount of Huntington mRNA was normalised to the amount of GAPDH mRNA in the same sample. The results are expressed relative to a control treated (without oligonucleotide) sample from fibroblasts (which was to 100%).
In the samples from fibroblasts transfected with either 100 or 200 nM of PS57, significantly lower levels of total Huntington mRNA levels were observed of approximately 53% and 66% compared to the levels in control-treated cells, respectively.
Thus, PS57, an oligonucleotide directed only to the (CAG) repeat, induces a decrease in Huntington mRNA levels and these results are consistent with a selective inhibition of mutant over normal Huntington mRNA.
TABLE 1
Overview oligonucleotides tested
Oligo
name
Modification
Sequence
Position
PS40
2′OMe RNA
GAGGGGCGUCC
intron
phosphorothioate/
AGGGAUCCG
14-exon 15
FAM
(SEQ ID NO: 1)
PS41
2′OMe RNA
GCGUCCAGGGA
intron
phosphorothioate
UCCGGACCG
14-exon 15
(SEQ ID NO: 2)
PS42
2′OMe RNA
CAGGGAUCCGG
intron
phosphorothioate
ACCGGAUAG
14-exon 15
(SEQ ID NO: 3)
PS56
DNA
CAGCAGCAGCA
repeat in
GCAGCAGCAG
exon 15
(SEQ ID NO: 4)
PS58
2′OMe RNA
CAGCAGCAGCA
repeat in
phosphorothioate/
GCAGCAGCAG
exon 15
FAM
(SEQ ID NO: 6)
PS59
2′OMe RNA
UGAGUUGGCCG
ESE exon 15
phosphorothioate
GCGUGGGCC
(SEQ ID NO: 7)
PS60
2′OMe RNA
UUCUAGGGUUC
ESE exon 15
phosphorothioate
AGGGAGCGCGG
(SEQ ID NO: 8)
PS61
2′OMe RNA
ACUGGAGCUGG
ESE exon 15
phosphorothioate
GCGGAGACCC
(SEQ ID NO: 9)
PS62
2′OMe RNA
CUCCCCGGCCG
ESE exon 15
phosphorothioate
CUAGGGGGC
(SEQ ID NO: 10)
PS113
DNA phospho
GAGCCGCCTCA
Exon 1
thioroate
GCCGCACCTC
(SEQ ID NO: 11)
PS114
DNA phospho-
GAAGTCGGCCA
Exon 1
thioroate
CGTACTTGTC
(SEQ ID NO: 12)
PS115
DNA phospho-
GGAGTCGAAGA
Exon 15
thioroate
CAGTTCTAGG
(SEQ ID NO: 13)
PS116
DNA phospho-
GGTACACAGGA
Exon 15
thioroate
CTGGAGCTGG
(SEQ ID NO: 14)
TABLE 2
Reduction of hDMPK mRNA after oligo transfection:
Oligo
Reduction hDMPK mRNA
SEQ ID No.'s
PS40
+
1
PS41
−
2
PS42
−
3
PS59
−
7
PS60
−
8
PS61
+/−
9
PS62
−
10
PS58
++++
6
PS56
−
4
PS113
−
11
PS114
−
12
PS115
+/−
13
PS116
+
14
(−) indicates no reduction, (+) indicates level of reduction in hDMPK mRNA.
TABLE 3
Oligonucleotides used in example 9
RNAse H
Substitution
breakdown
#
Length
(CAG)n
ribose
possible
PS58
21-mer
n = 7
2′O-Methyl
No
(SEQ ID NO: 6)
PS146
30-mer
n = 10
2′O-Methyl
No
(SEQ ID NO: 16)
PS147
15-mer
n = 5
2′O-Methyl
No
(SEQ ID NO: 17)
PS142
21-mer
n = 7
Deoxyribose
Yes
(SEQ ID NO: 15)
(DNA)
* all oligonucleotides full length phosphorothioate and substitution
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14809483
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Mar 27th, 2018 12:00AM
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Apr 26th, 2011 12:00AM
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https://www.uspto.gov?id=US09926557-20180327
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Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA
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The invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing an isolated muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. The invention further relates to such molecule used in the method.
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9926557
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1. A pharmaceutical composition comprising an antisense oligonucleotide and an excipient selected from the group consisting of polyethylenimine, and polypropylenimine, wherein said antisense oligonucleotide consists of the base sequence: 5′-UUUGCCGCUGCCCAAUGCCAUCCUG-3′ (SEQ ID: NO: 3), said oligonucleotide comprising a modification.
2. The pharmaceutical composition according to claim 1, wherein the oligonucleotide comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
3. The pharmaceutical composition according to claim 2, wherein the nucleotide analogue has a modified base.
4. The pharmaceutical composition according to claim 2, wherein the nucleotide analogue has a modified backbone.
5. The pharmaceutical composition according to claim 2, wherein the nucleotide analogue comprises one or more sugar moieties that are mono-or disubstituted at the 2′, 3′ and/or 5′ position.
6. The pharmaceutical composition according to claim 5, wherein said oligonucleotide comprises a 2′-O-substituted phosphorothioate antisense oligonucleotide.
7. The pharmaceutical composition according to claim 5, wherein said oligonucleotide comprises a 2′-O-methyl ribose.
8. The pharmaceutical composition according to claim 6, wherein all the sugar moieties are 2′-O-methyl substituted.
9. The pharmaceutical composition according to claim 4, wherein the modified backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.
10. The pharmaceutical composition according to claim 1, wherein the oligonucleotide comprises phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid, and/or locked nucleic acid.
11. The pharmaceutical composition according to claim 1, further comprising a molecule which is able to induce or promote skipping of exon 7, 44, 46, 51, 53, 59, or 67 of the pre-mRNA of the DMD gene of a patient.
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11
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PRIORITY
This application is a continuation of PCT/NL2009/050006, filed on Jan. 13, 2009, which is a continuation-in-part of PCT/NL2008/050673, filed on Oct. 27, 2008, the entirety of which is incorporated herein by reference.
FIELD
The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to human Duchenne Muscular Dystrophy.
BACKGROUND OF THE INVENTION
Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are mainly characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the DMD transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol. Ther. 2008; 10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, van Deutekom et al., N Engl J. Med. 2007; 357(26):2677-86).
Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol. Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet. 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat. Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol. Chem. 2008; 283: 5899-907).
As the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively large numbers of patients. The actual applicability of exon skipping can be determined for deletions, duplications and point mutations reported in DMD mutation databases such as the Leiden DMD mutation database available at www.dmd.nl. Therapeutic skipping of exon 45 of the DMD pre-mRNA would restore the open reading frame of DMD patients having deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA, occurring in a total of 16% of all DMD patients with a deletion (Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics) Research Focus, 2007 Nova Science Publishers, Inc). Furthermore, for some DMD patients the simultaneous skipping of one of more exons in addition to exon 45, such as exons 51 or 53 is required to restore the correct reading frame. None-limiting examples include patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.
Recently, a first-in-man study was successfully completed where an AON inducing the skipping of exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J. Med. 2007; 357: 2677-86).
Most AONs studied contain up to 20 nucleotides, and it has been argued that this relatively short size improves the tissue distribution and/or cell penetration of an AON. However, such short AONs will result in a limited specificity due to an increased risk for the presence of identical sequences elsewhere in the genome, and a limited target binding or target affinity due to a low free energy of the AON-target complex. Therefore the inventors decided to design new and optionally improved oligonucleotides that would not exhibit all of these drawbacks.
DESCRIPTION OF THE INVENTION
Method
In a first aspect, the invention provides a method for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.
Accordingly, a method is herewith provided for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.
It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method. As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. The DMD gene or protein corresponds to the dystrophin gene or protein.
A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background.
Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with sequences such as, for example, the splice donor or splice acceptor sequence that are both required for allowing the enzymatic process of splicing, or a molecule that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
Within the context of the invention inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.
Preferably, a method of the invention by inducing or promoting skipping of exon 45 of the DMD pre-mRNA in one or more cells of a patient provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient. Therefore a preferred method is a method, wherein a patient or a cell of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient or in a cell of said patient is decreased
Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immuno fluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.
Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the DMD gene that prevent synthesis of the complete dystrophin protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared to the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon—skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of the remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
A method of the invention may alleviate one or more characteristics of a muscle cell from a DMD patient comprising deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA of said patient (Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics) Research Focus, 2007 Nova Science Publishers, Inc) as well as from DMD patients requiring the simultaneous skipping of one of more exons in addition to exon 45 including but not limited to patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.
In a preferred method, one or more symptom(s) or characteristic(s) of a myogenic cell or muscle cell from a DMD patient is/are alleviated. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.
An alleviation of one or more symptoms or characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
The improvement of muscle fiber function, integrity and/or survival may also be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD patient before treatment.
A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD patient before treatment.
A detectable increase of the homogeneity of the diameter of muscle fibers is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of muscle fibers in a muscle biopsy cross-section of a same DMD patient before treatment.
An alleviation of one or more symptoms or characteristics may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
A treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. The frequency of administration of an oligonucleotide, composition, compound of the invention may depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.
Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD and may be administered directly in vivo, ex vivo or in vitro. An oligonucleotide as used herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne muscular dystrophy has a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably said cells comprise a gene encoding a mutant dystrophin protein. Preferably said cells are cells of an individual suffering from DMD.
A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipient are defined in the section entitled “pharmaceutical composition”. In vitro, we obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example.
In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of a distinct exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 7, 44, 46, 51, 53, 59, 67 of the dystrophin pre-mRNA of a patient. Molecules which can be used are depicted in table 2. Preferred molecules comprise or consist of any of the oligonucleotides as disclosed in table 2. Several oligonucleotides may also be used in combination. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping (Aartsma-Rus A, Janson AA, Kaman W E, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet. 2004; 74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006; 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other.
It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.
In case, more than one compounds are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.
Molecule
In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled “Method”. This molecule preferably comprises or consists of an oligonucleotide, Said oligonucleotide is preferably an antisense oligonucleotide (AON) or antisense oligoribonucleotide.
It was found by the present investigators that especially exon 45 is specifically skipped at a high frequency using a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. Although this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 21 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex. In a preferred embodiment, a molecule that binds to a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within said exon is used.
In a preferred embodiment, a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of exon 45 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100%. “A part of exon 45” preferably means a stretch of at least 21 nucleotides. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of exon 45 dystrophin pre-mRNA as defined herein. Alternatively, an oligonucleotide may comprise a sequence that is complementary to part of exon 45 dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Several types of flanking sequences may be used. Preferably, additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity. In another preferred embodiment, additional flanking sequences are complementary to sequences of the DMD pre-mRNA which are not present in exon 45. Such flanking sequences are preferably complementary to sequences comprising or consisting of the splice site acceptor or donor consensus sequences of exon 45. In a preferred embodiment, such flanking sequences are complementary to sequences comprising or consisting of sequences of an intron of the DMD pre-mRNA which is adjacent to exon 45; i.e. intron 44 or 45.
A continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within exon 45 is preferably selected from the sequence:
(SEQ ID NO 2)
5′-CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGAACAUUGAAUG
CAACUGGGGAAGAAAUAAUUCAGCAAUC-3′.
It was found that a molecule that binds to a nucleotide sequence comprising or consisting of a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides of SEQ ID NO. 2 results in highly efficient skipping of exon 45 in a cell provided with this molecule. Molecules that bind to a nucleotide sequence comprising a continuous stretch of less than 21 nucleotides of SEQ ID NO:2 were found to induce exon skipping in a less efficient way than the molecules of the invention. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 21, 25, 30, 35 nucleotides within SEQ ID NO:2. Contrary to what was generally thought, the inventors surprisingly found that a higher specificity and efficiency of exon skipping may be reached using an oligonucleotides having a length of at least 21 nucleotides. None of the indicated sequences is derived from conserved parts of splice-junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.
In one embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the indicated nucleotide sequence on a RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are for example disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein enclosed by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein enclosed by reference.
In a further embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA comprises an antisense oligonucleotide that is complementary to and can base-pair with the coding strand of the pre-mRNA of the DMD gene. Said antisense oligonucleotide preferably contains a RNA residue, a DNA residue, and/or a nucleotide analogue or equivalent, as will be further detailed herein below.
A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 21 and 50 nucleotides or bases, more preferred between 21 and 40 nucleotides, more preferred between 21 and 30 nucleotides, such as 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides.
A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.
In a preferred embodiment, a molecule of the invention binds to a continuous stretch of or is complementary to or is antisense to at least a continuous stretch of at least 21 nucleotides within the nucleotide sequence SEQ ID NO:2.
In a certain embodiment, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences as depicted in Table 1, except SEQ ID NO:68. A molecule of the invention that is antisense to the sequence of SEQ ID NO 2, which is present in exon 45 of the DMD gene preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66 and/or SEQ ID NO:67.
In a more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and/or SEQ ID NO 8.
In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3. It was found that this molecule is very efficient in modulating splicing of exon 45 of the DMD pre-mRNA in a muscle cell.
A nucleotide sequence of a molecule of the invention may contain a RNA residue, a DNA residue, a nucleotide analogue or equivalent as will be further detailed herein below. In addition, a molecule of the invention may encompass a functional equivalent of a molecule of the invention as defined herein.
It is preferred that a molecule of the invention comprises a or at least one residue that is modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, an antisense nucleotide sequence comprises a or at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
In a preferred embodiment, a nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
It is further preferred that the linkage between a residue in a backbone does not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of at least one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; O—, S—, or N-allyl; O-alkyl-β-alkyl, -methoxy, -aminopropoxy; aminoxy, methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or deoxyribose or derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
A preferred antisense oligonucleotide according to the invention comprises a 2′-O-alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
A most preferred antisense oligonucleotide according to the invention comprises a 2′-O-methyl phosphorothioate ribose.
A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 45 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 45 skipping and quantifying the amount of a functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analysis. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
It will also be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping of exon 45 of the human DMD pre-mRNA. In a preferred embodiment, a combination of at least two antisense oligonucleotides are used in a method of the invention, such as two distinct antisense oligonucleotides, three distinct antisense oligonucleotides, four distinct antisense oligonucleotides, or five distinct antisense oligonucleotides or even more. It is also encompassed by the present invention to combine several oligonucleotides or molecules as depicted in table 1 except SEQ ID NO:68.
An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably myogenic cells or muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
A preferred antisense oligonucleotide comprises a peptide-linked PMO.
A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for modulating skipping of exon 45 of the human DMD pre-mRNA.
A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 45 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by viral-based vector. Such a viral-based vector comprises an expression cassette that drives expression of an antisense molecule as defined herein. Preferred virus-based vectors include adenovirus- or adeno-associated virus-based vectors. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.
One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 45 of the DMD pre-mRNA.
A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 45 of the human DMD pre-mRNA.
Pharmaceutical Composition
If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.
Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.
A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector which is able to modulate skipping of exon 7, 44, 46, 51, 53, 59, 67 of the DMD pre-mRNA.
Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils, Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. We obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy in humans.
In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 □M is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. Molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, a compound preferably an oligonucleotide and an adjunct compound to be used in the invention to prevent, treat DMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
Use
In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for inducing and/or promoting splicing of the DMD pre-mRNA. The splicing is preferably modulated in a human myogenic cell or a muscle cell in vitro. More preferred is that splicing is modulated in human a myogenic cell or muscle cell in vivo.
Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for inducing and/or promoting splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD patient.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. In human control myotubes, a series of AONs (PS220 to PS225; SEQ ID NO: 3 to 8), all binding to a continuous stretch of at least 21 nucleotides within a specific sequence of exon 45 (i.e. SEQ ID NO:2), were tested at two different concentrations (200 and 500 nM). All six AONs were effective in inducing specific exon 45 skipping, as confirmed by sequence analysis (not shown). PS220 (SEQ ID NO:3) however, reproducibly induced highest levels of exon 45 skipping (see FIG. 2). (NT: non-treated cells, M: size marker).
FIG. 2. In human control myotubes, 25-mer PS220 (SEQ ID NO: 3) was tested at increasing concentration. Levels of exon 45 skipping of up to 75% (at 400 nM) were observed reproducibly, as assessed by Agilent LabChip Analysis.
FIG. 3. In human control myotubes, the efficiencies of a “short” 17-mer AON45-5 (SEQ ID NO:68) and its overlapping “long” 25-mer counterpart PS220 were directly compared at 200 nM and 500 nM. PS220 was markedly more efficient at both concentrations: 63% when compared to 3% obtained with 45-5. (NT: non-treated cells, M: size marker).
EXAMPLES
Examples 1 and 2
Materials and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program (Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 31, 3406-3415), and on (partly) overlapping putative SR-protein binding sites as predicted by numerous software programs such as ESEfinder (Cartegni, L. et al. (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res, 31, 3568-71; Smith, P. J. et al. (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum. Mol. Genet., 15, 2490-2508) that predicts binding sites for the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55). AONs were synthesized by Prosensa Therapeutics B. V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) were obtained as described previously (Aartsma-Rus et al. Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 0 to 500 nM of each AON. The transfection reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) was used according to manufacturer's instructions, with 2 μl PEI per μg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the Agilent DNA 1000 LabChip Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
A series of AONs targeting sequences within SEQ ID NO:2 within exon 45 were designed and tested in normal myotube cultures, by transfection and subsequent RT-PCR and sequence analysis of isolated RNA. PS220 (SEQ ID NO: 3) reproducibly induced highest levels of exon 45 skipping, when compared to PS221-PS225 (FIG. 1). High levels of exon 45 skipping of up to 75% were already obtained at 400 nM PS220 (FIG. 2). In a direct comparison, PS220 (a 25-mer) was reproducibly more efficient in inducing exon 45 skipping than its shorter 17-mer counterpart AON 45-5 (SEQ ID NO: 68; previously published as h45AON5 (Aartsma-Rus et al. Am J Hum Genet. 2004; 74: 83-92)), at both AON concentrations of 200 nM and 500 nM and with 63% versus 3% respectively at 500 nM (FIG. 3). This result is probably due to the fact that the extended length of PS220, in fact completely overlapping AON 45-5, increases the free energy of the AON-target complex such that the efficiency of inducing exon 45 skipping is also increased.
TABLE 1
AONs in exon 45
SEQ ID NO 3
UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID NO 36
GUUGCAUUCAAUGUUCUGACAACAG
(PS220)
SEQ ID NO 4
AUUCAAUGUUCUGACAACAGUUUGC
SEQ ID NO 37
UUGCAUUCAAUGUUCUGACAACAGU
(PS221)
SEQ ID NO 5
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID NO 38
UGCAUUCAAUGUUCUGACAACAGUU
(PS222)
SEQ ID NO 6
CAGUUGCAUUCAAUGUUCUGAC
SEQ ID NO 39
GCAUUCAAUGUUCUGACAACAGUUU
(PS223)
SEQ ID NO 7
AGUUGCAUUCAAUGUUCUGA
SEQ ID NO 40
CAUUCAAUGUUCUGACAACAGUUUG
(PS224)
SEQ ID NO 8
GAUUGCUGAAUUAUUUCUUCC
SEQ ID NO 41
AUUCAAUGUUCUGACAACAGUUUGC
(PS225)
SEQ ID NO 9
GAUUGCUGAAUUAUUUCUUCCCCAG
SEQ ID NO 42
UCAAUGUUCUGACAACAGUUUGCCG
SEQ ID NO 10
AUUGCUGAAUUAUUUCUUCCCCAGU
SEQ ID NO 43
CAAUGUUCUGACAACAGUUUGCCGC
SEQ ID NO 11
UUGCUGAAUUAUUUCUUCCCCAGUU
SEQ ID NO 44
AAUGUUCUGACAACAGUUUGCCGCU
SEQ ID NO 12
UGCUGAAUUAUUUCUUCCCCAGUUG
SEQ ID NO 45
AUGUUCUGACAACAGUUUGCCGCUG
SEQ ID NO 13
GCUGAAUUAUUUCUUCCCCAGUUGC
SEQ ID NO 46
UGUUCUGACAACAGUUUGCCGCUGC
SEQ ID NO 14
CUGAAUUAUUUCUUCCCCAGUUGCA
SEQ ID NO 47
GUUCUGACAACAGUUUGCCGCUGCC
SEQ ID NO 15
UGAAUUAUUUCUUCCCCAGUUGCAU
SEQ ID NO 48
UUCUGACAACAGUUUGCCGCUGCCC
SEQ ID NO 16
GAAUUAUUUCUUCCCCAGUUGCAUU
SEQ ID NO 49
UCUGACAACAGUUUGCCGCUGCCCA
SEQ ID NO 17
AAUUAUUUCUUCCCCAGUUGCAUUC
SEQ ID NO 50
CUGACAACAGUUUGCCGCUGCCCAA
SEQ ID NO 18
AUUAUUUCUUCCCCAGUUGCAUUCA
SEQ ID NO 51
UGACAACAGUUUGCCGCUGCCCAAU
SEQ ID NO 19
UUAUUUCUUCCCCAGUUGCAUUCAA
SEQ ID NO 52
GACAACAGUUUGCCGCUGCCCAAUG
SEQ ID NO 20
UAUUUCUUCCCCAGUUGCAUUCAAU
SEQ ID NO 53
ACAACAGUUUGCCGCUGCCCAAUGC
SEQ ID NO 21
AUUUCUUCCCCAGUUGCAUUCAAUG
SEQ ID NO 54
CAACAGUUUGCCGCUGCCCAAUGCC
SEQ ID NO 22
UUUCUUCCCCAGUUGCAUUCAAUGU
SEQ ID NO 55
AACAGUUUGCCGCUGCCCAAUGCCA
SEQ ID NO 23
UUCUUCCCCAGUUGCAUUCAAUGUU
SEQ ID NO 56
ACAGUUUGCCGCUGCCCAAUGCCAU
SEQ ID NO 24
UCUUCCCCAGUUGCAUUCAAUGUUC
SEQ ID NO 57
CAGUUUGCCGCUGCCCAAUGCCAUC
SEQ ID NO 25
CUUCCCCAGUUGCAUUCAAUGUUCU
SEQ ID NO 58
AGUUUGCCGCUGCCCAAUGCCAUCC
SEQ ID NO 26
UUCCCCAGUUGCAUUCAAUGUUCUG
SEQ ID NO 59
GUUUGCCGCUGCCCAAUGCCAUCCU
SEQ ID NO 27
UCCCCAGUUGCAUUCAAUGUUCUGA
SEQ ID NO 60
UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID NO 28
CCCCAGUUGCAUUCAAUGUUCUGAC
SEQ ID NO 61
UUGCCGCUGCCCAAUGCCAUCCUGG
SEQ ID NO 29
CCCAGUUGCAUUCAAUGUUCUGACA
SEQ ID NO 62
UGCCGCUGCCCAAUGCCAUCCUGGA
SEQ ID NO 30
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID NO 63
GCCGCUGCCCAAUGCCAUCCUGGAG
SEQ ID NO 31
CAGUUGCAUUCAAUGUUCUGACAAC
SEQ ID NO 64
CCGCUGCCCAAUGCCAUCCUGGAGU
SEQ ID NO 32
AGUUGCAUUCAAUGUUCUGACAACA
SEQ ID NO 65
CGCUGCCCAAUGCCAUCCUGGAGUU
SEQ ID NO 33
UCC UGU AGA AUA CUG GCA UC
SEQ ID NO 66
UGU UUU UGA GGA UUG CUG AA
SEQ ID NO 34
UGC AGA CCU CCU GCC ACC GCA
SEQ ID NO 67
UGUUCUGACAACAGUUUGCCGCUGCCCAAUGC
GAU UCA
CAUCCUGG
SEQ ID NO 35
UUGCAGACCUCCUGCCACCGCAGAUUCAG
SEQ ID NO 68
GCCCAAUGCCAUCCUGG
GCUUC
(45-5)
TABLE 2
AONs in exons 51, 53, 7, 44, 46, 59, and 67
DMD Gene Exon 51
SEQ ID NO 69
AGAGCAGGUACCUCCAACAUCAAGG
SEQ ID NO 91
UCAAGGAAGAUGGCAUUUCUAGUUU
SEQ ID NO 70
GAGCAGGUACCUCCAACAUCAAGGA
SEQ ID NO 92
UCAAGGAAGAUGGCAUUUCU
SEQ ID NO 71
AGCAGGUACCUCCAACAUCAAGGAA
SEQ ID NO 93
CAAGGAAGAUGGCAUUUCUAGUUUG
SEQ ID NO 72
GCAGGUACCUCCAACAUCAAGGAAG
SEQ ID NO 94
AAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID NO 73
CAGGUACCUCCAACAUCAAGGAAGA
SEQ ID NO 95
AGGAAGAUGGCAUUUCUAGUUUGGA
SEQ ID NO 74
AGGUACCUCCAACAUCAAGGAAGAU
SEQ ID NO 96
GGAAGAUGGCAUUUCUAGUUUGGAG
SEQ ID NO 75
GGUACCUCCAACAUCAAGGAAGAUG
SEQ ID NO 97
GAAGAUGGCAUUUCUAGUUUGGAGA
SEQ ID NO 76
GUACCUCCAACAUCAAGGAAGAUGG
SEQ ID NO 98
AAGAUGGCAUUUCUAGUUUGGAGAU
SEQ ID NO 77
UACCUCCAACAUCAAGGAAGAUGGC
SEQ ID NO 99
AGAUGGCAUUUCUAGUUUGGAGAUG
SEQ ID NO 78
ACCUCCAACAUCAAGGAAGAUGGCA
SEQ ID NO 100
GAUGGCAUUUCUAGUUUGGAGAUGG
SEQ ID NO 79
CCUCCAACAUCAAGGAAGAUGGCAU
SEQ ID NO 101
AUGGCAUUUCUAGUUUGGAGAUGGC
SEQ ID NO 80
CUCCAACAUCAAGGAAGAUGGCAUU
SEQ ID NO 102
UGGCAUUUCUAGUUUGGAGAUGGCA
SEQ ID NO 81
CUCCAACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 103
GGCAUUUCUAGUUUGGAGAUGGCAG
SEQ ID NO 82
UCCAACAUCAAGGAAGAUGGCAUUU
SEQ ID NO 104
GCAUUUCUAGUUUGGAGAUGGCAGU
SEQ ID NO 83
CCAACAUCAAGGAAGAUGGCAUUUC
SEQ ID NO 105
CAUUUCUAGUUUGGAGAUGGCAGUU
SEQ ID NO 84
CAACAUCAAGGAAGAUGGCAUUUCU
SEQ ID NO 106
AUUUCUAGUUUGGAGAUGGCAGUUU
SEQ ID NO 85
AACAUCAAGGAAGAUGGCAUUUCUA
SEQ ID NO 107
UUUCUAGUUUGGAGAUGGCAGUUUC
SEQ ID NO 86
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 108
UUCUAGUUUGGAGAUGGCAGUUUCC
SEQ ID NO 87
ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID NO 88
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 89
CAUCAAGGAAGAUGGCAUUUCUAGU
SEQ ID NO 90
AUCAAGGAAGAUGGCAUUUCUAGUU
DMD Gene Exon 53
SEQ ID NO 109
CCAUUGUGUUGAAUCCUUUAACAUU
SEQ ID NO 116
CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG
SEQ ID NO 110
CCAUUGUGUUGAAUCCUUUAAC
SEQ ID NO 117
CUGAAGGUGUUCUUGUACUUCAUCC
SEQ ID NO 111
AUUGUGUUGAAUCCUUUAAC
SEQ ID NO 118
UGUAUAGGGACCCUCCUUCCAUGACUC
SEQ ID NO 112
CCUGUCCUAAGACCUGCUCA
SEQ ID NO 119
AUCCCACUGAUUCUGAAUUC
SEQ ID NO 113
CUUUUGGAUUGCAUCUACUGUAUAG
SEQ ID NO 120
UUGGCUCUGGCCUGUCCUAAGA
SEQ ID NO 114
CAUUCAACUGUUGCCUCCGGUUCUG
SEQ ID NO 121
AAGACCUGCUCAGCUUCUUCCUUAGCUUCCAGCCA
SEQ ID NO 115
CUGUUGCCUCCGGUUCUGAAGGUG
DMD Gene Exon 7
SEQ ID NO 122
UGCAUGUUCCAGUCGUUGUGUGG
SEQ ID NO 124
AUUUACCAACCUUCAGGAUCGAGUA
SEQ ID NO 123
CACUAUUCCAGUCAAAUAGGUCUGG
SEQ ID NO 125
GGCCUAAAACACAUACACAUA
DMD Gene Exon 44
SEQ ID NO 126
UCAGCUUCUGUUAGCCACUG
SEQ ID NO 151
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 127
UUCAGCUUCUGUUAGCCACU
SEQ ID NO 152
CAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 128
UUCAGCUUCUGUUAGCCACUG
SEQ ID NO 153
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 129
UCAGCUUCUGUUAGCCACUGA
SEQ ID NO 154
AGCUUCUGUUAGCCACUGAU
SEQ ID NO 130
UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 155
GCUUCUGUUAGCCACUGAUU
SEQ ID NO 131
UCAGCUUCUGUUAGCCACUGA
SEQ ID NO 156
AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 132
UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 157
GCUUCUGUUAGCCACUGAUUA
SEQ ID NO 133
UCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 158
AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 134
UUCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 159
GCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 135
UCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 160
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 136
UUCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 161
GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 137
UCAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 162
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 138
UUCAGCUUCUGUUAGCCACUGAUA
SEQ ID NO 163
GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 139
UCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 164
CCAUUUGUAUUUAGCAUGUUCCC
SEQ ID NO 140
UUCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 165
AGAUACCAUUUGUAUUUAGC
SEQ ID NO 141
UCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 166
GCCAUUUCUCAACAGAUCU
SEQ ID NO 142
UUCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 167
GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 143
CAGCUUCUGUUAGCCACUG
SEQ ID NO 168
AUUCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 144
CAGCUUCUGUUAGCCACUGAU
SEQ ID NO 169
UCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 145
AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 170
GUUCAGCUUCUGUUAGCC
SEQ ID NO 146
CAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 171
CUGAUUAAAUAUCUUUAUAU C
SEQ ID NO 147
AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 172
GCCGCCAUUUCUCAACAG
SEQ ID NO 148
CAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 173
GUAUUUAGCAUGUUCCCA
SEQ ID NO 149
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 174
CAGGAAUUUGUGUCUUUC
SEQ ID NO 150
CAGCUUCUGUUAGCCACUGAUUAA
DMD Gene Exon 46
SEQ ID NO 175
GCUUUUCUUUUAGUUGCUGCUCUUU
SEQ ID NO 203
AGGUUCAAGUGGGAUACUAGCAAUG
SEQ ID NO 176
CUUUUCUUUUAGUUGCUGCUCUUUU
SEQ ID NO 204
GGUUCAAGUGGGAUACUAGCAAUGU
SEQ ID NO 177
UUUUCUUUUAGUUGCUGCUCUUUUC
SEQ ID NO 205
GUUCAAGUGGGAUACUAGCAAUGUU
SEQ ID NO 178
UUUCUUUUAGUUGCUGCUCUUUUCC
SEQ ID NO 206
UUCAAGUGGGAUACUAGCAAUGUUA
SEQ ID NO 179
UUCUUUUAGUUGCUGCUCUUUUCCA
SEQ ID NO 207
UCAAGUGGGAUACUAGCAAUGUUAU
SEQ ID NO 180
UCUUUUAGUUGCUGCUCUUUUCCAG
SEQ ID NO 208
CAAGUGGGAUACUAGCAAUGUUAUC
SEQ ID NO 181
CUUUUAGUUGCUGCUCUUUUCCAGG
SEQ ID NO 209
AAGUGGGAUACUAGCAAUGUUAUCU
SEQ ID NO 182
UUUUAGUUGCUGCUCUUUUCCAGGU
SEQ ID NO 210
AGUGGGAUACUAGCAAUGUUAUCUG
SEQ ID NO 183
UUUAGUUGCUGCUCUUUUCCAGGUU
SEQ ID NO 211
GUGGGAUACUAGCAAUGUUAUCUGC
SEQ ID NO 184
UUAGUUGCUGCUCUUUUCCAGGUUC
SEQ ID NO 212
UGGGAUACUAGCAAUGUUAUCUGCU
SEQ ID NO 185
UAGUUGCUGCUCUUUUCCAGGUUCA
SEQ ID NO 213
GGGAUACUAGCAAUGUUAUCUGCUU
SEQ ID NO 186
AGUUGCUGCUCUUUUCCAGGUUCAA
SEQ ID NO 214
GGAUACUAGCAAUGUUAUCUGCUUC
SEQ ID NO 187
GUUGCUGCUCUUUUCCAGGUUCAAG
SEQ ID NO 215
GAUACUAGCAAUGUUAUCUGCUUCC
SEQ ID NO 188
UUGCUGCUCUUUUCCAGGUUCAAGU
SEQ ID NO 216
AUACUAGCAAUGUUAUCUGCUUCCU
SEQ ID NO 189
UGCUGCUCUUUUCCAGGUUCAAGUG
SEQ ID NO 217
UACUAGCAAUGUUAUCUGCUUCCUC
SEQ ID NO 190
GCUGCUCUUUUCCAGGUUCAAGUGG
SEQ ID NO 218
ACUAGCAAUGUUAUCUGCUUCCUCC
SEQ ID NO 191
CUGCUCUUUUCCAGGUUCAAGUGGG
SEQ ID NO 219
CUAGCAAUGUUAUCUGCUUCCUCCA
SEQ ID NO 192
UGCUCUUUUCCAGGUUCAAGUGGGA
SEQ ID NO 220
UAGCAAUGUUAUCUGCUUCCUCCAA
SEQ ID NO 193
GCUCUUUUCCAGGUUCAAGUGGGAC
SEQ ID NO 221
AGCAAUGUUAUCUGCUUCCUCCAAC
SEQ ID NO 194
CUCUUUUCCAGGUUCAAGUGGGAUA
SEQ ID NO 222
GCAAUGUUAUCUGCUUCCUCCAACC
SEQ ID NO 195
UCUUUUCCAGGUUCAAGUGGGAUAC
SEQ ID NO 223
CAAUGUUAUCUGCUUCCUCCAACCA
SEQ ID NO 196
CUUUUCCAGGUUCAAGUGGGAUACU
SEQ ID NO 224
AAUGUUAUCUGCUUCCUCCAACCAU
SEQ ID NO 197
UUUUCCAGGUUCAAGUGGGAUACUA
SEQ ID NO 225
AUGUUAUCUGCUUCCUCCAACCAUA
SEQ ID NO 198
UUUCCAGGUUCAAGUGGGAUACUAG
SEQ ID NO 226
UGUUAUCUGCUUCCUCCAACCAUAA
SEQ ID NO 199
UUCCAGGUUCAAGUGGGAUACUAGC
SEQ ID NO 227
GUUAUCUGCUUCCUCCAACCAUAAA
SEQ ID NO 200
UCCAGGUUCAAGUGGGAUACUAGCA
SEQ ID NO 228
GCUGCUCUUUUCCAGGUUC
SEQ ID NO 201
CCAGGUUCAAGUGGGAUACUAGCAA
SEQ ID NO 229
UCUUUUCCAGGUUCAAGUGG
SEQ ID NO 202
CAGGUUCAAGUGGGAUACUAGCAAU
SEQ ID NO 230
AGGUUCAAGUGGGAUACUA
DMD Gene Exon 59
SEQ ID NO 231
CAAUUUUUCCCACUCAGUAUU
SEQ ID NO 233
UCCUCAGGAGGCAGCUCUAAAU
SEQ ID NO 232
UUGAAGUUCCUGGAGUCUU
DMD Gene Exon 67
SEQ ID NO 234
GCGCUGGUCACAAAAUCCUGUUGAAC
SEQ ID NO 236
GGUGAAUAACUUACAAAUUUGGAAGC
SEQ ID NO 235
CACUUGCUUGAAAAGGUCUACAAAGGA
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13094548
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Jan 14th, 2020 12:00AM
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Mar 24th, 2017 12:00AM
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https://www.uspto.gov?id=US10533171-20200114
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Oligonucleotide comprising an inosine for treating DMD
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The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the use of said oligonucleotide for preventing or treating DMD or BMD.
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10533171
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1. An isolated antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 557, provided that said isolated antisense oligonucleotide comprises at least one position wherein a guanosine base is substituted with an inosine base, wherein said isolated antisense oligonucleotide comprises a modification and induces skipping of said exon 45 of human dystrophin pre-mRNA.
2. The isolated antisense oligonucleotide of claim 1, said oligonucleotide comprising from one to four inosine bases.
3. The isolated antisense oligonucleotide of claim 1, wherein the modification is a base and/or sugar modification.
4. The isolated antisense oligonucleotide of claim 1, wherein said isolated antisense oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide.
5. The isolated antisense oligonucleotide of claim 1, which is a locked nucleic acid (LNA) oligonucleotide.
6. The isolated antisense oligonucleotide of claim 1, which is a peptide nucleic acid (PNA) oligonucleotide.
7. The isolated antisense oligonucleotide of claim 1, which is a phosphorodiamidate morpholino oligomer (PMO) oligonucleotide.
8. The isolated antisense oligonucleotide of claim 1, wherein the modification is a modified internucleoside linkage.
9. A pharmaceutical composition comprising the isolated antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.
10. A method for inducing skipping of exon 45 of human dystrophin pre-mRNA in a muscle cell, the method comprising contacting said cell with an isolated antisense oligonucleotide of claim 1 for a time and under conditions which permit exon skipping.
11. A method for inducing skipping of exon 45 of human dystrophin pre-mRNA in a human subject, the method comprising administering an isolated antisense oligonucleotide of claim 1 to said subject in an amount and for a time which is effective to induce exon skipping.
12. A method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual, the method comprising administering to said individual an isolated antisense oligonucleotide of claim 1.
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12
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RELATED APPLICATION
This application is a continuation application of U.S. application Ser. No. 15/168,662 filed May 31, 2016, which is a continuation application of U.S. application Ser. No. 14/678,517 filed Apr. 3, 2015, which is continuation application of U.S. application Ser. No. 13/266,110, filed Oct. 24, 2011, which is a U.S. national phase, pursuant to 35 U.S.C. § 371, of PCT international application Ser. No. PCT/NL2010/050230, filed Apr. 26, 2010, designating the United States and published in English on Oct. 28, 2010 as publication WO 2010/123369 A1, which claims priority to European application No. 09158731.1, filed Apr. 24, 2009 and U.S. provisional application Ser. No. 61/172,506 filed Apr. 24, 2009, the entire contents of each of which are incorporated herein by reference in their entirely.
SEQUENCE LISTING
The attached sequence listing, titled “3909 1065 seq listing.txt”, created on Mar. 24, 2017, and 141 kb in size, is herein incorporated by reference.
FIELD OF THE INVENTION
The invention relates to the fields of molecular biology and medicine.
BACKGROUND OF THE INVENTION
A muscle disorder is a disease that usually has a significant impact on the life of an individual. A muscle disorder can have either a genetic cause or a non-genetic cause. An important group of muscle diseases with a genetic cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD). These disorders are caused by defects in a gene for a muscle protein.
Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are genetic muscular dystrophies with a relatively high incidence. In both Duchenne and Becker muscular dystrophy the muscle protein dystrophin is affected. In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and/or the dystrophin present is abnormally formed. Both diseases are associated with recessive X-linked inheritance. DMD results from a frameshift mutation in the DMD gene. The frameshift in the DMD gene's transcript (mRNA) results in the production of a truncated non-functional dystrophin protein, resulting in progressive muscle wasting and weakness. BMD occurs as a mutation does not cause a frame-shift in the DMD transcript (mRNA). As in BMD some partly to largely functional dystrophin is present in contrast to DMD where dystrophin is absent, BMD has generally less severe symptoms then DMD. The onset of DMD is earlier than BMD. DMD usually manifests itself in early childhood, BMD in the teens or in early adulthood. The progression of BMD is slower and less predictable than DMD. Patients with BMD can survive into mid to late adulthood. Patients with DMD rarely survive beyond their thirties.
Dystrophin plays an important structural role in the muscle fiber, connecting the extracellular matrix and the cytoskeleton. The N-terminal region binds actin, whereas the C-terminal end is part of the dystrophin glycoprotein complex (DGC), which spans the sarcolemma. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis.
For most genetic muscular dystrophies no clinically applicable and effective therapies are currently available. Exon skipping techniques are nowadays explored in order to combat genetic muscular dystrophies. Promising results have recently been reported by us and others on a genetic therapy aimed at restoring the reading frame of the dystrophin pre-mRNA in cells from the mdx mouse, the GRMD dog (reference 59) and DMD patients1-11. By the targeted skipping of a specific exon, a DMD phenotype (lacking dystrophin) is converted into a milder BMD phenotype (partly to largely functional dystrophin). The skipping of an exon is preferably induced by the binding of antisense oligoribonucleotides (AONs) targeting either one or both of the splice sites, or exon-internal sequences. Since an exon will only be included in the mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been considered obvious targets for AONs. More preferably, one or more AONs are used which are specific for at least part of one or more exonic sequences involved in correct splicing of the exon. Using exon-internal AONs specific for an exon 46 sequence, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion11. Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also efficiently be induced in human control and patient muscle cells for 39 different DMD exons using exon-internal AONs1, 2, 11-15.
Hence, exon skipping techniques applied on the dystrophin gene result in the generation of at least partially functional—albeit shorter—dystrophin protein in DMD patients. Since DMD is caused by a dysfunctional dystrophin protein, it would be expected that the symptoms of DMD are sufficiently alleviated once a DMD patient has been provided with functional dystrophin protein. However, the present invention provides the insight that, even though exon skipping techniques are capable of inducing dystrophin synthesis, the oligonucleotide used for exon skipping technique can be improved any further by incorporating an inosine and/or a nucleotide containing a base able to form a wobble base pair in said oligonucleotide.
DESCRIPTION OF THE INVENTION
Oligonucleotide
In a first aspect, there is provided an oligonucleotide comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides.
The use of an inosine and/or a nucleotide containing a base able to form a wobble base pair in an oligonucleotide of the invention is very attractive as explained below. Inosine for example is a known modified base which can pair with three bases: uracil, adenine, and cytosine. Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a ß-N9-glycosidic bond. Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. A wobble base pair is a G-U and I-U/I-A/I-C pair fundamental in RNA secondary structure. Its thermodynamic stability is comparable to that of the Watson-Crick base pair. Wobble base pairs are critical for the proper translation of the genetic code. The genetic code makes up for disparities in the number of amino acids (20) for triplet codons (64), by using modified base pairs in the first base of the anti-codon. Similarly, when designing primers for polymerase chain reaction, inosine is useful in that it will indiscriminately pair with adenine, thymine, or cytosine. A first advantage of using such a base allows one to design a primer that spans a single nucleotide polymorphism (SNP), without worry that the polymorphism will disrupt the primer's annealing efficiency. Therefore in the invention, the use of such a base allows to design an oligonucleotide that may be used for an individual having a SNP within the dystrophin pre-mRNA stretch which is targeted by an oligonucleotide of the invention. A second advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is when said oligonucleotide would normally contain a CpG if one would have designed it as being complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. The presence of a CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (reference 60). This increased immunogenicity is undesired since it may induce the breakdown of muscle fibers. Replacing one, two or more CpG by the corresponding inosine and/or a base able to form a wobble base pair in said oligonucleotide is expected to provide an oligonucleotide with a decreased and/or acceptable level of immunogenicity. Immunogenicity may be assessed in an animal model by assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration in muscle biopsy of said animal.
Immunogenicity may also be assessed in blood of an animal or of a human being treated with an oligonucleotide of the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said oligonucleotide using a standard immunoassay known to the skilled person.
An increase in immunogenicity preferably corresponds to a detectable increase of at least one of these cell types by comparison to the amount of each cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having at least one inosine and/or a base able to form a wobble base pair. Alternatively, an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said oligonucleotide using a standard immunoassay.
A decrease in immunogenicity preferably corresponds to a detectable decrease of at least one of these cell types by comparison to the amount of corresponding cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having no inosine and/or a base able to form a wobble base pair. Alternatively a decrease in immunogenicity may be assessed by the absence of or a decreasing amount of said compound and/or neutralizing antibodies using a standard immunoassay.
A third advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to avoid or decrease a potential multimerisation or aggregation of oligonucleotides. It is for example known that an oligonucleotide comprising a G-quartet motif has the tendency to form a quadruplex, a multimer or aggregate formed by the Hoogsteen base-pairing of four single-stranded oligonucleotides (reference 61), which is of course not desired: as a result the efficiency of the oligonucleotide is expected to be decreased. Multimerisation or aggregation is preferably assessed by standard polyacrylamid non-denaturing gel electrophoresis techniques known to the skilled person. In a preferred embodiment, less than 20% or 15%, 10%, 7%, 5% or less of a total amount of an oligonucleotide of the invention has the capacity to multimerise or aggregate assessed using the assay mentioned above.
A fourth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is thus also to avoid quadruplex structures which have been associated with antithrombotic activity (reference 62) as well as with the binding to, and inhibition of, the macrophage scavenger receptor (reference 63).
A fifth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to allow to design an oligonucleotide with improved RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (http://www.unc.edu/˜cail/biotool/oligo/index.html) for single stranded RNA using the basic Tm and the nearest neighbour model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). If a Tm is too high, the oligonucleotide is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 65° C. and an acceptable free energy may be ranged between 15 and 45 kcal/mol.
The skilled person may therefore first choose an oligonucleotide as a potential therapeutic compound. In a second step, he may use the invention to further optimise said oligonucleotide by decreasing its immunogenicity and/or avoiding aggregation and/or quadruplex formation and/or by optimizing its Tm and/or free energy of the AON-target complex. He may try to introduce at least one inosine and/or a base able to form a wobble base pair in said oligonucleotide at a suitable position and assess how the immunogenicity and/or aggregation and/or quadruplex formation and/or Tm and/or free energy of the AON-target complex have been altered by the presence of said inosine and/or a base able to form a wobble base pair. If the alteration does not provide the desired alteration or decrease of immunogenicity and/or aggregation and/or quadruplex formation and/or its Tm and/or free energy of the AON-target complex he may choose to introduce a further inosine and/or a base able to form a wobble base pair in said oligonucleotide and/or to introduce a given inosine and/or a base able to form a wobble base pair at a distinct suitable position within said oligonucleotide.
An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair may be defined as an oligonucleotide wherein at least one nucleotide has been substituted with an inosine and/or a base able to form a wobble base pair. The skilled person knows how to test whether a nucleotide contains a base able to form a wobble base pair. Since for example inosine can form a base pair with uracil, adenine, and/or cytosine, it means that at least one nucleotide able to form a base pair with uracil, adenine and/or cytosine has been substituted with inosine. However, in order to safeguard specificity, the inosine containing oligonucleotide preferably comprises the substitution of at least one, two, three, four nucleotide(s) able to form a base pair with uracil or adenine or cytosine as long as an acceptable level of a functional activity of said oligonucleotide is retained as defined later herein.
An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair is preferably an olignucleotide, which is still able to exhibit an acceptable level of a functional activity of a corresponding oligonucleotide not comprising an inosine and/or a base able to form a wobble base pair. A functional activity of said oligonucleotide is preferably to provide an individual with a functional dystrophin protein and/or mRNA and/or at least in part decreasing the production of an aberrant dystrophin protein and/or mRNA. Each of these features are later defined herein. An acceptable level of such a functional activity is preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the functional activity of the corresponding oligonucleotide which does not comprise an inosine and/or a base able to form a wobble base pair. Such functional activity may be as measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to the functional activity of a corresponding oligonucleotides not comprising an inosine and/or a base able to form a wobble base pair. The assessment of the functionality may be carried out at the mRNA level, preferably using RT-PCR. The assessment of the functionality may be carried out at the protein level, preferably using western blot analysis or immunofluorescence analysis of cross-sections.
Within the context of the invention, an inosine and/or a base able to form a wobble base pair as present in an oligonucleotide is/are present in a part of said oligonucleotide which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. Therefore, in a preferred embodiment, an oligonucleotide comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides and wherein said inosine and/or a nucleotide containing a base able is/are present within the oligonucleotide sequence which is complementary to at least part of a dystrophin pre-mRNA as defined in previous sentence.
However, as later defined herein such inosine and/or a base able to form a wobble base pair may also be present in a linking moiety present in an oligonucleotide of the invention. Preferred linking moieties are later defined herein.
In a preferred embodiment, such oligonucleotide is preferably a medicament. More preferably, said medicament is for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or a patient. As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. A patient is preferably intended to mean a patient having DMD or BMD or a patient susceptible to develop DMD or BMD due to his or her genetic background. In the case of a DMD patient, an oligonucleotide used will preferably correct at least one of the DMD mutations as present in the DMD gene of said patient and therefore will preferably create a dystrophin that will look like a BMD dystrophin: said dystropin will preferably be a functional dystrophin as later defined herein.
In the case of a BMD patient, an oligonucleotide as used will preferably correct at least one of the BMD mutations as present in the DMD gene of said patient and therefore will preferably create a, or more of a, dystrophin, which will be more functional than the dystrophin which was originally present in said BMD patient. Even more preferably, said medicament provides an individual with a functional or more (of a) functional dystrophin protein and/or mRNA and/or at least in part decreases the production of an aberrant dystrophin protein and/or mRNA.
Preferably, a method of the invention by inducing and/or promoting skipping of at least one exon of the DMD pre-mRNA as identified herein in one or more cells, preferably muscle cells of a patient, provides said patient with an increased production of a more (of a) functional dystrophin protein and/or mRNA and/or decreases the production of an aberrant or less functional dystrophin protein and/or mRNA in said patient.
Providing a patient with a more functional dystrophin protein and/or mRNA and/or decreasing the production of an aberrant dystrophin protein and/or mRNA in said patient is typically applied in a DMD patient.
Increasing the production of a more functional or functional dystrophin and/or mRNA is typically applied in a BMD patient.
Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with an increased production of a more functional or functional dystrophin protein and/or mRNA and/or wherein the production of an aberrant dystrophin protein and/or mRNA in said patient is decreased, wherein the level of said aberrant or more functional dystrophin protein and/or mRNA is assessed by comparison to the level of said dystrophin and/or mRNA in said patient at the onset of the method.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO: 1. In another embodiment, a functional dystrophin is a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a wild type dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC)56. Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a biopsy of a muscle suspected to be dystrophic, as known to the skilled person.
Individuals suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevents synthesis of the complete protein, i.e a premature stop prevents the synthesis of the C-terminus of the protein. In Becker muscular dystrophy the dystrophin gene also comprises a mutation compared to the wild type but the mutation does typically not include a premature stop and the C-terminus of the protein is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as a wild type protein, although not necessarily the same amount of activity. In a preferred embodiment, a functional dystrophin protein means an in frame dystrophin gene. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin56. Exon-skipping for the treatment of DMD is preferably but not exclusively directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-domain shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated using an oligonucleotide as defined herein will be provided a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: preferably said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus et al (2006, ref 56). The central rod domain of wild type dystrophin comprises 24 spectrin-like repeats56. For example, a central rod shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC. Decreasing the production of an aberrant dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional or less to non-functional or semi-functional dystrophin mRNA or protein. A non-functional pre-mRNA dystrophin is preferably leads to an out of frame dystrophin protein, which means that no dystrophin protein will be produced and/or detected. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional or in frame dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional or in frame dystrophin mRNA.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
An increase or a decrease is preferably assessed in a muscular tissue or in a muscular cell of an individual or a patient by comparison to the amount present in said individual or patient before treatment with said molecule or composition of the invention. Alternatively, the comparison can be made with a muscular tissue or cell of said individual or patient, which has not yet been treated with said oligonucleotide or composition in case the treatment is local.
In a preferred method, one or more symptom(s) from a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of a muscle cell or tissue from a DMD or a BMD patient is/are alleviated using a molecule or a composition of the invention. Such symptoms may be assessed on the patient self. Such characteristics may be assessed at the cellular, tissue level of a given patient. An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual using a molecule or a composition of the invention may be assessed by any of the following assays: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (2008, ref 58) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a molecule or composition of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (2006, ref 57). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
An oligonucleotide as used herein preferably comprises an antisense oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied. Exon skipping interferes with the natural splicing processes occurring within a eukaryotic cell. In higher eukaryotes the genetic information for proteins in the DNA of the cell is encoded in exons which are separated from each other by intronic sequences. These introns are in some cases very long. The transcription machinery of eukaryotes generates a pre-mRNA which contains both exons and introns, while the splicing machinery, often already during the production of the pre-mRNA, generates the actual coding region for the protein by splicing together the exons present in the pre-mRNA.
Exon-skipping results in mature mRNA that lacks at least one skipped exon. Thus, when said exon codes for amino acids, exon skipping leads to the expression of an altered product. Technology for exon-skipping is currently directed towards the use of antisense oligonucleotides (AONs). Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy. The mdx mouse carries a nonsense mutation in exon 23. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. AONs directed to, respectively, the 3′ and/or 5′ splice sites of introns 22 and 23 in dystrophin pre-mRNA, have been shown to interfere with factors normally involved in removal of intron 23 so that also exon 23 was removed from the mRNA3, 5, 6, 39, 40.
By the targeted skipping of a specific exon, a DMD phenotype is converted into a milder BMD phenotype. The skipping of an exon is preferably induced by the binding of AONs targeting either one or both of the splice sites, or exon-internal sequences. An oligonucleotide directed toward an exon internal sequence typically exhibits no overlap with non-exon sequences. It preferably does not overlap with the splice sites at least not insofar, as these are present in the intron. An oligonucleotide directed toward an exon internal sequence preferably does not contain a sequence complementary to an adjacent intron. Further provided is thus an oligonucleotide according to the invention, wherein said oligonucleotide, or a functional equivalent thereof, is for inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA produced from splicing of said pre-mRNA. An exon skipping technique is preferably applied such that the absence of an exon from mRNA produced from dystrophin pre-mRNA generates a coding region for a more functional—albeit shorter—dystrophin protein. In this context, inhibiting inclusion of an exon preferably means that the detection of the original, aberrant dystrophin mRNA and/or protein is decreased as earlier defined herein.
Since an exon of a dystrophin pre-mRNA will only be included into the resulting mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been obvious targets for AONs. One embodiment therefore provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a non-exon region of a dystrophin pre mRNA. In one embodiment an AON is used which is solely complementary to a non-exon region of a dystrophin pre mRNA. This is however not necessary: it is also possible to use an AON which comprises an intron-specific sequence as well as exon-specific sequence. Such AON comprises a sequence which is complementary to a non-exon region of a dystrophin pre mRNA, as well as a sequence which is complementary to an exon region of a dystrophin pre mRNA. Of course, an AON is not necessarily complementary to the entire sequence of a dystrophin exon or intron. AONs, which are complementary to a part of such exon or intron are preferred. An AON is preferably complementary to at least part of a dystrohin exon and/or intron, said part having at least 8, 10, 13, 15, 20 nucleotides.
Splicing of a dystrophin pre-mRNA occurs via two sequential transesterification reactions. First, the 2′OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5′ splice site forming the lariat intermediate. Second, the 3′OH of the released 5′ exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3′ splice site thus joining the exons and releasing the intron lariat. The branch point and splice sites of an intron are thus involved in a splicing event. Hence, an oligonucleotide comprising a sequence, which is complementary to such branch point and/or splice site is preferably used for exon skipping. Further provided is therefore an oligonucleotide, or a functional equivalent thereof, which comprises a sequence which is complementary to a splice site and/or branch point of a dystrophin pre mRNA.
Since splice sites contain consensus sequences, the use of an oligonucleotide or a functional equivalent thereof (herein also called an AON) comprising a sequence which is complementary of a splice site involves the risk of promiscuous hybridization. Hybridization of AONs to other splice sites than the sites of the exon to be skipped could easily interfere with the accuracy of the splicing process. To overcome these and other potential problems related to the use of AONs which are complementary to an intron sequence, one preferred embodiment provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a dystrophin pre-mRNA exon. Preferably, said AON is capable of specifically inhibiting an exon inclusion signal of at least one exon in said dystrophin pre-mRNA. Interfering with an exon inclusion signal (EIS) has the advantage that such elements are located within the exon. By providing an AON for the interior of the exon to be skipped, it is possible to interfere with the exon inclusion signal thereby effectively masking the exon from the splicing apparatus. The failure of the splicing apparatus to recognize the exon to be skipped thus leads to exclusion of the exon from the final mRNA. This embodiment does not interfere directly with the enzymatic process of the splicing machinery (the joining of the exons). It is thought that this allows the method to be more specific and/or reliable. It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In this concept, it is the particular spatial conformation that enables the splicing machinery to recognize the exon. However, the invention is certainly not limited to this model. In a preferred embodiment, use is made of an oligonucleotide, which is capable of binding to an exon and is capable of inhibiting an EIS. An AON may specifically contact said exon at any point and still be able to specifically inhibit said EIS.
Within the context of the invention, a functional equivalent of an oligonucleotide preferably means an oligonucleotide as defined herein wherein one or more nucleotides have been substituted and wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by quantifying the amount of a functional dystrophin protein or by quantifying the amount of a functional dystrophin mRNA. A functional dystrophin protein (or a functional dystrophin mRNA) is herein preferably defined as being a dystrophin protein (or a dystrophin protein encoded by said mRNA) able to bind actin and members of the DGC protein. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR (m-RNA) or by immunofluorescence or Western blot analyses (protein). Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Such activity may be measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to an activity of a corresponding oligonucleotide of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
Hence, an oligonucleotide, or a functional equivalent thereof, comprising or consisting of a sequence which is complementary to a dystrophin pre-mRNA exon provides good DMD therapeutic results. In one preferred embodiment an oligonucleotide, or a functional equivalent thereof, is used which comprises or consists of a sequence which is complementary to at least part of either dystrophin pre-mRNA exons 2 to 75 said part having or comprising at least 13 nucleotides. However, said part may also have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. A part of dystrophin pre-mRNA to which an oligonucleotide is complementary may also be called a contiguous stretch of dystrophin pre-mRNA.
Most preferably an AON is used which comprises or consists of a sequence which is complementary to at least part of dystrophin pre-mRNA exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, 55, 2, 11, 17, 19, 21, 57, 59, 62, 63, 65, 66, 69, and/or 75 said part having or comprising at least 13 nucleotides. However, said part may also have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. More preferred oligonucleotides are represented by a sequence that comprises or consists of each of the following sequences SEQ ID NO: 2 to SEQ ID NO:539 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil. Accordingly, an even more preferred oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:2 to SEQ ID NO:486 or SEQ ID NO:539, even more preferably SEQ ID NO:2 to NO 237 or SEQ ID NO:539, most preferably SEQ ID NO:76 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil.
Accordingly, in another preferred embodiment, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:540 to SEQ ID NO:576. More preferably, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:557.
Said exons are listed in decreasing order of patient population applicability. Hence, the use of an AON comprising a sequence, which is complementary to at least part of dystrophin pre-mRNA exon 51 is suitable for use in a larger part of the DMD patient population as compared to an AON comprising a sequence which is complementary to dystrophin pre-mRNA exon 44, et cetera.
In a preferred embodiment, an oligonucleotide of the invention, which comprises a sequence that is complementary to part of dystrophin pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99%, or even more preferably 100%. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary to part of dystrophin pre-mRNA as defined herein. As an example, an oligonucleotide may comprise a sequence that is complementary to part of dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to the oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.
One preferred embodiment provides an oligonucleotide, or a functional equivalent thereof which comprises:
a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is hybridized to another part of a dystrophin pre-mRNA exon (closed structure), and
a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is not hybridized in said dystrophin pre-mRNA (open structure).
For this embodiment, reference is made to WO 2004/083432, which is incorporated by reference in its entirety. RNA molecules exhibit strong secondary structures, mostly due to base pairing of complementary or partly complementary stretches within the same RNA. It has long since been thought that structures in the RNA play a role in the function of the RNA. Without being bound by theory, it is believed that the secondary structure of the RNA of an exon plays a role in structuring the splicing process. The structure of an exon is one parameter which is believed to direct its inclusion into the mRNA. However, other parameters may also play a role therein. Herein this signaling function is referred to as an exon inclusion signal. A complementary oligonucleotide of this embodiment is capable of interfering with the structure of the exon and thereby capable of interfering with the exon inclusion signal of the exon. It has been found that many complementary oligonucleotides indeed comprise this capacity, some more efficient than others. Oligonucleotides of this preferred embodiment, i.e. those with the said overlap directed towards open and closed structures in the native exon RNA, are a selection from all possible oligonucleotides. The selection encompasses oligonucleotides that can efficiently interfere with an exon inclusion signal. Without being bound by theory it is thought that the overlap with an open structure improves the invasion efficiency of the oligonucleotide and prevents the binding of splicing factors (i.e. increases the efficiency with which the oligonucleotide can enter the structure), whereas the overlap with the closed structure subsequently increases the efficiency of interfering with the secondary structure of the RNA of the exon, and thereby interfere with the exon inclusion signal. It is found that the length of the partial complementarity to both the closed and the open structure is not extremely restricted. We have observed high efficiencies with oligonucleotides with variable lengths of complementarity in either structure. The term complementarity is used herein to refer to a stretch of nucleic acids that can hybridise to another stretch of nucleic acids under physiological conditions. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the oligonucleotide one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may, to some extent, be allowed, if under the circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridising to the complementary part. In this context, “sufficiently” preferably means that using a gel mobility shift assay as described in example 1 of EP 1 619 249, binding of an oligonucleotide is detectable. Optionally, said oligonucleotide may further be tested by transfection into muscle cells of patients. Skipping of the targeted exon may be assessed by RT-PCR (as described in EP 1 619 249). The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-)mRNA in the system. The risk that also one or more other pre-mRNA will be able to hybridise to the oligonucleotide decreases with increasing size of the oligonucleotide. It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain the capacity to hybridise to the targeted region(s) in the pre-mRNA, can be used in the present invention. However, preferably at least the complementary parts do not comprise such mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides having such mismatches in one or more complementary regions. It is thought, that higher hybridisation strengths, (i.e. increasing number of interactions with the opposing strand) are favourable in increasing the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the complementarity is between 90 and 100%. In general this allows for approximately 1 or 2 mismatch(es) in an oligonucleotide of around 20 nucleotides
The secondary structure is best analysed in the context of the pre-mRNA wherein the exon resides. Such structure may be analysed in the actual RNA. However, it is currently possible to predict the secondary structure of an RNA molecule (at lowest energy costs) quite well using structure-modeling programs. A non-limiting example of a suitable program is RNA mfold version 3.1 server41. A person skilled in the art will be able to predict, with suitable reproducibility, a likely structure of the exon, given the nucleotide sequence. Best predictions are obtained when providing such modeling programs with both the exon and flanking intron sequences. It is typically not necessary to model the structure of the entire pre-mRNA.
The open and closed structure to which the oligonucleotide is directed, are preferably adjacent to one another. It is thought, that in this way the annealing of the oligonucleotide to the open structure induces opening of the closed structure whereupon annealing progresses into this closed structure. Through this action the previously closed structure assumes a different conformation. The different conformation results in the disruption of the exon inclusion signal. However, when potential (cryptic) splice acceptor and/or donor sequences are present within the targeted exon, occasionally a new exon inclusion signal is generated defining a different (neo) exon, i.e. with a different 5′ end, a different 3′ end, or both. This type of activity is within the scope of the present invention as the targeted exon is excluded from the mRNA. The presence of a new exon, containing part of the targeted exon, in the mRNA does not alter the fact that the targeted exon, as such, is excluded. The inclusion of a neo-exon can be seen as a side effect, which occurs only occasionally. There are two possibilities when exon skipping is used to restore (part of) an open reading frame of dystrophin that is disrupted as a result of a mutation. One is that the neo-exon is functional in the restoration of the reading frame, whereas in the other case the reading frame is not restored. When selecting oligonucleotides for restoring dystrophin reading frames by means of exon-skipping it is of course clear that under these conditions only those oligonucleotides are selected that indeed result in exon-skipping that restores the dystrophin open reading frame, with or without a neo-exon.
Further provided is an oligonucleotide, or a functional equivalent thereof, comprising a sequence that is complementary to a binding site for a serine-arginine (SR) protein in RNA of an exon of a dystrophin pre-mRNA. In WO 2006/112705 we have disclosed the presence of a correlation between the effectivity of an exon-internal antisense oligonucleotide (AON) in inducing exon skipping and the presence of a (for example by ESE finder) predicted SR binding site in the target pre-mRNA site of said AON.
Therefore, in one embodiment an oligonucleotide is generated comprising determining a (putative) binding site for an SR (Ser-Arg) protein in RNA of a dystrophin exon and producing an oligonucleotide that is complementary to said RNA and that at least partly overlaps said (putative) binding site. The term “at least partly overlaps” is defined herein as to comprise an overlap of only a single nucleotide of an SR binding site as well as multiple nucleotides of said binding site as well as a complete overlap of said binding site. This embodiment preferably further comprises determining from a secondary structure of said RNA, a region that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide that at least partly overlaps said (putative) binding site and that overlaps at least part of said closed structure and overlaps at least part of said open structure. In this way we increase the chance of obtaining an oligonucleotide that is capable of interfering with the exon inclusion from the pre-mRNA into mRNA. It is possible that a first selected SR-binding region does not have the requested open-closed structure in which case another (second) SR protein binding site is selected which is then subsequently tested for the presence of an open-closed structure. This process is continued until a sequence is identified which contains an SR protein binding site as well as a(n) (partly overlapping) open-closed structure. This sequence is then used to design an oligonucleotide which is complementary to said sequence.
Such a method, for generating an oligonucleotide, is also performed by reversing the described order, i.e. first generating an oligonucleotide comprising determining, from a secondary structure of RNA from a dystrophin exon, a region that assumes a structure that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide, of which at least a part of said oligonucleotide is complementary to said closed structure and of which at least another part of said oligonucleotide is complementary to said open structure. This is then followed by determining whether an SR protein binding site at least overlaps with said open/closed structure. In this way the method of WO 2004/083432 is improved. In yet another embodiment the selections are performed simultaneously.
Without wishing to be bound by any theory it is currently thought that use of an oligonucleotide directed to an SR protein binding site results in (at least partly) impairing the binding of an SR protein to the binding site of an SR protein which results in disrupted or impaired splicing.
Preferably, an open/closed structure and an SR protein binding site partly overlap and even more preferred an open/closed structure completely overlaps an SR protein binding site or an SR protein binding site completely overlaps an open/closed structure. This allows for an improved disruption of exon inclusion.
Besides consensus splice sites sequences, many (if not all) exons contain splicing regulatory sequences such as exonic splicing enhancer (ESE) sequences to facilitate the recognition of genuine splice sites by the spliceosome42, 43. A subgroup of splicing factors, called the SR proteins, can bind to these ESEs and recruit other splicing factors, such as U1 and U2AF to (weakly defined) splice sites. The binding sites of the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55) have been analyzed in detail and these results are implemented in ESE finder, a web source that predicts potential binding sites for these SR proteins42, 43. There is a correlation between the effectiveness of an AON and the presence/absence of an SF2/ASF, SC35 and SRp40 binding site. In a preferred embodiment, the invention thus provides a combination as described above, wherein said SR protein is SF2/ASF or SC35 or SRp40.
In one embodiment an oligonucleotide, or a functional equivalent thereof is capable of specifically binding a regulatory RNA sequence which is required for the correct splicing of a dystrophin exon in a transcript. Several cis-acting RNA sequences are required for the correct splicing of exons in a transcript. In particular, supplementary elements such as intronic or exonic splicing enhancers (ISEs and ESEs) or silencers (ISSs and ESEs) are identified to regulate specific and efficient splicing of constitutive and alternative exons. Using sequence-specific antisense oligonucleotides (AONs) that bind to the elements, their regulatory function is disturbed so that the exon is skipped, as shown for DMD. Hence, in one preferred embodiment an oligonucleotide or functional equivalent thereof is used which is complementary to an intronic splicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronic splicing silencer (ISS) and/or an exonic splicing silencer (ESS). As already described herein before, a dystrophin exon is in one preferred embodiment skipped by an agent capable of specifically inhibiting an exon inclusion signal of said exon, so that said exon is not recognized by the splicing machinery as a part that needs to be included in the mRNA. As a result, a mRNA without said exon is formed.
An AON used herein is preferably complementary to a consecutive part or a contiguous stretch of between 8 and 50 nucleotides of dystrophin exon RNA or dystrophin intron RNA. In one embodiment an AON used herein is complementary to a consecutive part or a contiguous stretch of between 14 and 50 nucleotides of a dystrophin exon RNA or dystrophin intron RNA. Preferably, said AON is complementary to a consecutive part or contiguous stretch of between 14 and 25 nucleotides of said exon RNA. More preferably, an AON is used which comprises a sequence which is complementary to a consecutive part or a contiguous stretch of between 20 and 25 nucleotides of a dystrophin exon RNA or a dystrophin intron RNA.
Different types of nucleic acid may be used to generate an oligonucleotide. Preferably, said oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Since one of the aims of the exon skipping technique is to direct splicing in subjects it is preferred that the oligonucleotide RNA comprises a modification providing the RNA with an additional property, for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc. Preferably, said modification comprises a 2′-O-methyl-phosphorothioate oligoribonucleotide modification. Preferably, said modification comprises a 2′-O-methyl-phosphorothioate oligodeoxyribonucleotide modification. One embodiment thus provides an oligonucleotide is used which comprises RNA which contains a modification, preferably a 2′-O-methyl modified ribose (RNA) or deoxyribose (DNA) modification.
In one embodiment the invention provides a hybrid oligonucleotide comprising an oligonucleotide comprising a 2′-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification and locked nucleic acid. This particular oligonucleotide comprises better sequence specificity compared to an equivalent consisting of locked nucleic acid, and comprises improved effectivity when compared with an oligonucleotide consisting of 2′-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification.
With the advent of nucleic acid mimicking technology it has become possible to generate molecules that have a similar, preferably the same hybridisation characteristics in kind not necessarily in amount as nucleic acid itself. Such functional equivalents are of course also suitable for use in the invention. Preferred examples of functional equivalents of an oligonucleotide are peptide nucleic acid and/or locked nucleic acid. Most preferably, a morpholino phosphorodiamidate is used. Suitable but non-limiting examples of equivalents of oligonucleotides of the invention can be found in44-50. Hybrids between one or more of the equivalents among each other and/or together with nucleic acid are of course also suitable. In a preferred embodiment locked nucleic acid is used as a functional equivalent of an oligonucleotide, as locked nucleic acid displays a higher target affinity and reduced toxicity and therefore shows a higher efficiency of exon skipping.
In one embodiment an oligonucleotide, or a functional equivalent thereof, which is capable of inhibiting inclusion of a dystrophin exon into dystrophin mRNA is combined with at least one other oligonucleotide, or functional equivalent thereof, that is capable of inhibiting inclusion of another dystrophin exon into dystrophin mRNA. This way, inclusion of two or more exons of a dystrophin pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping2, 15. In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-exon skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other. Other preferred examples of multi-exon skipping are:
the use of an oligonucleotide targeting exon 17, and a second one exon 48 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 48.
the use of an oligonucleotide targeting exon 17, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 51.
the use of an oligonucleotide targeting exon 42, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 42 and exon 55.
the use of an oligonucleotide targeting exon 43, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 51.
the use of an oligonucleotide targeting exon 43, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 55.
the use of an oligonucleotide targeting exon 45, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 55.
the use of an oligonucleotide targeting exon 45, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 59.
the use of an oligonucleotide targeting exon 48, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 48 and exon 59.
the use of an oligonucleotide targeting exon 50, and a second one exon 51 which may result in the skipping of said both exons.
the use of an oligonucleotide targeting exon 51, and a second one exon 52 which may result in the skipping of said both exons.
Further provided is therefore an oligonucleotide which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first exon of a dystrophin pre-mRNA and wherein a nucleotide sequence is used which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon of said dystrophin pre-mRNA. Said first and said second exon may be the same.
In one preferred embodiment said first and said second exon are separated in said dystrophin pre-mRNA by at least one exon to which said oligonucleotide is not complementary. Alternatively, said first and said second exon are adjacent.
It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule. Further provided is therefore an oligonucleotide, or functional equivalent thereof which is complementary to at least two exons in a dystrophin pre-mRNA, said oligonucleotide or functional equivalent comprising at least two parts wherein a first part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first of said at least two exons and wherein a second part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon in said dystrophin pre-mRNA. The linkage may be through any means, but is preferably accomplished through a nucleotide linkage. In the latter case, the number of nucleotides that do not contain an overlap between one or the other complementary exon can be zero, but is preferably between 4 to 40 nucleotides. The linking moiety can be any type of moiety capable of linking oligonucleotides. Preferably, said linking moiety comprises at least 4 uracil nucleotides. Currently, many different compounds are available that mimic hybridisation characteristics of oligonucleotides. Such a compound, called herein a functional equivalent of an oligonucleotide, is also suitable for the present invention if such equivalent comprises similar hybridisation characteristics in kind not necessarily in amount. Suitable functional equivalents are mentioned earlier in this description. As mentioned, oligonucleotides of the invention do not have to consist of only oligonucleotides that contribute to hybridisation to the targeted exon. There may be additional material and/or nucleotides added.
The DMD gene is a large gene, with many different exons. Considering that the gene is located on the X-chromosome, it is mostly boys that are affected, although girls can also be affected by the disease, as they may receive a bad copy of the gene from both parents, or are suffering from a particularly biased inactivation of the functional allele due to a particularly biased X chromosome inactivation in their muscle cells. The protein is encoded by a plurality of exons (79) over a range of at least 2.4 Mb. Defects may occur in any part of the DMD gene. Skipping of a particular exon or particular exons can, very often, result in a restructured mRNA that encodes a shorter than normal but at least partially functional dystrophin protein. A practical problem in the development of a medicament based on exon-skipping technology is the plurality of mutations that may result in a deficiency in functional dystrophin protein in the cell. Despite the fact that already multiple different mutations can be corrected for by the skipping of a single exon, this plurality of mutations, requires the generation of a series of different pharmaceuticals as for different mutations different exons need to be skipped. An advantage of an oligonucleotide or of a composition comprising at least two distinct oligonucleotide as later defined herein capable of inducing skipping of two or more exons, is that more than one exon can be skipped with a single pharmaceutical. This property is not only practically very useful in that only a limited number of pharmaceuticals need to be generated for treating many different DMD or particular, severe BMD mutations. Another option now open to the person skilled in the art is to select particularly functional restructured dystrophin proteins and produce compounds capable of generating these preferred dystrophin proteins. Such preferred end results are further referred to as mild phenotype dystrophins.
Dose ranges of oligonucleotide according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg.
In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 μM is used. Preferably, this range is for in vitro use in a cellular model such as muscular cells or muscular tissue. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. If several oligonucleotides are used, this concentration or dose may refer to the total concentration or dose of oligonucleotides or the concentration or dose of each oligonucleotide added.
The ranges of concentration or dose of oligonucleotide(s) as given above are preferred concentrations or doses for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of oligonucleotide(s) used may further vary and may need to be optimised any further.
An oligonucleotide as defined herein for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD or BMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne and Becker muscular dystrophy have a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably, said cells comprise a gene encoding a mutant dystrophin protein. Preferably, said cells are cells of an individual suffering from DMD or BMD.
An oligonucleotide of the invention may be indirectly administrated using suitable means known in the art. An oligonucleotide may for example be provided to an individual or a cell, tissue or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of a molecule as identified herein. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector4, 51, 52 and the like. Also, plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from PolIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are PolIII driven transcripts. Preferably, in the form of a fusion transcript with an U1 or U7 transcript4, 51, 52. Such fusions may be generated as described53, 54. The oligonucleotide may be delivered as is. However, the oligonucleotide may also be encoded by the viral vector. Typically, this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.
Improvements in means for providing an individual or a cell, tissue, organ of said individual with an oligonucleotide and/or an equivalent thereof, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. An oligonucleotide and/or an equivalent thereof can be delivered as is to an individual, a cell, tissue or organ of said individual. When administering an oligonucleotide and/or an equivalent thereof, it is preferred that an oligonucleotide and/or an equivalent thereof is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a muscle cell. Preferred are excipients capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives,
synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as antisense nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an oligonucleotide across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an oligonucleotide for use in the current invention to deliver it for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
In addition, an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, an oligonucleotide is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said oligonucleotide to a cell and/or enhancing its intracellular delivery. It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an oligonucleotide and a further adjunct compound as later defined herein.
A preferred oligonucleotide is for preventing or treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in an individual. An individual, which may be treated using an oligonucleotide of the invention may already have been diagnosed as having a DMD or a BMD. Alternatively, an individual which may be treated using an oligonucleotide of the invention may not have yet been diagnosed as having a DMD or a BMD but may be an individual having an increased risk of developing a DMD or a BMD in the future given his or her genetic background. A preferred individual is a human being.
Composition
In a further aspect, there is provided a composition comprising an oligonucleotide as defined herein. Preferably, said composition comprises at least two distinct oligonucleotide as defined herein. More preferably, these two distinct oligonucleotides are designed to skip distinct two or more exons as earlier defined herein for multi-exon skipping.
In a preferred embodiment, said composition being preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Such a pharmaceutical composition may comprise any pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000. Each feature of said composition has earlier been defined herein.
If several oligonucleotides are used, concentration or dose already defined herein may refer to the total concentration or dose of all oligonucleotides used or the concentration or dose of each oligonucleotide used or added. Therefore in one embodiment, there is provided a composition wherein each or the total amount of oligonucleotide used is dosed in an amount ranged between 0.5 mg/kg and 10 mg/kg.
A preferred composition additionally comprises:
a) an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or
b) an adjunct compound for improving muscle fiber function, integrity and/or survival and/or
c) a compound exhibiting readthrough activity.
It has surprisingly been found that the skipping frequency of a dystrophin exon from a pre-MRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon, is enhanced if cells expressing said pre-mRNA are also provided with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
According to the present invention, even when a dystrophin protein deficiency has been restored in a DMD patient by administering an oligonucleotide of the invention, the presence of tissue inflammation and damaged muscle cells still continues to contribute to the symptoms of DMD. Hence, even though the cause of DMD—i.e. a dysfunctional dystrophin protein—is alleviated, treatment of DMD is still further improved by additionally using an adjunct therapy according to the present invention. Furthermore, the present invention provides the insight that a reduction of inflammation does not result in significant reduction of AON uptake by muscle cells. This is surprising because, in general, inflammation enhances the trafficking of cells, blood and other compounds. As a result, AON uptake/delivery is also enhanced during inflammation. Hence, before the present invention it would be expected that an adjunct therapy counteracting inflammation involves the risk of negatively influencing AON therapy. This, however, appears not to be the case.
An adjunct compound for reducing inflammation comprises any therapy which is capable of at least in part reducing inflammation, preferably inflammation caused by damaged muscle cells. Said adjunct compound is most preferably capable of reducing muscle tissue inflammation. Inflammation is preferably assessed by detecting an increase in the number of infiltrating immune cells such as neutrophils and/or mast cells and/or dendritic cells and/or lymphocytes in muscle tissue suspected to be dystrophic. This assessment is preferably carried out in cross-sections of a biopsy57 of muscle tissue suspected to be dystrophic after having specifically stained immune cells as identified above. The quantification is preferably carried out under the microscope. Reducing inflammation is therefore preferably assessed by detecting a decrease in the number of immune cells in a cross-section of muscle tissue suspected to be dystrophic. Detecting a decrease preferably means that the number of at least one sort of immune cells as identified above is decreased of at least 1%, 2%, 3%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the number of a corresponding immune cell in a same individual before treatment. Most preferably, no infiltrating immune cells are detected in cross-sections of said biopsy.
An adjunct compound for improving muscle fiber function, integrity and/or survival comprises any therapy, which is capable of measurably enhancing muscle fiber function, integrity and/or survival as compared to an otherwise similar situation wherein said adjunct compound is not present. The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
Creatine kinase may be detected in blood as described in 57. A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same individual before treatment.
A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in 57 using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same individual before treatment.
A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in 57.
In one embodiment, an adjunct compound for increasing turnover of damaged muscle cells is used. An adjunct compound for increasing turnover of damaged muscle cells comprises any therapy, which is capable of at least in part inducing and/or increasing turnover of damaged muscle cells.
Damaged muscle cells are muscle cells, which have significantly less clinically measurable functionality than a healthy, intact muscle cell. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis, resulting in damaged muscle cells. Increasing turnover of damaged muscle cells means that damaged muscle cells are more quickly broken down and/or removed as compared to a situation wherein turnover of damaged muscle cells is not increased. Turnover of damaged muscle cells is preferably assessed in a muscle biopsy, more preferably as described in 57 using a cross-section of a biopsy. A detectable increase of turnover may be an increase of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein turnover has been identified using a biopsy cross-section. The increase is measured by comparison to the turnover as assessed in a same individual before treatment.
Without wishing to be bound to theory, it is believed that increasing turnover of muscle cells is preferred because this reduces inflammatory responses.
According to the present invention, a composition of the invention further comprising an adjunct therapy for reducing inflammation, preferably for reducing muscle tissue inflammation in an individual, is particularly suitable for use as a medicament. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy as compared to a combination not comprising said adjunct compound. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
Further provided is therefore a composition further comprising an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, for use as a medicament, preferably for treating or preventing counteracting DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein or altered or truncated dystrophin mRNA or protein is formed which is not sufficiently functional.
Preferred adjunct compound for reducing inflammation include a steroid, a TNFα inhibitor, a source of mIGF-1 and/or an antioxidant. However, any other compound able to reduce inflammation as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for improving muscle fiber function, integrity and/or survival.
Furthermore, a composition comprising an adjunct therapy for improving muscle fiber function, integrity and/or survival in an individual is particularly suitable for use as a medicament, preferably for treating or preventing DMD. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy as compared to a composition not comprising said adjunct compound.
Preferred adjunct compounds for improving muscle fiber function, integrity and/or survival include an ion channel inhibitor, a protease inhibitor, L-arginine and/or an angiotensin II type I receptor blocker. However, any other compound able to improving muscle fiber function, integrity and/or survival as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for reducing inflammation.
In a particularly preferred embodiment, a composition further comprises a steroid. Such composition results in significant alleviation of DMD symptoms. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
A steroid is a terpenoid lipid characterized by a carbon skeleton with four fused rings, generally arranged in a 6-6-6-5 fashion. Steroids vary by the functional groups attached to these rings and the oxidation state of the rings. Steroids include hormones and drugs, which are usually used to relieve swelling and inflammation, such as for instance prednisone, dexamethasone and vitamin D.
According to the present invention, supplemental effects of adjunct steroid therapy in DMD patients include reduction of tissue inflammation, suppression of cytotoxic cells, and improved calcium homeostasis. Most positive results are obtained in younger boys. Preferably, the steroid is a corticosteroid, more preferably, a glucocorticosteroid. Preferably, prednisone steroids such as prednisone, prednizolone or deflazacort are used in a combination according to the invention21. Dose ranges of steroid or of a glucocorticosteroid to be used in the therapeutic applications as described herein are designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist. The usual doses are 0.5-1.0 mg/kg/day, preferably 0.75 mg/kg/day for prednisone and prednisolone, and 0.4-1.4 mg/kg/day, preferably 0.9 mg/kg/day for deflazacort.
In one embodiment, a steroid is administered to said individual prior to administering a composition as earlier defined herein. In this embodiment, it is preferred that said steroid is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering said composition.
In another preferred embodiment, a combination further comprises a tumour necrosis factor-alpha (TNFα) inhibitor. Tumour necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that stimulates the inflammatory response. Pharmacological blockade of TNFα activity with the neutralising antibody infliximab (Remicade) is highly effective clinically at reducing symptoms of inflammatory diseases. In mdx mice, both infliximab and etanercept delay and reduce the necrosis of dystrophic muscle24, 25, with additional physiological benefits on muscle strength, chloride channel function and reduced CK levels being demonstrated in chronically treated exercised adult mdx mice26. Such highly specific anti-inflammatory drugs designed for use in other clinical conditions, are attractive alternatives to the use of steroids for DMD. In one embodiment, the use of a TNFα inhibitor is limited to periods of intensive muscle growth in boys when muscle damage and deterioration are especially pronounced.
A composition further comprising a TNFα inhibitor for use as a medicament is also provided. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. A preferred TNFα inhibitor is a dimeric fusion protein consisting of the extracellular ligand-binding domain of the human p75 receptor of TNFα linked to the Fc portion of human IgG1. A more preferred TNFα inhibitor is ethanercept (Amgen, America)26. The usual doses of ethanercept is about 0.2 mg/kg, preferably about 0.5 mg/kg twice a week. The administration is preferably subcutaneous.
In another preferred embodiment, a composition of the invention further comprises a source of mIGF-1. As defined herein, a source of IGF-1 preferably encompasses mIGF-1 itself, a compound able of enhancing mIGF-1 expression and/or activity. Enhancing is herein synonymous with increasing. Expression of mIGF-1 is synonymous with amount of mIGF-1. mIGF-1 promotes regeneration of muscles through increase in satellite cell activity, and reduces inflammation and fibrosis27. Local injury of muscle results in increased mIGF-1 expression. In transgenic mice with extra IGF-1 genes, muscle hypertrophy and enlarged muscle fibers are observed27. Similarly, transgenic mdx mice show reduced muscle fiber degeneration28. Upregulation of the mIGF-1 gene and/or administration of extra amounts of mIGF-1 protein or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]) thus promotes the effect of other, preferably genetic, therapies for DMD, including antisense-induced exon skipping. The additional mIGF-1 levels in the above mentioned transgenic mice do not induce cardiac problems nor promote cancer, and have no pathological side effects. As stated before, the amount of mIGF-1 is for instance increased by enhancing expression of the mIGF-1 gene and/or by administration of mIGF-1 protein and/or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]). A composition of the invention further preferably comprises mIGF-1, a compound capable of enhancing mIGF-1 expression and/or an mIGF-1 activity, for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, such composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Within the context of the invention, an increased amount or activity of mIGF-1 may be reached by increasing the gene expression level of an IGF-1 gene, by increasing the amount of a corresponding IGF-1 protein and/or by increasing an activity of an IGF1-protein. A preferred mIGF-1 protein has been earlier defined herein. An increase of an activity of said protein is herein understood to mean any detectable change in a biological activity exerted by said protein or in the steady state level of said protein as compared to said activity or steady-state in a individual who has not been treated. Increased amount or activity of mIGF-1 is preferably assessed by detection of increased expression of muscle hypertrophy biomarker GATA-2 (as described in 27).
Gene expression level is preferably assessed using classical molecular biology techniques such as (real time) PCR, arrays or Northern analysis. A steady state level of a protein is determined directly by quantifying the amount of a protein. Quantifying a protein amount may be carried out by any known technique such as Western blotting or immunoassay using an antibody raised against a protein. The skilled person will understand that alternatively or in combination with the quantification of a gene expression level and/or a corresponding protein, the quantification of a substrate of a corresponding protein or of any compound known to be associated with a function or activity of a corresponding protein or the quantification of said function or activity of a corresponding protein using a specific assay may be used to assess the alteration of an activity or steady state level of a protein.
In the invention, an activity or steady-state level of a said protein may be altered at the level of the protein itself, e.g. by providing a protein to a cell from an exogenous source.
Preferably, an increase or an up-regulation of the expression level of a said gene means an increase of at least 5% of the expression level of said gene using arrays. More preferably, an increase of the expression level of said gene means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. In another preferred embodiment, an increase of the expression level of said protein means an increase of at least 5% of the expression level of said protein using Western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a protein means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.
In another preferred embodiment, an increase of a polypeptide activity means an increase of at least 5% of a polypeptide activity using a suitable assay. More preferably, an increase of a polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. The increase is preferably assessed by comparison to corresponding activity in the individual before treatment.
A preferred way of providing a source of mIGF1 is to introduce a transgene encoding mIGF1, preferably an mIGF-1 Ea isoform (as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577), more preferably in an AAV vector as later defined herein. Such source of mIGF1 is specifically expressed in muscle tissue as described in mice in 27.
In another preferred embodiment, a composition further comprises an antioxidant. Oxidative stress is an important factor in the progression of DMD and promotes chronic inflammation and fibrosis29. The most prevalent products of oxidative stress, the peroxidized lipids, are increased by an average of 35% in Duchenne boys. Increased levels of the enzymes superoxide dismutase and catalase reduce the excessive amount of free radicals causing these effects. In fact, a dietary supplement Protandim® (LifeVantage) was clinically tested and found to increase levels of superoxide dismutase (up to 30%) and catalase (up to 54%), which indeed significantly inhibited the peroxidation of lipids in 29 healthy persons30. Such effective management of oxidative stress thus preserves muscle quality and so promotes the positive effect of DMD therapy. Idebenone is another potent antioxidant with a chemical structure derived from natural coenzyme Q10. It protects mitochondria where adenosine triphosphate, ATP, is generated by oxidative phosphorylation. The absence of dystrophin in DMD negatively affects this process in the heart, and probably also in skeletal muscle. Idebenone was recently applied in clinical trials in the US and Europe demonstrating efficacy on neurological aspects of Friedreich's Ataxia31. A phase-IIa double-blind, placebo-controlled randomized clinical trial with Idebenone has recently been started in Belgium, including 21 Duchenne boys at 8 to 16 years of age. The primary objective of this study is to determine the effect of Idebenone on heart muscle function. In addition, several different tests will be performed to detect the possible functional benefit on muscle strength in the patients. When effective, Idebenone is a preferred adjunct compound for use in a combination according to the present invention in order to enhance the therapeutic effect of DMD therapy, especially in the heart. A composition further comprising an antioxidant for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the antioxidant, the skilled person will know which quantities are preferably used. An antioxidant may include bacoside, silymarin, curcumin and/or a polyphenol. Preferably, a polyphenol is or comprises epigallocatechin-3-gallate (EGCG). Preferably, an antioxidant is a mixture of antioxidants as the dietary supplement Protandim® (LifeVantage). A daily capsule of 675 mg of Protandim® comprises 150 mg of B. monniera (45% bacosides), 225 mg of S. marianum (70-80% silymarin), 150 mg of W. somnifera powder, 75 mg green tea (98% polyphenols wherein 45% EGCG) and 75 mg turmeric (95% curcumin).
In another preferred embodiment, a composition further comprises an ion channel inhibitor. The presence of damaged muscle membranes in DMD disturbs the passage of calcium ions into the myofibers, and the consequently disrupted calcium homeostasis activates many enzymes, e.g. proteases, that cause additional damage and muscle necrosis. Ion channels that directly contribute to the pathological accumulation of calcium in dystrophic muscle are potential targets for adjunct compounds to treat DMD. There is evidence that some drugs, such as pentoxifylline, block exercise-sensitive calcium channels32 and antibiotics that block stretch activated channels reduce myofibre necrosis in mdx mice and CK levels in DMD boys33. A composition further comprising an ion channel inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Preferably, an ion channel inhibitor of the class of xanthines is used.
More preferably, said xanthines are derivatives of methylxanthines, and most preferably, said methylxanthine derivates are chosen from the group consisting of pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof. Most preferred is the use of pentoxifylline. Ion channel inhibitors of the class of xanthines enhance the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
Depending on the identity of the ion channel inhibitor, the skilled person will know which quantities are preferably used. Suitable dosages of pentoxifylline are between 1 mg/kg/day to 100 mg/kg/day, preferred dosages are between 10 mg/kg/day to 50 mg/kg/day. Typical dosages used in humans are 20 mg/kg/day.
In one embodiment, an ion channel inhibitor is administered to said individual prior to administering a composition comprising an oligonucleotide. In this embodiment, it is preferred that said ion channel inhibitor is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering a composition comprising an oligonucleotide.
In another preferred embodiment, a composition further comprises a protease inhibitor. Calpains are calcium-activated proteases that are increased in dystrophic muscle and account for myofiber degeneration. Calpain inhibitors such as calpastatin, leupeptin34, calpeptin, calpain inhibitor III, or PD150606 are therefore applied to reduce the degeneration process. A new compound, BN 82270 (Ipsen) that has dual action as both a calpain inhibitor and an antioxidant increased muscle strength, decreased serum CK and reduced fibrosis of the mdx diaphragm, indicating a therapeutic effect with this new compound35. Another compound of Leupeptin/Carnitine (Myodur) has recently been proposed for clinical trials in DMD patients.
MG132 is another proteasomal inhibitor that has shown to reduce muscle membrane damage, and to ameliorate the histopathological signs of muscular dystrophy36. MG-132 (CBZ-leucyl-leucyl-leucinal) is a cell-permeable, proteasomal inhibitor (Ki=4 nM), which inhibits NFkappaB activation by preventing IkappaB degradation (IC50=3 μM). In addition, it is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis by binding to and inactivating 20S and 26S proteasomes. MG-132 has shown to inhibit the proteasomal degradation of dystrophin-associated proteins in the dystrophic mdx mouse model36. This compound is thus also suitable for use as an adjunct pharmacological compound for DMD. A composition further comprising a protease inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the protease inhibitor, the skilled person will know which quantities are preferably used.
In another preferred embodiment, a composition further comprises L-arginine. Dystrophin-deficiency is associated with the loss of the DGC-complex at the fiber membranes, including neuronal nitric oxide synthase (nNOS). Expression of a nNOS transgene in mdx mice greatly reduced muscle membrane damage. Similarly, administration of L-arginine (the substrate for nitric oxide synthase) increased NO production and upregulated utrophin expression in mdx mice. Six weeks of L-arginine treatment improved muscle pathology and decreased serum CK in mdx mice37. The use of L-arginine as a further constituent in a composition of the invention has not been disclosed.
A composition further comprising L-arginine for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
In another preferred embodiment, a composition further comprises angiotensin II type 1 receptor blocker Losartan, which normalizes muscle architecture, repair and function, as shown in the dystrophin-deficient mdx mouse model23. A composition further comprising angiotensin II type 1 receptor blocker Losartan for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the angiotensin II type 1 receptor blocker, the skilled person will know which quantities are preferably used.
In another preferred embodiment, a composition further comprises an angiotensin-converting enzyme (ACE) inhibitor, preferably perindopril. ACE inhibitors are capable of lowering blood pressure. Early initiation of treatment with perindopril is associated with a lower mortality in DMD patients22. A composition further comprising an ACE inhibitor, preferably perindopril for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of an ACE inhibitor, preferably perindopril are about 2 to 4 mg/day22.
In a more preferred embodiment, an ACE inhibitor is combined with at least one of the previously identified adjunct compounds.
In another preferred embodiment, a composition further comprises a compound exhibiting a readthrough activity. A compound exhibiting a readthrough activity may be any compound, which is able to suppress a stop codon. For 20% of DMD patients, the mutation in the dystrophin gene is comprising a point mutation, of which 13% is a nonsense mutation. A compound exhibiting a readthrough activity or which is able to suppress a stop codon is a compound which is able to provide an increased amount of a functional dystrophin mRNA or protein and/or a decreased amount of an aberrant or truncated dystrophin mRNA or protein. Increased preferably means increased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. Decreased preferably means decreased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. An increase or a decrease of said protein is preferably assessed in a muscular tissue or in a muscular cell of an individual by comparison to the amount present in said individual before treatment with said compound exhibiting a readthrough activity.
Alternatively, the comparison can be made with a muscular tissue or cell of said individual, which has not yet been treated with said compound in case the treatment is local. The assessment of an amount at the protein level is preferably carried out using western blot analysis.
Preferred compounds exhibiting a readthrough activity comprise or consist of aminoglycosides, including, but not limited to, geneticin (G418), paromomycin, gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), and derivatives thereof (references 64, 65). A more preferred compound exhibiting a readthrough activity comprises or consists of PTC124™, and/or a functional equivalent thereof. PTC124™ is a registered trademark of PTC Therapeutics, Inc. South Plainfield, N.J. 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124™ (references 16, 17) belongs to a new class of small molecules that mimics at lower concentrations the readthrough activity of gentamicin (reference 55). A functional equivalent of 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin is a compound which is able to exhibit a readthrough activity as earlier defined herein. Most preferably, a compound exhibiting a readthrough activity comprises or consists of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124™. A composition further comprising a compound exhibiting a readthrough activity, preferably comprising or consisting of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of a compound exhibiting a readthrough activity, preferably 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin are ranged between 3 mg/kg/day to 200 mg/kg/day, preferred dosages are between 10 mg/kg to 50 mg/kg per day or twice a day.
In a more preferred embodiment, a compound exhibiting a readthrough activity is combined with at least one of the previously identified adjunct compounds.
In another preferred embodiment, a composition further comprises a compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing. Small chemical compounds, such as for instance specific indole derivatives, have been shown to selectively inhibit spliceosome assembly and splicing38, for instance by interfering with the binding of serine- and arginine-rich (SR) proteins to their cognate splicing enhancers (ISEs or ESEs) and/or by interfering with the binding of splicing repressors to silencer sequences (ESSs or ISSs). These compounds are therefore suitable for applying as adjunct compounds that enhance exon skipping. A composition further comprising a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing, the skilled person will know which quantities are preferably used. In a more preferred embodiment, a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing is combined with a ACE inhibitor and/or with any adjunct compounds as identified earlier herein.
The invention thus provides a composition further comprising an adjunct compound, wherein said adjunct compound comprises a steroid, an ACE inhibitor (preferably perindopril), angiotensin II type 1 receptor blocker Losartan, a tumour necrosis factor-alpha (TNFα) inhibitor, a source of mIGF-1, preferably mIGF-1, a compound for enhancing mIGF-1 expression, a compound for enhancing mIGF-1 activity, an antioxidant, an ion channel inhibitor, a protease inhibitor, L-arginine, a compound exhibiting a readthrough activity and/or inhibiting spliceosome assembly and/or splicing.
In one embodiment an individual is further provided with a functional dystrophin protein using a vector, preferably a viral vector, comprising a micro-mini-dystrophin gene. Most preferably, a recombinant adeno-associated viral (rAAV) vector is used. AAV is a single-stranded DNA parvovirus that is non-pathogenic and shows a helper-dependent life cycle. In contrast to other viruses (adenovirus, retrovirus, and herpes simplex virus), rAAV vectors have demonstrated to be very efficient in transducing mature skeletal muscle. Application of rAAV in classical DMD “gene addition” studies has been hindered by its restricted packaging limits (<5 kb). Therefore, rAAV is preferably applied for the efficient delivery of a much smaller micro- or mini-dystrophin gene. Administration of such micro- or mini-dystrophin gene results in the presence of an at least partially functional dystrophin protein. Reference is made to18-20.
Each constituent of a composition can be administered to an individual in any order. In one embodiment, each constituent is administered simultaneously (meaning that each constituent is administered within 10 hours, preferably within one hour). This is however not necessary. In one embodiment at least one adjunct compound is administered to an individual in need thereof before administration of an oligonucleotide. Alternatively, an oligonucleotide is administered to an individual in need thereof before administration of at least one adjunct compound.
Use
In a further aspect, there is provided the use of a oligoucleotide or of a composition as defined herein for the manufacture of a medicament for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual. Each feature of said use has earlier been defined herein.
A treatment in a use or in a method according to the invention is at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an oligonucleotide, composition, compound or adjunct compound of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (i.e. dose), the formulation of said molecule. The frequency may be ranged between at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.
Method
In a further aspect, there is provided a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or alleviate one or more characteristic(s) of a myogenic or muscle cell of said individual, the method comprising administering to said individual an oligonucleotide or a composition as defined herein.
There is further provided a method for enhancing, inducing or promoting skipping of an exon from a dystrophin pre-mRNA in a cell expressing said pre-mRNA in an individual suffering from Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the method comprising administering to said individual an oligonucleotide or a composition as defined herein. Further provided is a method for increasing the production of a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in a cell, said cell comprising a pre-mRNA of a dystrophin gene encoding an aberrant dystrophin protein, the method comprising providing said cell with an oligonucleotide or composition of the invention and allowing translation of mRNA produced from splicing of said pre-mRNA. In one embodiment, said method is performed in vitro, for instance using a cell culture. Preferably, said method is in vivo.
In this context, increasing the production of a functional dystrophin protein has been earlier defined herein.
Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Each embodiment as identified herein may be combined together unless otherwise indicated.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. In human control myotubes, PS220 and PS305 both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies. PS220 reproducibly induced highest levels of exon 45 skipping (up to 73%), whereas with PS305 maximum exon 45 skipping levels of up to 46% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells)
FIG. 2. Graph showing relative exon 45 skipping levels of inosine-containing AONs as assessed by RT-PCR analysis. In human control myotubes, a series of new AONs, all targeting exon 45 and containing one inosine for guanosine substitution were tested for relative exon 45 skipping efficiencies when compared with PS220 and PS305 (see FIG. 1). All new inosine-containing AONs were effective, albeit at variable levels (between 4% and 25%). PS220 induced highest levels of exon 45 skipping (up to 72%), whereas with PS305 maximum exon 45 skipping levels of up to 63% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells).
EXAMPLES
Example 1
Materials and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200 nM for each AON (PS220 and PS305). Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per μg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
DMD exon 45 skipping.
Two AONs, PS220 (SEQ ID NO: 76; 5′-UUUGCCGCUGCCCAAUGCCAUCCUG-3′) and PS305 (SEQ ID NO: 557; 5′-UUUGCCICUGCCCAAUGCCAUCCUG-3′) both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies in healthy control myotube cultures.
Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that both AONs were indeed capable of inducing exon 45 skipping. PS220, consisting a GCCGC stretch, reproducibly induced highest levels of exon 45 skipping (up to 73%), as shown in FIG. 1. However, PS305, which is identical to PS220 but containing an inosine for a G substitution at position 4 within that stretch is also effective and leading to exon 45 skipping levels of up to 46%. No exon 45 skipping was observed in non-treated cells (NT).
Example 2
Materials and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon 45 RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA, full-length phosphorothioate (PS) backbones and one inosine for guanosine substitution.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200 nM for each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per μg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
DMD exon 45 skipping.
An additional series of AONs targeting exon 45 and containing one inosine-substitution were tested in healthy control myotube cultures for exon 45 skipping efficiencies, and directly compared to PS220 (without inosine; SEQ ID NO: 76)) and PS305 (identical sequence as PS220 but with inosine substitution; SEQ ID NO: 557). Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that all new AONs (PS309 to PS316) were capable of inducing exon 45 skipping between 4% (PS311) and 25% (PS310) as shown in FIG. 2. When compared to PS220 and PS305, PS220 induced highest levels of exon 45 skipping (up to 72%). Of the new inosine-containing AONs PS305 was most effective, showing exon 45 skipping levels of up to 63%. No exon 45 skipping was observed in non-treated cells (NT).
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56. Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144.
57. Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.
58. Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.
59.Yokota T. et al., Mar. 13, 2009, e-publication: Efficacy of systemic morpholino exon-skipping in duchennes dystrophy dogs. Ann. Neurol. 2009
60. Dorn and Kippenberger, Curr Opin Mol Ther 2008 10(1) 10-20
61. Cheng and Van Dyke, Gene. 1997 Sep. 15; 197(1-2):253-60
62. Macaya et al., Biochemistry. 1995 4; 34(13):4478-92.
63. Suzuki et al., Eur J Biochem. 1999, 260(3):855-6
64. Howard et al., Ann Neurol 2004 55(3): 422-6;
65. Nudelman et al., 2006, Bioorg Med Chem Lett 16(24), 6310-5
Sequence Listing
DMD Gene Amino Acid Sequence
SEQ ID NO 1:
MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRL
LDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIV
DGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRN
YPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAF
NIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQE
VEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYA
YTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEE
VLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNIL
QLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLM
DLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDL
EQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDRWANICRWTEDR
WVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSL
QKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARC
WDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILVKHAQ
EELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIF
RKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADS
IKQASEQLNSRWIEFCQLLSERLNWLEYQNNHAFYNQLQQLEQMTTTAEN
WLKIQPTTPSEPTAIKSQLKICKDEVNRLSGLQPQIERLKIQSIALKEKG
QGPMFLDADFVAFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAI
RTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLST
TVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLR
KIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTI
QPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEA
LKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMK
RAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQWLC
TRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEE
ISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRW
RELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYIADKV
DAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKL
QDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQEVVQSQ
LNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLH
YNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVE
GMPSNLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVED
KLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTL
LDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRK
LVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEI
QQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQ
QLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLA
SLPEPRDERKIKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQ
LSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYL
TEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGR
IDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDR
SVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDG
IGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSD
RKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLE
QVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQ
WIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQL
EIYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKR
KLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVVT
KETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVG
DLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIIT
DRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARA
KLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDY
SADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKF
LAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDV
YHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLE
ASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAF
KRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVT
RLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLR
QAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQ
LTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH
FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNV
RFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQII
NCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKT
GIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEV
ASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV
AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHK
MHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVL
EGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSN
GSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESE
ERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQ
SPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQ
AEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQD
TSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM
DMD Gene Exon 51
SEQ ID
GUACCUCCAACAUCAAGGAAGAUGG
SEQ ID
GAGAUGGCAGUUUCCUUAGUAACCA
NO 2
NO 39
SEQ ID
UACCUCCAACAUCAAGGAAGAUGGC
SEQ ID
AGAUGGCAGUUUCCUUAGUAACCAC
NO 3
NO 40
SEQ ID
ACCUCCAACAUCAAGGAAGAUGGCA
SEQ ID
GAUGGCAGUUUCCUUAGUAACCACA
NO 4
NO 41
SEQ ID
CCUCCAACAUCAAGGAAGAUGGCAU
SEQ ID
AUGGCAGUUUCCUUAGUAACCACAG
NO 5
NO 42
SEQ ID
CUCCAACAUCAAGGAAGAUGGCAUU
SEQ ID
UGGCAGUUUCCUUAGUAACCACAGG
NO 6
NO 43
SEQ ID
UCCAACAUCAAGGAAGAUGGCAUUU
SEQ ID
GGCAGUUUCCUUAGUAACCACAGGU
NO 7
NO 44
SEQ ID
CCAACAUCAAGGAAGAUGGCAUUUC
SEQ ID
GCAGUUUCCUUAGUAACCACAGGUU
NO 8
NO 45
SEQ ID
CAACAUCAAGGAAGAUGGCAUUUCU
SEQ ID
CAGUUUCCUUAGUAACCACAGGUUG
NO 9
NO 46
SEQ ID
AACAUCAAGGAAGAUGGCAUUUCUA
SEQ ID
AGUUUCCUUAGUAACCACAGGUUGU
NO 10
NO 47
SEQ ID
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID
GUUUCCUUAGUAACCACAGGUUGUG
NO 11
NO 48
SEQ ID
CAUCAAGGAAGAUGGCAUUUCUAGU
SEQ ID
UUUCCUUAGUAACCACAGGUUGUGU
NO 12
NO 49
SEQ ID
AUCAAGGAAGAUGGCAUUUCUAGUU
SEQ ID
UUCCUUAGUAACCACAGGUUGUGUC
NO 13
NO 50
SEQ ID
UCAAGGAAGAUGGCAUUUCUAGUUU
SEQ ID
UCCUUAGUAACCACAGGUUGUGUCA
NO 14
NO 51
SEQ ID
CAAGGAAGAUGGCAUUUCUAGUUUG
SEQ ID
CCUUAGUAACCACAGGUUGUGUCAC
NO 15
NO 52
SEQ ID
AAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID
CUUAGUAACCACAGGUUGUGUCACC
NO 16
NO 53
SEQ ID
AGGAAGAUGGCAUUUCUAGUUUGGA
SEQ ID
UUAGUAACCACAGGUUGUGUCACCA
NO 17
NO 54
SEQ ID
GGAAGAUGGCAUUUCUAGUUUGGAG
SEQ ID
UAGUAACCACAGGUUGUGUCACCAG
NO 18
NO 55
SEQ ID
GAAGAUGGCAUUUCUAGUUUGGAGA
SEQ ID
AGUAACCACAGGUUGUGUCACCAGA
NO 19
NO 56
SEQ ID
AAGAUGGCAUUUCUAGUUUGGAGAU
SEQ ID
GUAACCACAGGUUGUGUCACCAGAG
NO 20
NO 57
SEQ ID
AGAUGGCAUUUCUAGUUUGGAGAUG
SEQ ID
UAACCACAGGUUGUGUCACCAGAGU
NO 21
NO 58
SEQ ID
GAUGGCAUUUCUAGUUUGGAGAUGG
SEQ ID
AACCACAGGUUGUGUCACCAGAGUA
NO 22
NO 59
SEQ ID
AUGGCAUUUCUAGUUUGGAGAUGGC
SEQ ID
ACCACAGGUUGUGUCACCAGAGUAA
NO 23
NO 60
SEQ ID
UGGCAUUUCUAGUUUGGAGAUGGCA
SEQ ID
CCACAGGUUGUGUCACCAGAGUAAC
NO 24
NO 61
SEQ ID
GGCAUUUCUAGUUUGGAGAUGGCAG
SEQ ID
CACAGGUUGUGUCACCAGAGUAACA
NO 25
NO 62
SEQ ID
GCAUUUCUAGUUUGGAGAUGGCAGU
SEQ ID
ACAGGUUGUGUCACCAGAGUAACAG
NO 26
NO 63
SEQ ID
CAUUUCUAGUUUGGAGAUGGCAGUU
SEQ ID
CAGGUUGUGUCACCAGAGUAACAGU
NO 27
NO 64
SEQ ID
AUUUCUAGUUUGGAGAUGGCAGUUU
SEQ ID
AGGUUGUGUCACCAGAGUAACAGUC
NO 28
NO 65
SEQ ID
UUUCUAGUUUGGAGAUGGCAGUUUC
SEQ ID
GGUUGUGUCACCAGAGUAACAGUCU
NO 29
NO 66
SEQ ID
UUCUAGUUUGGAGAUGGCAGUUUCC
SEQ ID
GUUGUGUCACCAGAGUAACAGUCUG
NO 30
NO 67
SEQ ID
UCUAGUUUGGAGAUGGCAGUUUCCU
SEQ ID
UUGUGUCACCAGAGUAACAGUCUGA
NO 31
NO 68
SEQ ID
CUAGUUUGGAGAUGGCAGUUUCCUU
SEQ ID
UGUGUCACCAGAGUAACAGUCUGAG
NO 32
NO 69
SEQ ID
UAGUUUGGAGAUGGCAGUUUCCUUA
SEQ ID
GUGUCACCAGAGUAACAGUCUGAGU
NO 33
NO 70
SEQ ID
AGUUUGGAGAUGGCAGUUUCCUUAG
SEQ ID
UGUCACCAGAGUAACAGUCUGAGUA
NO 34
NO 71
SEQ ID
GUUUGGAGAUGGCAGUUUCCUUAGU
SEQ ID
GUCACCAGAGUAACAGUCUGAGUAG
NO 35
NO 72
SEQ ID
UUUGGAGAUGGCAGUUUCCUUAGUA
SEQ ID
UCACCAGAGUAACAGUCUGAGUAGG
NO 36
NO 73
SEQ ID
UUGGAGAUGGCAGUUUCCUUAGUAA
SEQ ID
CACCAGAGUAACAGUCUGAGUAGGA
NO 37
NO 74
SEQ ID
UGGAGAUGGCAGUUUCCUUAGUAAC
SEQ ID
ACCAGAGUAACAGUCUGAGUAGGAG
NO 38
NO 75
SEQ ID
UCAAGGAAGAUGGCAUUUCU
SEQ ID
UCAAGGAAGAUGGCAUIUCU
NO 539
NO 548
SEQ ID
UCAAIGAAGAUGGCAUUUCU
SEQ ID
UCAAGGAAGAUGGCAUUICU
NO 540
NO 549
SEQ ID
UCAAGIAAGAUGGCAUUUCU
SEQ ID
UCAAGGAAGAUGGCAUUUCI
NO 541
NO 550
SEQ ID
UCAAGGAAIAUGGCAUUUCU
SEQ ID
UCIAGGAAGAUGGCAUUUCU
NO 542
NO 551
SEQ ID
UCAAGGAAGAUIGCAUUUCU
SEQ ID
UCAIGGAAGAUGGCAUUUCU
NO 543
NO 552
SEQ ID
UCAAGGAAGAUGICAUUUCU
SEQ ID
UCAAGGIAGAUGGCAUUUCU
NO 544
NO 553
SEQ ID
ICAAGGAAGAUGGCAUUUCU
SEQ ID
UCAAGGAIGAUGGCAUUUCU
NO 545
NO 554
SEQ ID
UCAAGGAAGAIGGCAUUUCU
SEQ ID
UCAAGGAAGIUGGCAUUUCU
NO 546
NO 555
SEQ ID
UCAAGGAAGAUGGCAIUUCU
SEQ ID
UCAAGGAAGAUGGCIUUUCU
NO 547
NO 556
DMD Gene Exon 45
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID
GUUGCAUUCAAUGUUCUGACAACAG
NO 76
NO 109
PS220
SEQ ID
AUUCAAUGUUCUGACAACAGUUUGC
SEQ ID
UUGCAUUCAAUGUUCUGACAACAGU
NO 77
NO 110
SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID
UGCAUUCAAUGUUCUGACAACAGUU
NO 78
NO 111
SEQ ID
CAGUUGCAUUCAAUGUUCUGAC
SEQ ID
GCAUUCAAUGUUCUGACAACAGUUU
NO 79
NO 112
SEQ ID
AGUUGCAUUCAAUGUUCUGA
SEQ ID
CAUUCAAUGUUCUGACAACAGUUUG
NO 80
NO 113
SEQ ID
GAUUGCUGAAUUAUUUCUUCC
SEQ ID
AUUCAAUGUUCUGACAACAGUUUGC
NO 81
NO 114
SEQ ID
GAUUGCUGAAUUAUUUCUUCCCCAG
SEQ ID
UCAAUGUUCUGACAACAGUUUGCCG
NO 82
NO 115
SEQ ID
AUUGCUGAAUUAUUUCUUCCCCAGU
SEQ ID
CAAUGUUCUGACAACAGUUUGCCGC
NO 83
NO 116
SEQ ID
UUGCUGAAUUAUUUCUUCCCCAGUU
SEQ ID
AAUGUUCUGACAACAGUUUGCCGCU
NO 84
NO 117
SEQ ID
UGCUGAAUUAUUUCUUCCCCAGUUG
SEQ ID
AUGUUCUGACAACAGUUUGCCGCUG
NO 85
NO 118
SEQ ID
GCUGAAUUAUUUCUUCCCCAGUUGC
SEQ ID
UGUUCUGACAACAGUUUGCCGCUGC
NO 86
NO 119
SEQ ID
CUGAAUUAUUUCUUCCCCAGUUGCA
SEQ ID
GUUCUGACAACAGUUUGCCGCUGCC
NO 87
NO 120
SEQ ID
UGAAUUAUUUCUUCCCCAGUUGCAU
SEQ ID
UUCUGACAACAGUUUGCCGCUGCCC
NO 88
NO 121
SEQ ID
GAAUUAUUUCUUCCCCAGUUGCAUU
SEQ ID
UCUGACAACAGUUUGCCGCUGCCCA
NO 89
NO 122
SEQ ID
AAUUAUUUCUUCCCCAGUUGCAUUC
SEQ ID
CUGACAACAGUUUGCCGCUGCCCAA
NO 90
NO 123
SEQ ID
AUUAUUUCUUCCCCAGUUGCAUUCA
SEQ ID
UGACAACAGUUUGCCGCUGCCCAAU
NO 91
NO 124
SEQ ID
UUAUUUCUUCCCCAGUUGCAUUCAA
SEQ ID
GACAACAGUUUGCCGCUGCCCAAUG
NO 92
NO 125
SEQ ID
UAUUUCUUCCCCAGUUGCAUUCAAU
SEQ ID
ACAACAGUUUGCCGCUGCCCAAUGC
NO 93
NO 126
SEQ ID
AUUUCUUCCCCAGUUGCAUUCAAUG
SEQ ID
CAACAGUUUGCCGCUGCCCAAUGCC
NO 94
NO 127
SEQ ID
UUUCUUCCCCAGUUGCAUUCAAUGU
SEQ ID
AACAGUUUGCCGCUGCCCAAUGCCA
NO 95
NO 128
SEQ ID
UUCUUCCCCAGUUGCAUUCAAUGUU
SEQ ID
ACAGUUUGCCGCUGCCCAAUGCCAU
NO 96
NO 129
SEQ ID
UCUUCCCCAGUUGCAUUCAAUGUUC
SEQ ID
CAGUUUGCCGCUGCCCAAUGCCAUC
NO 97
NO 130
SEQ ID
CUUCCCCAGUUGCAUUCAAUGUUCU
SEQ ID
AGUUUGCCGCUGCCCAAUGCCAUCC
NO 98
NO 131
SEQ ID
UUCCCCAGUUGCAUUCAAUGUUCUG
SEQ ID
GUUUGCCGCUGCCCAAUGCCAUCCU
NO 99
NO 132
SEQ ID
UCCCCAGUUGCAUUCAAUGUUCUGA
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG
NO 100
NO 133
SEQ ID
CCCCAGUUGCAUUCAAUGUUCUGAC
SEQ ID
UUGCCGCUGCCCAAUGCCAUCCUGG
NO 101
NO 134
SEQ ID
CCCAGUUGCAUUCAAUGUUCUGACA
SEQ ID
UGCCGCUGCCCAAUGCCAUCCUGGA
NO 102
NO 135
SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID
GCCGCUGCCCAAUGCCAUCCUGGAG
NO 103
NO 136
SEQ ID
CAGUUGCAUUCAAUGUUCUGACAAC
SEQ ID
CCGCUGCCCAAUGCCAUCCUGGAGU
NO 104
NO 137
SEQ ID
AGUUGCAUUCAAUGUUCUGACAACA
SEQ ID
CGCUGCCCAAUGCCAUCCUGGAGUU
NO 105
NO 138
SEQ ID
UCC UGU AGA AUA CUG GCA UC
SEQ ID
UGUUUUUGAGGAUUGCUGAA
NO 106
NO 139
SEQ ID
UGCAGACCUCCUGCCACCGCAGAUU
SEQ ID
UGUUCUGACAACAGUUUGCCGCUGCC
NO 107
CA
NO 140
CAAUGCCAUCCUGG
SEQ ID
UUGCAGACCUCCUGCCACCGCAGAU
SEQ ID
UUUGCCICUGCCCAAUGCCAUCCUG
NO 108
UCAGGCUUC
NO 557
PS305
SEQ ID
UUUGCCGCUICCCAAUGCCAUCCUG
SEQ ID
UUUGCCGCUGCCCAIUGCCAUCCUG
NO 558
NO 566
SEQ ID
UUUGCCGCUGCCCAAUICCAUCCUG
SEQ ID
UUUGCCGCUGCCCAAUGCCIUCCUG
NO 559
NO 567
SEQ ID
UUUICCGCUGCCCAAUGCCAUCCUG
SEQ ID
UUUICCICUGCCCAAUGCCAUCCUG
NO 560
NO 568
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUI
SEQ ID
UUUGCCGCUGCCCAAIGCCAUCCUG
NO 561
NO 569
SEQ ID
IUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID
UUUGCCGCUGCCCAAUGCCAICCUG
NO 562
NO 570
SEQ ID
UIUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCIG
NO 563
NO 571
SEQ ID
UUIGCCGCUGCCCAAUGCCAUCCUG
SEQ ID
UUUGCCGCUGCCCIAUGCCAUCCUG
NO 564
NO 572
SEQ ID
UUUGCCGCIGCCCAAUGCCAUCCUG
NO 565
DMD Gene Exon 53
SEQ ID
CUCUGGCCUGUCCUAAGACCUGCUC
SEQ ID
CAGCUUCUUCCUUAGCUUCCAGCCA
NO 141
NO 165
SEQ ID
UCUGGCCUGUCCUAAGACCUGCUCA
SEQ ID
AGCUUCUUCCUUAGCUUCCAGCCAU
NO 142
NO 166
SEQ ID
CUGGCCUGUCCUAAGACCUGCUCAG
SEQ ID
GCUUCUUCCUUAGCUUCCAGCCAUU
NO 143
NO 167
SEQ ID
UGGCCUGUCCUAAGACCUGCUCAGC
SEQ ID
CUUCUUCCUUAGCUUCCAGCCAUUG
NO 144
NO 168
SEQ ID
GGCCUGUCCUAAGACCUGCUCAGCU
SEQ ID
UUCUUCCUUAGCUUCCAGCCAUUGU
NO 145
NO 169
SEQ ID
GCCUGUCCUAAGACCUGCUCAGCUU
SEQ ID
UCUUCCUUAGCUUCCAGCCAUUGUG
NO 146
NO 170
SEQ ID
CCUGUCCUAAGACCUGCUCAGCUUC
SEQ ID
CUUCCUUAGCUUCCAGCCAUUGUGU
NO 147
NO 171
SEQ ID
CUGUCCUAAGACCUGCUCAGCUUCU
SEQ ID
UUCCUUAGCUUCCAGCCAUUGUGUU
NO 148
NO 172
SEQ ID
UGUCCUAAGACCUGCUCAGCUUCUU
SEQ ID
UCCUUAGCUUCCAGCCAUUGUGUUG
NO 149
NO 173
SEQ ID
GUCCUAAGACCUGCUCAGCUUCUUC
SEQ ID
CCUUAGCUUCCAGCCAUUGUGUUGA
NO 150
NO 174
SEQ ID
UCCUAAGACCUGCUCAGCUUCUUCC
SEQ ID
CUUAGCUUCCAGCCAUUGUGUUGAA
NO 151
NO 175
SEQ ID
CCUAAGACCUGCUCAGCUUCUUCCU
SEQ ID
UUAGCUUCCAGCCAUUGUGUUGAAU
NO 152
NO 176
SEQ ID
CUAAGACCUGCUCAGCUUCUUCCUU
SEQ ID
UAGCUUCCAGCCAUUGUGUUGAAUC
NO 153
NO 177
SEQ ID
UAAGACCUGCUCAGCUUCUUCCUUA
SEQ ID
AGCUUCCAGCCAUUGUGUUGAAUCC
NO 154
NO 178
SEQ ID
AAGACCUGCUCAGCUUCUUCCUUAG
SEQ ID
GCUUCCAGCCAUUGUGUUGAAUCCU
NO 155
NO 179
SEQ ID
AGACCUGCUCAGCUUCUUCCUUAGC
SEQ ID
CUUCCAGCCAUUGUGUUGAAUCCUU
NO 156
NO 180
SEQ ID
GACCUGCUCAGCUUCUUCCUUAGCU
SEQ ID
UUCCAGCCAUUGUGUUGAAUCCUUU
NO 157
NO 181
SEQ ID
ACCUGCUCAGCUUCUUCCUUAGCUU
SEQ ID
UCCAGCCAUUGUGUUGAAUCCUUUA
NO 158
NO 182
SEQ ID
CCUGCUCAGCUUCUUCCUUAGCUUC
SEQ ID
CCAGCCAUUGUGUUGAAUCCUUUAA
NO 159
NO 183
SEQ ID
CUGCUCAGCUUCUUCCUUAGCUUCC
SEQ ID
CAGCCAUUGUGUUGAAUCCUUUAAC
NO 160
NO 184
SEQ ID
UGCUCAGCUUCUUCCUUAGCUUCCA
SEQ ID
AGCCAUUGUGUUGAAUCCUUUAACA
NO 161
NO 185
SEQ ID
GCUCAGCUUCUUCCUUAGCUUCCAG
SEQ ID
GCCAUUGUGUUGAAUCCUUUAACAU
NO 162
NO 186
SEQ ID
CUCAGCUUCUUCCUUAGCUUCCAGC
SEQ ID
CCAUUGUGUUGAAUCCUUUAACAUU
NO 163
NO 187
SEQ ID
UCAGCUUCUUCCUUAGCUUCCAGCC
SEQ ID
CAUUGUGUUGAAUCCUUUAACAUUU
NO 164
NO 188
DMD Gene Exon 44
SEQ ID
UCAGCUUCUGUUAGCCACUG
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 189
NO 214
SEQ ID
UUCAGCUUCUGUUAGCCACU
SEQ ID
CAGCUUCUGUUAGCCACUGAUUAA
NO 190
NO 215
A
SEQ ID
UUCAGCUUCUGUUAGCCACUG
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 191
NO 216
SEQ ID
UCAGCUUCUGUUAGCCACUGA
SEQ ID
AGCUUCUGUUAGCCACUGAU
NO 192
NO 217
SEQ ID
UUCAGCUUCUGUUAGCCACUGA
SEQ ID
GCUUCUGUUAGCCACUGAUU
NO 193
NO 218
SEQ ID
UCAGCUUCUGUUAGCCACUGA
SEQ ID
AGCUUCUGUUAGCCACUGAUU
NO 194
NO 219
SEQ ID
UUCAGCUUCUGUUAGCCACUGA
SEQ ID
GCUUCUGUUAGCCACUGAUUA
NO 195
NO 220
SEQ ID
UCAGCUUCUGUUAGCCACUGAU
SEQ ID
AGCUUCUGUUAGCCACUGAUUA
NO 196
NO 221
SEQ ID
UUCAGCUUCUGUUAGCCACUGAU
SEQ ID
GCUUCUGUUAGCCACUGAUUAA
NO 197
NO 222
SEQ ID
UCAGCUUCUGUUAGCCACUGAUU
SEQ ID
AGCUUCUGUUAGCCACUGAUUAA
NO 198
NO 223
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUU
SEQ ID
GCUUCUGUUAGCCACUGAUUAAA
NO 199
NO 224
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUA
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 200
NO 225
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUA
SEQ ID
GCUUCUGUUAGCCACUGAUUAAA
NO 201
NO 226
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID
CCAUUUGUAUUUAGCAUGUUCCC
NO 202
NO 227
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID
AGAUACCAUUUGUAUUUAGC
NO 203
NO 228
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID
GCCAUUUCUCAACAGAUCU
NO 204
NO 229
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID
GCCAUUUCUCAACAGAUCUGUCA
NO 205
NO 230
SEQ ID
CAGCUUCUGUUAGCCACUG
SEQ ID
AUUCUCAGGAAUUUGUGUCUUUC
NO 206
NO 231
SEQ ID
CAGCUUCUGUUAGCCACUGAU
SEQ ID
UCUCAGGAAUUUGUGUCUUUC
NO 207
NO 232
SEQ ID
AGCUUCUGUUAGCCACUGAUU
SEQ ID
GUUCAGCUUCUGUUAGCC
NO 208
NO 233
SEQ ID
CAGCUUCUGUUAGCCACUGAUU
SEQ ID
CUGAUUAAAUAUCUUUAUAU C
NO 209
NO 234
SEQ ID
AGCUUCUGUUAGCCACUGAUUA
SEQ ID
GCCGCCAUUUCUCAACAG
NO 210
NO 235
SEQ ID
CAGCUUCUGUUAGCCACUGAUUA
SEQ ID
GUAUUUAGCAUGUUCCCA
NO 211
NO 236
SEQ ID
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID
CAGGAAUUUGUGUCUUUC
NO 212
NO 237
SEQ ID
CAGCUUCUGUUAGCCACUGAUUAA
SEQ ID
UCAICUUCUGUUAGCCACUG
NO 213
NO 575
SEQ ID
UCAGCUUCUIUUAGCCACUG
SEQ ID
UCAGCUUCUGUUAGCCACUI
NO 573
NO 576
SEQ ID
UCAGCUUCUGUUAICCACUG
NO 574
DMD Gene Exon 46
SEQ ID
GCUUUUCUUUUAGUUGCUGCUCUUU
SEQ ID
CCAGGUUCAAGUGGGAUACUAGCAA
NO 238
NO 265
SEQ ID
CUUUUCUUUUAGUUGCUGCUCUUUU
SEQ ID
CAGGUUCAAGUGGGAUACUAGCAAU
NO 239
NO 266
SEQ ID
UUUUCUUUUAGUUGCUGCUCUUUUC
SEQ ID
AGGUUCAAGUGGGAUACUAGCAAUG
NO 240
NO 267
SEQ ID
UUUCUUUUAGUUGCUGCUCUUUUCC
SEQ ID
GGUUCAAGUGGGAUACUAGCAAUGU
NO 241
NO 268
SEQ ID
UUCUUUUAGUUGCUGCUCUUUUCCA
SEQ ID
GUUCAAGUGGGAUACUAGCAAUGUU
NO 242
NO 269
SEQ ID
UCUUUUAGUUGCUGCUCUUUUCCAG
SEQ ID
UUCAAGUGGGAUACUAGCAAUGUUA
NO 243
NO 270
SEQ ID
CUUUUAGUUGCUGCUCUUUUCCAGG
SEQ ID
UCAAGUGGGAUACUAGCAAUGUUAU
NO 244
NO 271
SEQ ID
UUUUAGUUGCUGCUCUUUUCCAGGU
SEQ ID
CAAGUGGGAUACUAGCAAUGUUAUC
NO 245
NO 272
SEQ ID
UUUAGUUGCUGCUCUUUUCCAGGUU
SEQ ID
AAGUGGGAUACUAGCAAUGUUAUCU
NO 246
NO 273
SEQ ID
UUAGUUGCUGCUCUUUUCCAGGUUC
SEQ ID
AGUGGGAUACUAGCAAUGUUAUCUG
NO 247
NO 274
SEQ ID
UAGUUGCUGCUCUUUUCCAGGUUCA
SEQ ID
GUGGGAUACUAGCAAUGUUAUCUGC
NO 248
NO 275
SEQ ID
AGUUGCUGCUCUUUUCCAGGUUCAA
SEQ ID
UGGGAUACUAGCAAUGUUAUCUGCU
NO 249
NO 276
SEQ ID
GUUGCUGCUCUUUUCCAGGUUCAAG
SEQ ID
GGGAUACUAGCAAUGUUAUCUGCUU
NO 250
NO 277
SEQ ID
UUGCUGCUCUUUUCCAGGUUCAAGU
SEQ ID
GGAUACUAGCAAUGUUAUCUGCUUC
NO 251
NO 278
SEQ ID
UGCUGCUCUUUUCCAGGUUCAAGUG
SEQ ID
GAUACUAGCAAUGUUAUCUGCUUCC
NO 252
NO 279
SEQ ID
GCUGCUCUUUUCCAGGUUCAAGUGG
SEQ ID
AUACUAGCAAUGUUAUCUGCUUCCU
NO 253
NO 280
SEQ ID
CUGCUCUUUUCCAGGUUCAAGUGGG
SEQ ID
UACUAGCAAUGUUAUCUGCUUCCUC
NO 254
NO 281
SEQ ID
UGCUCUUUUCCAGGUUCAAGUGGGA
SEQ ID
ACUAGCAAUGUUAUCUGCUUCCUCC
NO 255
NO 282
SEQ ID
GCUCUUUUCCAGGUUCAAGUGGGAC
SEQ ID
CUAGCAAUGUUAUCUGCUUCCUCCA
NO 256
NO 283
SEQ ID
CUCUUUUCCAGGUUCAAGUGGGAUA
SEQ ID
UAGCAAUGUUAUCUGCUUCCUCCAA
NO 257
NO 284
SEQ ID
UCUUUUCCAGGUUCAAGUGGGAUAC
SEQ ID
AGCAAUGUUAUCUGCUUCCUCCAAC
NO 258
NO 285
SEQ ID
UCUUUUCCAGGUUCAAGUGG
SEQ ID
GCAAUGUUAUCUGCUUCCUCCAACC
NO 259
NO 286
SEQ ID
CUUUUCCAGGUUCAAGUGGGAUACU
SEQ ID
CAAUGUUAUCUGCUUCCUCCAACCA
NO 260
NO 287
SEQ ID
UUUUCCAGGUUCAAGUGGGAUACUA
SEQ ID
AAUGUUAUCUGCUUCCUCCAACCAU
NO 261
NO 288
SEQ ID
UUUCCAGGUUCAAGUGGGAUACUAG
SEQ ID
AUGUUAUCUGCUUCCUCCAACCAUA
NO 262
NO 289
SEQ ID
UUCCAGGUUCAAGUGGGAUACUAGC
SEQ ID
UGUUAUCUGCUUCCUCCAACCAUAA
NO 263
NO 290
SEQ ID
UCCAGGUUCAAGUGGGAUACUAGCA
NO 264
DMD Gene Exon 52
SEQ ID
AGCCUCUUGAUUGCUGGUCUUGUUU
SEQ ID
UUGGGCAGCGGUAAUGAGUUCUUCC
NO 291
NO 326
SEQ ID
GCCUCUUGAUUGCUGGUCUUGUUUU
SEQ ID
UGGGCAGCGGUAAUGAGUUCUUCCA
NO 292
NO 327
SEQ ID
CCUCUUGAUUGCUGGUCUUGUUUUU
SEQ ID
GGGCAGCGGUAAUGAGUUCUUCCAA
NO 293
NO 328
SEQ ID
CCUCUUGAUUGCUGGUCUUG
SEQ ID
GGCAGCGGUAAUGAGUUCUUCCAAC
NO 294
NO 329
SEQ ID
CUCUUGAUUGCUGGUCUUGUUUUUC
SEQ ID
GCAGCGGUAAUGAGUUCUUCCAACU
NO 295
NO 330
SEQ ID
UCUUGAUUGCUGGUCUUGUUUUUCA
SEQ ID
CAGCGGUAAUGAGUUCUUCCAACUG
NO 296
NO 331
SEQ ID
CUUGAUUGCUGGUCUUGUUUUUCAA
SEQ ID
AGCGGUAAUGAGUUCUUCCAACUGG
NO 297
NO 332
SEQ ID
UUGAUUGCUGGUCUUGUUUUUCAAA
SEQ ID
GCGGUAAUGAGUUCUUCCAACUGGG
NO 298
NO 333
SEQ ID
UGAUUGCUGGUCUUGUUUUUCAAAU
SEQ ID
CGGUAAUGAGUUCUUCCAACUGGGG
NO 299
NO 334
SEQ ID
GAUUGCUGGUCUUGUUUUUCAAAUU
SEQ ID
GGUAAUGAGUUCUUCCAACUGGGGA
NO 300
NO 335
SEQ ID
GAUUGCUGGUCUUGUUUUUC
SEQ ID
GGUAAUGAGUUCUUCCAACUGG
NO 301
NO 336
SEQ ID
AUUGCUGGUCUUGUUUUUCAAAUUU
SEQ ID
GUAAUGAGUUCUUCCAACUGGGGAC
NO 302
NO 337
SEQ ID
UUGCUGGUCUUGUUUUUCAAAUUUU
SEQ ID
UAAUGAGUUCUUCCAACUGGGGACG
NO 303
NO 338
SEQ ID
UGCUGGUCUUGUUUUUCAAAUUUUG
SEQ ID
AAUGAGUUCUUCCAACUGGGGACGC
NO 304
NO 339
SEQ ID
GCUGGUCUUGUUUUUCAAAUUUUGG
SEQ ID
AUGAGUUCUUCCAACUGGGGACGCC
NO 305
NO 340
SEQ ID
CUGGUCUUGUUUUUCAAAUUUUGGG
SEQ ID
UGAGUUCUUCCAACUGGGGACGCCU
NO 306
NO 341
SEQ ID
UGGUCUUGUUUUUCAAAUUUUGGGC
SEQ ID
GAGUUCUUCCAACUGGGGACGCCUC
NO 307
NO 342
SEQ ID
GGUCUUGUUUUUCAAAUUUUGGGCA
SEQ ID
AGUUCUUCCAACUGGGGACGCCUCU
NO 308
NO 343
SEQ ID
GUCUUGUUUUUCAAAUUUUGGGCAG
SEQ ID
GUUCUUCCAACUGGGGACGCCUCUG
NO 309
NO 344
SEQ ID
UCUUGUUUUUCAAAUUUUGGGCAGC
SEQ ID
UUCUUCCAACUGGGGACGCCUCUGU
NO 310
NO 345
SEQ ID
CUUGUUUUUCAAAUUUUGGGCAGCG
SEQ ID
UCUUCCAACUGGGGACGCCUCUGUU
NO 311
NO 346
SEQ ID
UUGUUUUUCAAAUUUUGGGCAGCGG
SEQ ID
CUUCCAACUGGGGACGCCUCUGUUC
NO 312
NO 347
SEQ ID
UGUUUUUCAAAUUUUGGGCAGCGGU
SEQ ID
UUCCAACUGGGGACGCCUCUGUUCC
NO 313
NO 348
SEQ ID
GUUUUUCAAAUUUUGGGCAGCGGUA
SEQ ID
UCCAACUGGGGACGCCUCUGUUCCA
NO 314
NO 349
SEQ ID
UUUUUCAAAUUUUGGGCAGCGGUAA
SEQ ID
CCAACUGGGGACGCCUCUGUUCCAA
NO 315
NO 350
SEQ ID
UUUUCAAAUUUUGGGCAGCGGUAAU
SEQ ID
CAACUGGGGACGCCUCUGUUCCAAA
NO 316
NO 351
SEQ ID
UUUCAAAUUUUGGGCAGCGGUAAUG
SEQ ID
AACUGGGGACGCCUCUGUUCCAAAU
NO 317
NO 352
SEQ ID
UUCAAAUUUUGGGCAGCGGUAAUGA
SEQ ID
ACUGGGGACGCCUCUGUUCCAAAUC
NO 318
NO 353
SEQ ID
UCAAAUUUUGGGCAGCGGUAAUGAG
SEQ ID
CUGGGGACGCCUCUGUUCCAAAUCC
NO 319
NO 354
SEQ ID
CAAAUUUUGGGCAGCGGUAAUGAGU
SEQ ID
UGGGGACGCCUCUGUUCCAAAUCCU
NO 320
NO 355
SEQ ID
AAAUUUUGGGCAGCGGUAAUGAGUU
SEQ ID
GGGGACGCCUCUGUUCCAAAUCCUG
NO 321
NO 356
SEQ ID
AAUUUUGGGCAGCGGUAAUGAGUUC
SEQ ID
GGGACGCCUCUGUUCCAAAUCCUGC
NO 322
NO 357
SEQ ID
AUUUUGGGCAGCGGUAAUGAGUUCU
SEQ ID
GGACGCCUCUGUUCCAAAUCCUGCA
NO 323
NO 358
SEQ ID
UUUUGGGCAGCGGUAAUGAGUUCUU
SEQ ID
GACGCCUCUGUUCCAAAUCCUGCAU
NO 324
NO 359
SEQ ID
UUUGGGCAGCGGUAAUGAGUUCUUC
NO 325
DMD Gene Exon 50
SEQ ID
CCAAUAGUGGUCAGUCCAGGAGCUA
SEQ ID
CUAGGUCAGGCUGCUUUGCCCUCAG
NO 360
NO 386
SEQ ID
CAAUAGUGGUCAGUCCAGGAGCUAG
SEQ ID
UAGGUCAGGCUGCUUUGCCCUCAGC
NO 361
NO 387
SEQ ID
AAUAGUGGUCAGUCCAGGAGCUAGG
SEQ ID
AGGUCAGGCUGCUUUGCCCUCAGCU
NO 362
NO 388
SEQ ID
AUAGUGGUCAGUCCAGGAGCUAGGU
SEQ ID
GGUCAGGCUGCUUUGCCCUCAGCUC
NO 363
NO 389
SEQ ID
AUAGUGGUCAGUCCAGGAGCU
SEQ ID
GUCAGGCUGCUUUGCCCUCAGCUCU
NO 364
NO 390
SEQ ID
UAGUGGUCAGUCCAGGAGCUAGGUC
SEQ ID
UCAGGCUGCUUUGCCCUCAGCUCUU
NO 365
NO 391
SEQ ID
AGUGGUCAGUCCAGGAGCUAGGUCA
SEQ ID
CAGGCUGCUUUGCCCUCAGCUCUUG
NO 366
NO 392
SEQ ID
GUGGUCAGUCCAGGAGCUAGGUCAG
SEQ ID
AGGCUGCUUUGCCCUCAGCUCUUGA
NO 367
NO 393
SEQ ID
UGGUCAGUCCAGGAGCUAGGUCAGG
SEQ ID
GGCUGCUUUGCCCUCAGCUCUUGAA
NO 368
NO 394
SEQ ID
GGUCAGUCCAGGAGCUAGGUCAGGC
SEQ ID
GCUGCUUUGCCCUCAGCUCUUGAAG
NO 369
NO 395
SEQ ID
GUCAGUCCAGGAGCUAGGUCAGGCU
SEQ ID
CUGCUUUGCCCUCAGCUCUUGAAGU
NO 370
NO 396
SEQ ID
UCAGUCCAGGAGCUAGGUCAGGCUG
SEQ ID
UGCUUUGCCCUCAGCUCUUGAAGUA
NO 371
NO 397
SEQ ID
CAGUCCAGGAGCUAGGUCAGGCUGC
SEQ ID
GCUUUGCCCUCAGCUCUUGAAGUAA
NO 372
NO 398
SEQ ID
AGUCCAGGAGCUAGGUCAGGCUGCU
SEQ ID
CUUUGCCCUCAGCUCUUGAAGUAAA
NO 373
NO 399
SEQ ID
GUCCAGGAGCUAGGUCAGGCUGCUU
SEQ ID
UUUGCCCUCAGCUCUUGAAGUAAAC
NO 374
NO 400
SEQ ID
UCCAGGAGCUAGGUCAGGCUGCUUU
SEQ ID
UUGCCCUCAGCUCUUGAAGUAAACG
NO 375
NO 401
SEQ ID
CCAGGAGCUAGGUCAGGCUGCUUUG
SEQ ID
UGCCCUCAGCUCUUGAAGUAAACGG
NO 376
NO 402
SEQ ID
CAGGAGCUAGGUCAGGCUGCUUUGC
SEQ ID
GCCCUCAGCUCUUGAAGUAAACGGU
NO 377
NO 403
SEQ ID
AGGAGCUAGGUCAGGCUGCUUUGCC
SEQ ID
CCCUCAGCUCUUGAAGUAAACGGUU
NO 378
NO 404
SEQ ID
GGAGCUAGGUCAGGCUGCUUUGCCC
SEQ ID
CCUCAGCUCUUGAAGUAAAC
NO 379
NO 405
SEQ ID
GAGCUAGGUCAGGCUGCUUUGCCCU
SEQ ID
CCUCAGCUCUUGAAGUAAACG
NO 380
NO 406
SEQ ID
AGCUAGGUCAGGCUGCUUUGCCCUC
SEQ ID
CUCAGCUCUUGAAGUAAACG
NO 381
NO 407
SEQ ID
GCUAGGUCAGGCUGCUUUGCCCUCA
SEQ ID
CCUCAGCUCUUGAAGUAAACGGUUU
NO 382
NO 408
SEQ ID
CUCAGCUCUUGAAGUAAACGGUUUA
SEQ ID
UCAGCUCUUGAAGUAAACGGUUUAC
NO 383
NO 409
SEQ ID
CAGCUCUUGAAGUAAACGGUUUACC
SEQ ID
AGCUCUUGAAGUAAACGGUUUACCG
NO 384
NO 410
SEQ ID
GCUCUUGAAGUAAACGGUUUACCGC
SEQ ID
CUCUUGAAGUAAACGGUUUACCGCC
NO 385
NO 411
DMD Gene Exon 43
SEQ ID
CCACAGGCGUUGCACUUUGCAAUGC
SEQ ID
UCUUCUUGCUAUGAAUAAUGUCAAU
NO 412
NO 443
SEQ ID
CACAGGCGUUGCACUUUGCAAUGCU
SEQ ID
CUUCUUGCUAUGAAUAAUGUCAAUC
NO 413
NO 444
SEQ ID
ACAGGCGUUGCACUUUGCAAUGCUG
SEQ ID
UUCUUGCUAUGAAUAAUGUCAAUCC
NO 414
NO 445
SEQ ID
CAGGCGUUGCACUUUGCAAUGCUGC
SEQ ID
UCUUGCUAUGAAUAAUGUCAAUCCG
NO 415
NO 446
SEQ ID
AGGCGUUGCACUUUGCAAUGCUGCU
SEQ ID
CUUGCUAUGAAUAAUGUCAAUCCGA
NO 416
NO 447
SEQ ID
GGCGUUGCACUUUGCAAUGCUGCUG
SEQ ID
UUGCUAUGAAUAAUGUCAAUCCGAC
NO 417
NO 448
SEQ ID
GCGUUGCACUUUGCAAUGCUGCUGU
SEQ ID
UGCUAUGAAUAAUGUCAAUCCGACC
NO 418
NO 449
SEQ ID
CGUUGCACUUUGCAAUGCUGCUGUC
SEQ ID
GCUAUGAAUAAUGUCAAUCCGACCU
NO 419
NO 450
SEQ ID
CGUUGCACUUUGCAAUGCUGCUG
SEQ ID
CUAUGAAUAAUGUCAAUCCGACCUG
NO 420
NO 451
SEQ ID
GUUGCACUUUGCAAUGCUGCUGUCU
SEQ ID
UAUGAAUAAUGUCAAUCCGACCUGA
NO 421
NO 452
SEQ ID
UUGCACUUUGCAAUGCUGCUGUCUU
SEQ ID
AUGAAUAAUGUCAAUCCGACCUGAG
NO 422
NO 453
SEQ ID
UGCACUUUGCAAUGCUGCUGUCUUC
SEQ ID
UGAAUAAUGUCAAUCCGACCUGAGC
NO 423
NO 454
SEQ ID
GCACUUUGCAAUGCUGCUGUCUUCU
SEQ ID
GAAUAAUGUCAAUCCGACCUGAGCU
NO 424
NO 455
SEQ ID
CACUUUGCAAUGCUGCUGUCUUCUU
SEQ ID
AAUAAUGUCAAUCCGACCUGAGCUU
NO 425
NO 456
SEQ ID
ACUUUGCAAUGCUGCUGUCUUCUUG
SEQ ID
AUAAUGUCAAUCCGACCUGAGCUUU
NO 426
NO 457
SEQ ID
CUUUGCAAUGCUGCUGUCUUCUUGC
SEQ ID
UAAUGUCAAUCCGACCUGAGCUUUG
NO 427
NO 458
SEQ ID
UUUGCAAUGCUGCUGUCUUCUUGCU
SEQ ID
AAUGUCAAUCCGACCUGAGCUUUGU
NO 428
NO 459
SEQ ID
UUGCAAUGCUGCUGUCUUCUUGCUA
SEQ ID
AUGUCAAUCCGACCUGAGCUUUGUU
NO 429
NO 460
SEQ ID
UGCAAUGCUGCUGUCUUCUUGCUAU
SEQ ID
UGUCAAUCCGACCUGAGCUUUGUUG
NO 430
NO 461
SEQ ID
GCAAUGCUGCUGUCUUCUUGCUAUG
SEQ ID
GUCAAUCCGACCUGAGCUUUGUUGU
NO 431
NO 462
SEQ ID
CAAUGCUGCUGUCUUCUUGCUAUGA
SEQ ID
UCAAUCCGACCUGAGCUUUGUUGUA
NO 432
NO 463
SEQ ID
AAUGCUGCUGUCUUCUUGCUAUGAA
SEQ ID
CAAUCCGACCUGAGCUUUGUUGUAG
NO 433
NO 464
SEQ ID
AUGCUGCUGUCUUCUUGCUAUGAAU
SEQ ID
AAUCCGACCUGAGCUUUGUUGUAGA
NO 434
NO 465
SEQ ID
UGCUGCUGUCUUCUUGCUAUGAAUA
SEQ ID
AUCCGACCUGAGCUUUGUUGUAGAC
NO 435
NO 466
SEQ ID
GCUGCUGUCUUCUUGCUAUGAAUAA
SEQ ID
UCCGACCUGAGCUUUGUUGUAGACU
NO 436
NO 467
SEQ ID
CUGCUGUCUUCUUGCUAUGAAUAAU
SEQ ID
CCGACCUGAGCUUUGUUGUAGACUA
NO 437
NO 468
SEQ ID
UGCUGUCUUCUUGCUAUGAAUAAUG
SEQ ID
CGACCUGAGCUUUGUUGUAG
NO 438
NO 469
SEQ ID
GCUGUCUUCUUGCUAUGAAUAAUGU
SEQ ID
CGACCUGAGCUUUGUUGUAGACUAU
NO 439
NO 470
SEQ ID
CUGUCUUCUUGCUAUGAAUAAUGUC
SEQ ID
GACCUGAGCUUUGUUGUAGACUAUC
NO 440
NO 471
SEQ ID
UGUCUUCUUGCUAUGAAUAAUGUCA
SEQ ID
ACCUGAGCUUUGUUGUAGACUAUCA
NO 441
NO 472
SEQ ID
GUCUUCUUGCUAUGAAUAAUGUCAA
SEQ ID
CCUGA GCUUU GUUGU AGACU AUC
NO 442
NO 473
DMD Gene Exon 6
SEQ ID
CAUUUUUGACCUACAUGUGG
SEQ ID
AUUUUUGACCUACAUGGGAAA G
NO 474
NO 479
SEQ ID
UUUGACCUACAUGUGGAAAG
SEQ ID
UACGAGUUGAUUGUCGGACCCAG
NO 475
NO 480
SEQ ID
UACAUUUUUGACCUACAUGUGGAAA
SEQ ID
GUGGUCUCCUUACCUAUGACUGUGG
NO 476
G
NO 481
SEQ ID
GGUCUCCUUACCUAUGA
SEQ ID
UGUCUCAGUAAUCUUCUUACCUAU
NO 477
NO 482
SEQ ID
UCUUACCUAUGACUAUGGAUGAGA
NO 478
DMD Gene Exon 7
SEQ ID
UGCAUGUUCCAGUCGUUGUGUGG
SEQ ID
AUUUACCAACCUUCAGGAUCGAGUA
NO 483
NO 485
SEQ ID
CACUAUUCCAGUCAAAUAGGUCUGG
SEQ ID
GGCCUAAAACACAUACACAUA
NO 484
NO 486
DMD Gene Exon 8
SEQ ID
GAUAGGUGGUAUCAACAUCUGUAA
SEQ ID
UGUUGUUGUUUAUGCUCAUU
NO 487
NO 490
SEQ ID
GAUAGGUGGUAUCAACAUCUG
SEQ ID
GUACAUUAAGAUGGACUUC
NO 488
NO 491
SEQ ID
CUUCCUGGAUGGCUUGAAU
NO 489
DMD Gene Exon 55
SEQ ID
CUGUUGCAGUAAUCUAUGAG
SEQ ID
UGCCAUUGUUUCAUCAGCUCUUU
NO 492
NO 495
SEQ ID
UGCAGUAAUCUAUGAGUUUC
SEQ ID
UCCUGUAGGACAUUGGCAGU
NO 493
NO 496
SEQ ID
GAGUCUUCUAGGAGCCUU
SEQ ID
CUUGGAGUCUUCUAGGAGCC
NO 494
NO 497
DMD Gene Exon 2
SEQ ID
CCAUUUUGUGAAUGUUUUCUUUUG
SEQ ID
GAAAAUUGUGCAUUUACCCAUUUU
NO 498
AACAUC
NO 500
SEQ ID
CCCAUUUUGUGAAUGUUUUCUUUU
SEQ ID
UUGUGCAUUUACCCAUUUUGUG
NO 499
NO 501
DMD Gene Exon 11
SEQ ID
CCCUGAGGCAUUCCCAUCUUGAAU
SEQ ID
CUUGAAUUUAGGAGAUUCAUCUG
NO 502
NO 504
SEQ ID
AGGACUUACUUGCUUUGUUU
SEQ ID
CAUCUUCUGAUAAUUUUCCUGUU
NO 503
NO 505
DMD Gene Exon 17
SEQ ID
CCAUUACAGUUGUCUGUGUU
SEQ ID
UAAUCUGCCUCUUCUUUUGG
NO 506
NO 508
SEQ ID
UGACAGCCUGUGAAAUCUGUGAG
NO 507
DMD Gene Exon 19
SEQ ID
CAGCAGUAGUUGUCAUCUGC
SEQ ID
GCCUGAGCUGAUCUGCUGGCAUCUUG
NO 509
NO 511
CAGUU
SEQ ID
GCCUGAGCUGAUCUGCUGGCAUCUUGC
SEQ ID
UCUGCUGGCAUCUUGC
NO 510
NO 512
DMD Gene Exon 21
SEQ ID
GCCGGUUGACUUCAUCCUGUGC
SEQ ID
CUGCAUCCAGGAACAUGGGUCC
NO 513
NO 516
SEQ ID
GUCUGCAUCCAGGAACAUGGGUC
SEQ ID
GUUGAAGAUCUGAUAGCCGGUUGA
NO 514
NO 517
SEQ ID
UACUUACUGUCUGUAGCUCUUUCU
NO 515
DMD Gene Exon 57
SEQ ID
UAGGUGCCUGCCGGCUU
SEQ ID
CUGAACUGCUGGAAAGUCGCC
NO 518
NO 520
SEQ ID
UUCAGCUGUAGCCACACC
SEQ ID
CUGGCUUCCAAAUGGGACCUGAAAAAGAAC
NO 519
NO 521
DMD Gene Exon 59
SEQ ID
CAAUUUUUCCCACUCAGUAUU
SEQ ID
UCCUCAGGAGGCAGCUCUAAAU
NO 522
NO 524
SEQ ID
UUGAAGUUCCUGGAGUCUU
NO 523
DMD Gene Exon 62
SEQ ID
UGGCUCUCUCCCAGGG
SEQ ID
GGGCACUUUGUUUGGCG
NO 525
NO 527
SEQ ID
GAGAUGGCUCUCUCCCAGGGACCCUGG
NO 526
DMD Gene Exon 63
SEQ ID
GGUCCCAGCAAGUUGUUUG
SEQ ID
GUAGAGCUCUGUCAUUUUGGG
NO 528
NO 530
SEQ ID
UGGGAUGGUCCCAGCAAGUUGUUUG
NO 529
DMD Gene Exon 65
SEQ ID
GCUCAAGAGAUCCACUGCAAAAAAC
SEQ ID
UCUGCAGGAUAUCCAUGGGCUGGUC
NO 531
NO 533
SEQ ID
GCCAUACGUACGUCAUCAUAAACAUUC
NO 532
DMD Gene Exon 66
SEQ ID
GAUCCUCCCUGUUCGUCCCCUAUUAUG
NO 534
DMD Gene Exon 69
SEQ ID
UGCUUUAGACUCCUGUACCUGAUA
NO 535
DMD Gene Exon 75
SEQ ID
GGCGGCCUUUGUGUUGAC
SEQ ID
CCUUUAUGUUCGUGCUGCU
NO 536
NO 538
SEQ ID
GGACAGGCCUUUAUGUUCGUGCUGC
NO 537
Human IGF-1 Isoform 4 Amino Acid Sequence
SEQ ID NO 577:
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAG
PETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFR
SCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAG
NKNYRM
|
15468239
|
biomarin technologies b.v.
|
USA
|
B2
|
Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
|
Open
|
|
|
Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Mar 16th, 2021 12:00AM
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Jan 26th, 2018 12:00AM
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https://www.uspto.gov?id=USRE048468-20210316
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Means and methods for counteracting muscle disorders
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The invention provides means and methods for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy and/or Becker Muscular Dystrophy. Therapies using compounds for providing patients with functional muscle proteins are combined with at least one adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or at least one adjunct compound for improving muscle fiber function, integrity and/or survival.
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RE48468
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1. A composition comprising:
a first compound that increases the level of a functional dystrophin protein produced in a muscle cell of a Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) individual,
wherein said first compound is an antisense oligonucleotide that induces skipping of exon 51 of human dystrophin pre-mRNA of said individual;
and a second compound comprising a steroid;
wherein, upon administration to a DMD or BMD patient, the composition increases the ratio of said dystrophin to laminin-α2 in muscle tissue of said patient as compared to the ratio of said dystrophin to laminin-α2 in muscle tissue of a patient administered with said first compound and not said second compound; and
wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 13 to 50 nucleotides in length and wherein said oligonucleotide comprises a non naturally-occurring modification.
2. The composition of claim 1, wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 14 to 25 nucleotides in length.
3. The composition of claim 1, wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 20 to 25 nucleotides in length.
4. The composition of claim 1, wherein said oligonucleotide comprises one or more ribonucleotides, and wherein a said ribonucleotide contains a modification.
5. The composition of claim 4, wherein said modification is a 2′-O-methyl modified ribose.
6. The composition of claim 1, wherein said modification is selected from the group consisting of at least one of a peptide nucleic acid, a locked nucleic acid, and morpholino phosphorodiamidate.
7. A method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual, the method comprising administering to a DMD or BMD patient:
a first compound that increases the level of a functional dystrophin protein produced in a muscle cell of said individual in said individual,
wherein said first compound is an antisense oligonucleotide that induces skipping of exon 51 of dystrophin pre-mRNA of said individual, and
a second compound, comprising a steroid;
wherein, upon administration to a DMD or BMD patient, the composition increases the ratio of said dystrophin to laminin-α2 in muscle tissue of said patient as compared to the ratio of said dystrophin to laminin-α2 in muscle tissue of a patient administered with said first compound and not said second compound; and
wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 13 to 50 nucleotides in length and wherein said oligonucleotide comprises a non naturally-occurring modification.
8. The method of claim 7, wherein said oligonucleotide comprises one or more ribonucleotides, and wherein a said ribonucleotide contains a modification.
9. The method of claim 8, wherein said modification is selected from the group consisting of a 2′-O-methyl modified ribose.
10. The method of claim 7, wherein said modification is selected from the group consisting of at least one of a peptide nucleic acid, a locked nucleic acid, and morpholino phosphorodiamidate.
11. A method for increasing the production of a functional dystrophin protein in a cell, said cell comprising pre-mRNA of a dystrophin gene encoding an aberrant dystrophin protein comprising:
providing said cell with a first compound for inhibiting inclusion of exon 51 into mRNA produced from splicing of said dystrophin pre-mRNA, wherein said first compound is an antisense oligonucleotide that induces the skipping of exon 51 of the human dystrophin pre-mRNA, and providing said cell with a second compound comprising a steroid,
said method further comprising allowing translation of mRNA produced from splicing of said pre-mRNA;
wherein, upon administration to a DMD or BMD patient, the composition increases the ratio of said dystrophin to laminin-α2 in muscle tissue of said patient as compared to the ratio of said dystrophin to laminin-α2 in muscle tissue of a patient administered with said first compound and not said second compound; and
wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 13 to 50 nucleotides in length and wherein said oligonucleotide comprises a non naturally-occurring modification.
12. A pharmaceutical preparation comprising:
said first compound according to claim 1,
said second compound according to claim 1, comprising a steroid,
and a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.
13. A kit comprising:
said first compound according to claim 1,
and said second compound according to claim 1.
14. The kit of claim 13, further comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.
15. The kit of claim 13, further comprising packaging means thereof.
16. The composition according to claim 1, wherein the oligonucleotide comprises a phosphorothioate internucleotide linkage, a 2′-O-methyl ribose and/or a LNA.
17. The kit according to claim 13, wherein the oligonucleotide comprises a phosphorothioate internucleotide linkage, a 2′-O-methyl ribose and/or a LNA.
18. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier, adjuvant, diluent, and/or excipient.
19. The method of claim 7 wherein said steroid is a glucocorticosteroid.
20. The method of claim 19 wherein said glucocorticosteroid is selected from a group consisting of prednisone, dexamethasone, prednizolone and deflazacort.
21. The method of claim 20 wherein said prednisone is present at a dosage of 0.5-1.0 mg/kg.
22. The method of claim 20 wherein said deflazacort is present at a dosage of 0.4-1.4 mg/kg.
23. A method for alleviating one or more symptoms of Duchenne muscular dystrophy in a human patient, comprising administering to the patient an antisense oligonucleotide that is: (a) 100% complementary to a portion of exon 51 of the human dystrophin pre-mRNA and (b) 30 nucleotides in length,
wherein the antisense oligonucleotide comprises the sequence 5′-CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG-3′ (SEQ ID NO:193),
wherein the antisense oligonucleotide is a morpholino phosphorodiamidate,
wherein the antisense oligonucleotide is administered intravenously,
wherein the antisense oligonucleotide induces skipping of exon 51 of dystrophin pre-mRNA, and
wherein the patient is receiving glucocorticosteroid treatment.
24. The method of claim 23, wherein the patient was receiving the glucocorticosteroid treatment prior to the administration of the antisense oligonucleotide.
25. The method of claim 24, wherein the prior glucocorticosteroid treatment was for a period of at least three weeks.
26. The method of claim 23, wherein the glucocorticosteroid is selected from the group consisting of prednisone, dexamethasone, prednisolone, and deflazacort.
27. The method of claim 26, wherein the glucocorticosteroid is prednisone.
28. The method of claim 27, wherein the patient is receiving the prednisolone at a dose of about 0.5 mg/kg/day to about 1.0 mg/kg/day.
29. The method of claim 26, wherein the glucocorticosteroid is deflazacort.
30. The method of claim 29, wherein the patient is receiving the deflazacort at a dose of about 0.4 mg/kg/day to about 1.4 mg/kg/day.
31. The method of claim 23, wherein the method increases the ratio of dystrophin to laminin-α2 in muscle tissue of the patient as compared to the ratio of dystrophin to laminin-α2 in muscle tissue of a similar patient treated with the antisense oligonucleotide and not the glucocorticosteroid.
32. A method for alleviating one or more symptoms of Duchenne muscular dystrophy in a human patient, comprising administering to the patient an antisense oligonucleotide that is: (a) 100% complementary to a portion of exon 51 of the human dystrophin pre-mRNA and (b) 30 nucleotides in length,
wherein the antisense oligonucleotide is a functional equivalent of an oligonucleotide comprising the sequence 5′-CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG-3′ (SEQ ID NO:193),
wherein the antisense oligonucleotide is a morpholino phosphorodiamidate,
wherein the antisense oligonucleotide is administered intravenously,
wherein the antisense oligonucleotide induces skipping of exon 51 of dystrophin pre-mRNA, and
wherein the patient is receiving glucocorticosteroid treatment.
33. A method for alleviating one or more symptoms of Duchenne muscular dystrophy in a human patient, comprising administering to the patient an antisense oligonucleotide that is 100% complementary to the portion of exon 51 of the human dystrophin pre-mRNA to which the sequence 5′-CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG-3′ (SEQ ID NO:193) is complementary,
wherein the antisense oligonucleotide is 30 nucleotides in length,
wherein the antisense oligonucleotide is a morpholino phosphorodiamidate,
wherein the antisense oligonucleotide is administered intravenously,
wherein the antisense oligonucleotide induces skipping of exon 51 of dystrophin pre-mRNA, and
wherein the patient is receiving glucocorticosteroid treatment.
34. The method of claim 33, wherein the patient has received the glucocorticosteroid treatment prior to the administration of the antisense oligonucleotide.
35. The method of claim 34, wherein the prior glucocorticosteroid treatment was for a period of at least three weeks.
36. The method of claim 33, wherein the glucocorticosteroid is selected from the group consisting of prednisone, dexamethasone, prednisolone, and deflazacort.
37. The method of claim 36, wherein the glucocorticosteroid is prednisone.
38. The method of claim 37, wherein the patient is receiving the prednisone at a dose of about 0.5 mg/kg/day to about 1.0 mg/kg/day.
39. The method of claim 36, wherein the glucocorticosteroid is deflazacort.
40. The method of claim 39, wherein the patient is receiving the deflazacort at a dose of about 04. Mg/kg/day to about 1.4 mg/kg/day.
41. The method of claim 33, wherein the method increases the ratio of dystrophin to laminin-α2 in muscle tissue of the patient as compared to the ratio of dystrophin to laminin-α2 in muscle tissue of a similar patient treated with the antisense oligonucleotide and not the glucocorticosteroid.
42. A method for alleviating one or more symptoms of Duchenne muscular dystrophy in a human patient, comprising administering to the patient an antisense oligonucleotide that is 100% complementary to the portion of exon 51 of the human dystrophin pre-mRNA to which the sequence 5′-CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG-3′ (SEQ ID NO:193) is complementary,
wherein the antisense oligonucleotide is 30 nucleotides in length,
wherein the antisense oligonucleotide is a morpholino phosphorodiamidate,
wherein the antisense oligonucleotide is administered intravenously,
wherein the antisense oligonucleotide induces skipping of exon 51 of dystrophin pre-mRNA,
wherein the patient is receiving glucocorticosteroid treatment, wherein the glucocorticosteroid is selected from prednisone and deflazacort, and
wherein the method increases the ratio of dystrophin to laminin-α2 in muscle tissue of the patient as compared to the ratio of dystrophin to laminin-α2 in muscle tissue of a similar patient treated with the antisense oligonucleotide and not the glucocorticosteroid.
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Notice: More than one reissue application has been filed for the reissue of U.S. Pat. No. 9,243,245. Besides the instant application, other reissue applications include two reissue applications (U.S. application Ser. No. 15/881,604 and U.S. application Ser. No. 15/881,610), which were both filed on Jan. 26, 2018; two continuation reissue applications (U.S. application Ser. No. 16/249,759 and U.S. application Ser. No. 16/249,777) which were both filed on Jan. 16, 2019; one continuation reissue application (U.S. application Ser. No. 16/353,978) which was filed on Mar. 14, 2019; and two divisional reissue applications (U.S. application Ser. No. 16/373,429 and U.S. application Ser. No. 16/373,459), which were both filed on Apr. 2, 2019.
This application is a reissue of U.S. Pat. No. 9,243,245, filed as U.S. application Ser. No. 12/767,702 on Apr. 26, 2010, which is a continuation application of and claims priority to of PCT/NL2008/050673, filed on Oct. 27, 2008, which claims priority to EPO Application No. 07119351.0, filed on Oct. 26, 2007, and U.S. Provisional Application No. 61/000,670, filed on Oct. 26, 2007, the. The contents of which each of the above applications are hereby incorporated by reference in their entirety by this reference.
The invention relates to the fields of molecular biology and medicine. A muscle disorder is a disease that usually has a significant impact on the life of an individual. A muscle disorder can either have a genetic cause or a non-genetic cause. An important group of muscle diseases with a genetic cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD). These disorders are caused by defects in a gene for a muscle protein.
Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are genetic muscular dystrophies with a relatively high incidence. In both Duchenne and Becker muscular dystrophy the muscle protein dystrophin is affected. In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and/or the dystrophin present is abnormally formed. Both diseases are associated with recessive X-linked inheritance. DMD results from a frameshift mutation in the DMD gene. The frameshift in the DMD gene results in the production of a truncated non-functional dystrophin protein, resulting in progressive muscle wasting and weakness. BMD occurs as a mutation does not cause a frame-shift in the DMD gene. As in BMD some dystrophin is present in contrast to DMD where dystrophin is absent, BMD has less severe symptoms then DMD. The onset of DMD is earlier than BMD. DMD usually manifests itself in early childhood, BMD in the teens or in early adulthood. The progression of BMD is slower and less predictable than DMD. Patients with BMD can survive into mid to late adulthood. Patients with DMD rarely survive beyond their thirties.
Dystrophin plays an important structural role in the muscle fiber, connecting the extracellular matrix and the cytoskeleton. The N-terminal region binds actin, whereas the C-terminal end is part of the dystrophin glycoprotein complex (DGC), which spans the sarcolemma. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis.
For most genetic muscular dystrophies no clinically applicable and effective therapies are currently available. Exon skipping techniques are nowadays explored in order to combat genetic muscular dystrophies. Promising results have recently been reported by us and others on a genetic therapy aimed at restoring the reading frame of the dystrophin pre-mRNA in cells from the mdx mouse and DMD patients1-11. By the targeted skipping of a specific exon, a DMD phenotype (lacking dystrophin) is converted into a milder BMD phenotype (partly to largely functional dystrophin). The skipping of an exon is preferably induced by the binding of antisense oligoribonucleotides (AONs) targeting either one or both of the splice sites, or exon-internal sequences. Since an exon will only be included in the mRNA when both the splice sites are recognised by the spliceosome complex, splice sites are obvious targets for AONs. Alternatively, or additionally, one or more AONs are used which are specific for at least part of one or more exonic sequences. Using exon-internal AONs specific for an exon 46 sequence, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion11. Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also efficiently be induced in human control and patient muscle cells for 39 different DMD exons using exon-internal AONs1,2,11-15.
Hence, exon skipping techniques applied on the dystrophin gene result in the generation of at least partially functional—albeit shorter—dystrophin protein in DMD patients. Since DMD is caused by a dysfunctional dystrophin protein, it would be expected that the symptoms of DMD are sufficiently alleviated once a DMD patient has been provided with functional dystrophin protein. However, the present invention provides the insight that, even though exon skipping techniques are capable of inducing dystrophin synthesis, DMD symptom(s) is/are still further alleviated by administering to a DMD patient an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. According to the present invention, even when a dystrophin protein deficiency has been restored in a DMD patient, the presence of tissue inflammation and damaged muscle cells still continues to contribute to the symptoms of DMD. Hence, even though the cause of DMD—i.e. a dysfunctional dystrophin protein—is alleviated, treatment of DMD is still further improved by additionally using an adjunct therapy according to the present invention. Furthermore, the present invention provides the insight that a reduction of inflammation does not result in significant reduction of AON uptake by muscle cells. This is surprising because, in general, inflammation enhances the trafficking of cells, blood and other compounds. As a result, AON uptake/delivery is also enhanced during inflammation. Hence, before the present invention it would be expected that an adjunct therapy counteracting inflammation involves the risk of negatively influencing AON therapy. This, however, appears not to be the case.
The present invention therefore provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual, the method comprising:
administering to said individual a compound for providing said individual with a (at least partially) functional dystrophin protein, and
administering to said individual an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival.
In another preferred embodiment the method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual comprises administering to said individual an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival.
It has surprisingly been found that the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon, is enhanced if cells expressing said pre-mRNA are also provided with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
The present invention further provides a method for enhancing skipping of an exon from a dystrophin pre-mRNA in cells expressing said pre-mRNA, said method comprising
contacting said pre-mRNA in said cells with an oligonucleotide for skipping said exon and,
contacting said cells with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival.
As Duchenne and Becker muscular dystrophy have a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells. Preferably said cells comprise a gene encoding a mutant dystrophin protein. Preferably said cells are cells of an individual suffering from DMD or BMD.
The present invention further provides a method for enhancing skipping of an exon from a dystrophin pre-mRNA in cells expressing said pre-mRNA in an individual suffering from Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the method comprising:
administering to said individual a compound for providing said individual with a (at least partially) functional dystrophin protein, and
administering to said individual an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival
An individual is provided with a functional dystrophin protein in various ways. In one embodiment an exon skipping technique is applied. However, alternative methods are available, such as for instance stop codon suppression by gentamycin or PTC12416,17 (also known as 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), and/or adeno-associated virus (AAV)-mediated gene delivery of a functional mini- or micro-dystrophin gene18-20. PTC124™ is a registered trademark of PTC Therapeutics, Inc. South Plainfield, N.J.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin “At least to some extent” preferably means at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC)56. Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a biopsy of a muscle suspected to be dystrophic, as known to the skilled person.
Individuals suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevent synthesis of the complete protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the dystrophin gene also comprises a mutation compared tot the wild type but the mutation does typically not include a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin56. Exon—skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-domain shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchennes patient or is suspected to be a Duchennes patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus et al (2006, ref 56). The central rod domain of wild type dystrophin comprises 24 spectrin-like repeats56. For example, a central rod shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual in a method of the invention may be assessed by any of the following assays: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (2008, ref 58) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (2006, ref 57). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
An adjunct compound for reducing inflammation comprises any therapy which is capable of at least in part reducing inflammation, preferably inflammation caused by damaged muscle cells. Said adjunct compound is most preferably capable of reducing muscle tissue inflammation. Inflammation is preferably assessed by detecting an increase in the number of infiltrating immune cells such as neutrophils and/or mast cells and/or dendritic cells and/or lymphocytes in muscle tissue suspected to be dystrophic. This assessment is preferably carried out in cross-sections of a biopsy57 of muscle tissue suspected to be dystrophic after having specifically stained immune cells as identified above. The quantification is preferably carried out under the microscope. Reducing inflammation is therefore preferably assessed by detecting a decrease in the number of immune cells in a cross-section of muscle tissue suspected to be dystrophic. Detecting a decrease preferably means that the number of at least one sort of immune cells as identified above is decreased of at least 1%, 2%, 3%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the number of a corresponding immune cell in a same individual before treatment. Most preferably, no infiltrating immune cells are detected in cross-sections of said biopsy.
An adjunct compound for improving muscle fiber function, integrity and/or survival comprises any therapy which is capable of measurably enhancing muscle fiber function, integrity and/or survival as compared to an otherwise similar situation wherein said adjunct compound is not present. The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
Creatine kinase may be detected in blood as described in 57. A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same individual before treatment.
A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in 57 using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same individual before treatment.
A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in 57.
A treatment in a method according to the invention is about at least one week, about at least one month, about at least several months, about at least one year, about at least 2, 3, 4, 5, 6 years or more.
In one embodiment an adjunct compound for increasing turnover of damaged muscle cells is used. An adjunct compound for increasing turnover of damaged muscle cells comprises any therapy which is capable of at least in part inducing and/or increasing turnover of damaged muscle cells. Damaged muscle cells are muscle cells which have significantly less clinically measurable functionality than a healthy, intact muscle cell. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis, resulting in damaged muscle cells. Increasing turnover of damaged muscle cells means that damaged muscle cells are more quickly broken down and/or removed as compared to a situation wherein turnover of damaged muscle cells is not increased. Turnover of damaged muscle cells is preferably assessed in a muscle biopsy, more preferably as described in 57 using a cross-section of a biopsy. A detectable increase of turnover may be an increase of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein turnover has been identified using a biopsy cross-section. The increase is measured by comparison to the turnover as assessed in a same individual before treatment.
Without wishing to be bound to theory, it is believed that increasing turnover of muscle cells is preferred because this reduces inflammatory responses.
According to the present invention, a combination of a therapy for providing an individual with a functional dystrophin protein, together with an adjunct therapy for reducing inflammation, preferably for reducing muscle tissue inflammation in an individual, is particularly suitable for use as a medicament. Such combination is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy as compared to a sole therapy for providing an individual with a functional dystrophin protein. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
Further provided is therefore a combination of a compound for providing an individual with a functional dystrophin protein, and an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, for use as a medicament. Since said combination is particularly suitable for counteracting DMD, the invention also provides a use of a compound for providing an individual with a functional dystrophin protein, and an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, for the preparation of a medicament for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Preferred adjunct compound for reducing inflammation include a steroid, a TNF□ inhibitor, a source of mIGF-1 and/or an antioxidant. However, any other compound able to reduce inflammation as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for improving muscle fiber function, integrity and/or survival.
Furthermore, a combination of a therapy for providing an individual with a functional dystrophin protein, together with an adjunct therapy for improving muscle fiber function, integrity and/or survival in an individual is particularly suitable for use as a medicament. Such combination is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy as compared to a sole therapy for providing an individual with a functional dystrophin protein.
Further provided is therefore a combination of a compound for providing an individual with a functional dystrophin protein, and an adjunct compound for improving muscle fiber function, integrity and/or survival in said individual, for use as a medicament. This combination is also particularly suitable for counteracting DMD. A use of a compound for providing an individual with a functional dystrophin protein, and an adjunct compound for improving muscle fiber function, integrity and/or survival in said individual, for the preparation of a medicament for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy is therefore also provided. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Preferred adjunct compounds for improving muscle fiber function, integrity and/or survival include a ion channel inhibitor, a protease inhibitor, L-arginine and/or an angiotensin II type I receptor blocker. However, any other compound able to improving muscle fiber function, integrity and/or survival as defined herein is also encompassed within the present invention. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for reducing inflammation.
In one embodiment a pharmaceutical preparation is made which comprises at least one of the above mentioned combinations comprising a compound for providing an individual with a functional dystrophin protein together with an adjunct compound according to the invention. Further provided is therefore a pharmaceutical preparation comprising:
a compound for providing an individual with a functional dystrophin protein, and
an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival in said individual, and
a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Examples of suitable carriers and adjuvants are well known in the art and for instance comprise a saline solution. Dose ranges of compounds used in a pharmaceutical preparation according to the invention are designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist.
In a particularly preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with a steroid. As shown in the Examples, such combination results in significant alleviation of DMD symptoms. One preferred embodiment of the present invention therefore provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual a steroid and a compound for providing said individual with a functional dystrophin protein. A combination of a steroid and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of a steroid and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
A steroid is a terpenoid lipid characterized by a carbon skeleton with four fused rings, generally arranged in a 6-6-6-5 fashion. Steroids vary by the functional groups attached to these rings and the oxidation state of the rings. Steroids include hormones and drugs which are usually used to relieve swelling and inflammation, such as for instance prednisone, dexamethasone and vitamin D.
According to the present invention, supplemental effects of adjunct steroid therapy in DMD patients include reduction of tissue inflammation, suppression of cytotoxic cells, and improved calcium homeostasis. Most positive results are obtained in younger boys. Preferably the steroid is a corticosteroid (glucocorticosteroid). Preferably, prednisone steroids (such as prednisone, prednizolone or deflazacort) are used in a method according to the invention21. Dose ranges of (glucocortico)steroids to be used in the therapeutic applications as described herein are designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist. The usual doses are about 0.5-1.0 mg/kg/day, preferably about 0.75 mg/kg/day for prednisone and prednisolone, and about 0.4-1.4 mg/kg/day, preferably about 0.9 mg/kg/day for deflazacort.
In one embodiment, a steroid is administered to said individual prior to administering a compound for providing an individual with a functional dystrophin protein. In this embodiment, it is preferred that said steroid is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering a compound for providing said individual with a functional dystrophin protein.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with a tumour necrosis factor-alpha (TNFα) inhibitor. Tumour necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that stimulates the inflammatory response. Pharmacological blockade of TNFα activity with the neutralising antibody infliximab (Remicade) is highly effective clinically at reducing symptoms of inflammatory diseases. In mdx mice, both infliximab and etanercept delay and reduce the necrosis of dystrophic muscle24,25, with additional physiological benefits on muscle strength, chloride channel function and reduced CK levels being demonstrated in chronically treated exercised adult mdx mice26. Such highly specific anti-inflammatory drugs designed for use in other clinical conditions, are attractive alternatives to the use of steroids for DMD. In one embodiment, the use of a TNFα inhibitor is limited to periods of intensive muscle growth in boys when muscle damage and deterioration are especially pronounced.
One aspect of the present invention thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual a TNFα inhibitor and a compound for providing said individual with a functional dystrophin protein. A combination of a TNFα inhibitor and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of a TNFα inhibitor and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. A preferred TNFα inhibitor is a dimeric fusion protein consisting of the extracellular ligand-binding domain of the human p75 receptor of TNFα linked to the Fc portion of human IgGl. A more preferred TNFα inhibitor is ethanercept (Amgen, America)26. The usual doses of ethanercept is about 0.2 mg/kg, preferably about 0.5 mg/kg twice a week. The administration is preferably subcutaneous.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with a source of mIGF-1. As defined herein, a source of IGF-1 preferably encompasses mIGF-1 itself, a compound able of enhancing mIGF-1 expression and/or activity. Enhancing is herein synonymous with increasing. Expression of mIGF-1 is synonymous with amount of mIGF-1. mIGF-1 promotes regeneration of muscles through increase in satellite cell activity, and reduces inflammation and fibrosis27. Local injury of muscle results in increased mIGF-1 expression. In transgenic mice with extra IGF-1 genes, muscle hypertrophy and enlarged muscle fibers are observed27. Similarly, transgenic mdx mice show reduced muscle fiber degeneration28. Upregulation of the mIGF-1 gene and/or administration of extra amounts of mIGF-1 protein or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 2]) thus promotes the effect of other, preferably genetic, therapies for DMD, including antisense-induced exon skipping. The additional mIGF-1 levels in the above mentioned transgenic mice do not induce cardiac problems nor promote cancer, and have no pathological side effects. One aspect of the present invention thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual a compound for providing said individual with a functional dystrophin protein, and providing said individual with a source of mIGF-1, preferably mIGF-1 itself, a compound able of increasing mIGF-1 expression and/or activity. As stated before, the amount of mIGF-1 is for instance increased by enhancing expression of the mIGF-1 gene and/or by administration of mIGF-1 protein and/or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 2]). A combination of mIGF-1, or a compound capable of enhancing mIGF-1 expression or an mIGF-1 activity, and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of mIGF-1, or a compound capable of enhancing mIGF-1 expression or mIGF-1 activity, and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, such combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Within the context of the invention, an increased amount or activity of mIGF-1 may be reached by increasing the gene expression level of an IGF-1 gene, by increasing the amount of a corresponding IGF-1 protein and/or by increasing an activity of an IGF1-protein. A preferred mIGF-1 protein has been earlier defined herein. An increase of an activity of said protein is herein understood to mean any detectable change in a biological activity exerted by said protein or in the steady state level of said protein as compared to said activity or steady-state in a individual who has not been treated. Increased amount or activity of mIGF-1 is preferably assessed by detection of increased expression of muscle hypertrophy biomarker GATA-2 (as described in 27).
Gene expression level is preferably assessed using classical molecular biology techniques such as (real time) PCR, arrays or Northern analysis. A steady state level of a protein is determined directly by quantifying the amount of a protein. Quantifying a protein amount may be carried out by any known technique such as Western blotting or immunoassay using an antibody raised against a protein. The skilled person will understand that alternatively or in combination with the quantification of a gene expression level and/or a corresponding protein, the quantification of a substrate of a corresponding protein or of any compound known to be associated with a function or activity of a corresponding protein or the quantification of said function or activity of a corresponding protein using a specific assay may be used to assess the alteration of an activity or steady state level of a protein.
In a method of the invention, an activity or steady-state level of a said protein may be altered at the level of the protein itself, e.g. by providing a protein to a cell from an exogenous source.
Preferably, an increase or an upregulation of the expression level of a said gene means an increase of at least 5% of the expression level of said gene using arrays. More preferably, an increase of the expression level of said gene means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. In another preferred embodiment, an increase of the expression level of said protein means an increase of at least 5% of the expression level of said protein using western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a protein means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.
In another preferred embodiment, an increase of a polypeptide activity means an increase of at least 5% of a polypeptide activity using a suitable assay. More preferably, an increase of a polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. The increase is preferably assessed by comparison to corresponding activity in the individual before treatment.
A preferred way of providing a source of mIGF1 is to introduce a transgene encoding mIGF1, preferably an mIGF-1 Ea isoform (as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 2), more preferably in an AAV vector as later defined herein. Such source of mIGF1 is specifically expressed in muscle tissue as described in mice in 27.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with an antioxidant. Oxidative stress is an important factor in the progression of DMD and promotes chronic inflammation and fibrosis29. The most prevalent products of oxidative stress, the peroxidized lipids, are increased by an average of 35% in Duchenne boys. Increased levels of the enzymes superoxide dismutase and catalase reduce the excessive amount of free radicals causing these effects. In fact, a dietary supplement Protandim® (LifeVantage) was clinically tested and found to increase levels of superoxide dismutase (up to 30%) and catalase (up to 54%), which indeed significantly inhibited the peroxidation of lipids in 29 healthy persons30. Such effective management of oxidative stress thus preserves muscle quality and so promotes the positive effect of DMD therapy. Idebenone is another potent antioxidant with a chemical structure derived from natural coenzyme Q10. It protects mitochondria where adenosine triphosphate, ATP, is generated by oxidative phosphorylation. The absence of dystrophin in DMD negatively affects this process in the heart, and probably also in skeletal muscle. Idebenone was recently applied in clinical trials in the US and Europe demonstrating efficacy on neurological aspects of Friedreich's Ataxia31. A phase-IIa double-blind, placebo-controlled randomized clinical trial with Idebenone has recently been started in Belgium, including 21 Duchenne boys at 8 to 16 years of age. The primary objective of this study is to determine the effect of Idebenone on heart muscle function. In addition several different tests will be performed to detect the possible functional benefit on muscle strength in the patients. When effective, Idebenone is a preferred adjunct compound for use in a method according to the present invention in order to enhance the therapeutic effect of DMD therapy, especially in the heart. One aspect of the present invention thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual an antioxidant and a compound for providing said individual with a functional dystrophin protein. A combination of an antioxidant and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of an antioxidant and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the antioxidant, the skilled person will know which quantities are preferably used. An antioxidant may include bacoside, silymarin, curcumin, a polyphenol, preferably epigallocatechin-3-gallate (EGCG). Preferably, an anti-oxidant is a mixture of antioxidants as the dietary supplement Protandim® (LifeVantage). A daily capsule of 675 mg of Protandim® comprises 150 mg of B. monniera (45% bacosides), 225 mg of S. marianum (70-80% silymarin), 150 mg of W. somnifera powder, 75 mg green tea (98% polyphenols wherein 45% EGCG) and 75 mg turmeric (95% curcumin).
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with an ion channel inhibitor. The presence of damaged muscle membranes in DMD disturbs the passage of calcium ions into the myofibers, and the consequently disrupted calcium homeostasis activates many enzymes, e.g. proteases, that cause additional damage and muscle necrosis. Ion channels that directly contribute to the pathological accumulation of calcium in dystrophic muscle are potential targets for adjunct compounds to treat DMD. There is evidence that some drugs, such as pentoxifylline, block exercise-sensitive calcium channels32 and antibiotics that block stretch activated channels reduce myofibre necrosis in mdx mice and CK levels in DMD boys33. One embodiment thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual an ion channel inhibitor and a compound for providing said individual with a functional dystrophin protein. A combination of an ion channel inhibitor and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of an ion channel inhibitor and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
Preferably, ion channel inhibitors of the class of xanthines are used. More preferably, said xanthines are derivatives of methylxanthines, and most preferably, said methylxanthine derivates are chosen from the group consisting of pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof. Most preferred is the use of pentoxifylline. Ion channel inhibitors of the class of xanthines enhance the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
Depending on the identity of the ion channel inhibitor, the skilled person will know which quantities are preferably used. Suitable dosages of pentoxifylline are between about 1 mg/kg/day to about 100 mg/kg/day, preferred dosages are between about 10 mg/kg/day to 50 mg/kg/day. Typical dosages used in humans are 20 mg/kg/day.
In one embodiment, an ion channel inhibitor is administered to said individual prior to administering a compound for providing an individual with a functional dystrophin protein. In this embodiment, it is preferred that said ion channel inhibitor is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering a compound for providing said individual with a functional dystrophin protein.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with a protease inhibitor. Calpains are calcium activated proteases that are increased in dystrophic muscle and account for myofiber degeneration. Calpain inhibitors such as calpastatin, leupeptin34, calpeptin, calpain inhibitor III, or PD150606 are therefore applied to reduce the degeneration process. A new compound, BN 82270 (Ipsen) that has dual action as both a calpain inhibitor and an antioxidant increased muscle strength, decreased serum CK and reduced fibrosis of the mdx diaphragm, indicating a therapeutic effect with this new compound35. Another compound of Leupeptin/Carnitine (Myodur) has recently been proposed for clinical trials in DMD patients.
MG132 is another proteasomal inhibitor that has shown to reduce muscle membrane damage, and to ameliorate the histopathological signs of muscular dystrophy36. MG-132 (CBZ-leucyl-leucyl-leucinal) is a cell-permeable, proteasomal inhibitor (Ki=4 nM) which inhibits NFkappaB activation by preventing IkappaB degradation (IC50=3 □M). In addition, it is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis by binding to and inactivating 20S and 26S proteasomes. MG-132 has shown to inhibit the proteasomal degradation of dystrophin-associated proteins in the dystrophic mdx mouse model36. This compound is thus also suitable for use as an adjunct pharmacological compound for DMD. Further provided is therefore a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual a protease inhibitor and a compound for providing said individual with a functional dystrophin protein. A combination of a protease inhibitor and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of a protease inhibitor and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the protease inhibitor, the skilled person will know which quantities are preferably used.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with L-arginine. Dystrophin-deficiency is associated with the loss of the DGC-complex at the fiber membranes, including neuronal nitric oxide synthase (nNOS). Expression of a nNOS transgene in mdx mice greatly reduced muscle membrane damage. Similarly, administration of L-arginine (the substrate for nitric oxide synthase) increased NO production and upregulated utrophin expression in mdx mice. Six weeks of L-arginine treatment improved muscle pathology and decreased serum CK in mdx mice37. The use of L-arginine as an adjunct therapy in combination with a compound for providing said individual with a functional dystrophin protein has not been disclosed.
Further provided is therefore a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual L-arginine and a compound for providing said individual with a functional dystrophin protein. A combination of L-arginine and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of L-arginine and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with angiotensin II type 1 receptor blocker Losartan which normalizes muscle architecture, repair and function, as shown in the dystrophin-deficient mdx mouse model23. One aspect of the present invention thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual angiotensin II type 1 receptor blocker Losartan, and a compound for providing said individual with a functional dystrophin protein. A combination of angiotensin II type 1 receptor blocker Losartan and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of angiotensin II type 1 receptor blocker Losartan and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the angiotensin II type 1 receptor blocker, the skilled person will know which quantities are preferably used.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with an angiotensin-converting enzyme (ACE) inhibitor, preferably perindopril. ACE inhibitors are capable of lowering blood pressure. Early initiation of treatment with perindopril is associated with a lower mortality in DMD patients22. One aspect of the present invention thus provides a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual an ACE inhibitor, preferably perindopril, and a compound for providing said individual with a functional dystrophin protein. A combination of an ACE inhibitor, preferably perindopril, and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of an ACE inhibitor, preferably perindopril, and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of an ACE inhibitor, preferably perindopril are about 2 to 4 mg/day22.
In a more preferred embodiment, an ACE inhibitor is combined with at least one of the previously identified adjunct compounds.
In another preferred embodiment, a compound for providing an individual with a functional dystrophin protein is combined with a compound which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing. Small chemical compounds, such as for instance specific indole derivatives, have been shown to selectively inhibit spliceosome assembly and splicing38, for instance by interfering with the binding of serine- and arginine-rich (SR) proteins to their cognate splicing enhancers (ISEs or ESEs) and/or by interfering with the binding of splicing repressors to silencer sequences (ESSs or ISSs). These compounds are therefore suitable for applying as adjunct compounds that enhance exon skipping.
Further provided is therefore a method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy in an individual, the method comprising administering to said individual a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing, and a compound for providing said individual with a functional dystrophin protein. A combination of a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing and a compound for providing an individual with a functional dystrophin protein for use as a medicament is also provided, as well as a use of a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing and a compound for providing an individual with a functional dystrophin protein for the preparation of a medicament for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the compound which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing, the skilled person will know which quantities are preferably used. In a more preferred embodiment, a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing is combined with a ACE inhibitor and/or with any adjunct compounds as identified earlier herein.
A pharmaceutical preparation comprising a compound for providing an individual with a functional dystrophin protein, any of the above mentioned adjunct compounds, and a pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
The invention thus provides a method, combination, use or pharmaceutical preparation according to the invention, wherein said adjunct compound comprises a steroid, an ACE inhibitor (preferably perindopril), angiotensin II type 1 receptor blocker Losartan, a tumour necrosis factor-alpha (TNFα) inhibitor, a source of mIGF-1, preferably mIGF-1, a compound for enhancing mIGF-1 expression, a compound for enhancing mIGF-1 activity, an antioxidant, an ion channel inhibitor, a protease inhibitor, L-arginine and/or a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing.
As described herein before, an individual is provided with a functional dystrophin protein in various ways, for instance by stop codon suppression by gentamycin or PTC12416,17, or by adeno-associated virus (AAV)-mediated gene delivery of a functional mini- or micro-dystrophin gene18-20.
Preferably, however, said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, for at least in part decreasing the production of an aberrant dystrophin protein in said individual. Decreasing the production of an aberrant dystrophin mRNA, or aberrant dystrophin protein, preferably means that 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, or aberrant dystrophin protein, is still detectable by RT PCR (mRNA) or immunofluorescence or western blot analysis (protein). An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein. Said oligonucleotide preferably comprises an antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied.
Exon skipping interferes with the natural splicing processes occurring within a eukaryotic cell. In higher eukaryotes the genetic information for proteins in the DNA of the cell is encoded in exons which are separated from each other by intronic sequences. These introns are in some cases very long. The transcription machinery of eukaryotes generates a pre-mRNA which contains both exons and introns, while the splicing machinery, often already during the production of the pre-mRNA, generates the actual coding region for the protein by splicing together the exons present in the pre-mRNA.
Exon-skipping results in mature mRNA that lacks at least one skipped exon. Thus, when said exon codes for amino acids, exon skipping leads to the expression of an altered product. Technology for exon-skipping is currently directed towards the use of antisense oligonucleotides (AONs). Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy. The mdx mouse, which carries a non-sense mutation in exon 23 of the dystrophin gene, has been used as an animal model of DMD. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. AONs directed to, respectively, the 3′ and/or 5′ splice sites of introns 22 and 23 in dystrophin pre-mRNA, have been shown to interfere with factors normally involved in removal of intron 23 so that also exon 23 was removed from the mRNA3,5,6,39,40.
By the targeted skipping of a specific exon, a DMD phenotype is converted into a milder BMD phenotype. The skipping of an exon is preferably induced by the binding of AONs targeting either one or both of the splice sites, or exon-internal sequences. An oligonucleotide directed toward an exon internal sequence typically exhibits no overlap with non-exon sequences. It preferably does not overlap with the splice sites at least not insofar as these are present in the intron. An oligonucleotide directed toward an exon internal sequence preferably does not contain a sequence complementary to an adjacent intron. Further provided is thus a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, for inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA produced from splicing of said pre-mRNA. An exon skipping technique is preferably applied such that the absence of an exon from mRNA produced from dystrophin pre-mRNA generates a coding region for a functional—albeit shorter—dystrophin protein. In this context, inhibiting inclusion of an exon preferably means that the detection of the original, aberrant dystrophin mRNA is decreased of at least about 10% as assessed by RT-PCR or that a corresponding aberrant dystrophin protein is decreased of at least about 10% as assessed by immunofluorescence or western blot analysis. The decrease is preferably of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
Once a DMD patient is provided with a functional dystrophin protein, the cause of DMD is taken away. Hence, it would then be expected that the symptoms of DMD are sufficiently alleviated. However, as already described before, the present invention provides the insight that, even though exon skipping techniques are capable of providing a functional dystrophin protein, a symptom of DMD is still further alleviated by administering to a DMD patient an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. Moreover, the present invention provides the insight that an adjunct therapy counteracting inflammation does not negatively influence AON therapy. The present invention further provides the insight that the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon is enhanced, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.
Since an exon of a dystrophin pre-mRNA will only be included into the resulting mRNA when both the splice sites are recognised by the spliceosome complex, splice sites are obvious targets for AONs. One embodiment therefore provides a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a non-exon region of a dystrophin pre mRNA. In one embodiment an AON is used which is solely complementary to a non-exon region of a dystrophin pre mRNA. This is however not necessary: it is also possible to use an AON which comprises an intron-specific sequence as well as exon-specific sequence. Such AON comprises a sequence which is complementary to a non-exon region of a dystrophin pre mRNA, as well as a sequence which is complementary to an exon region of a dystrophin pre mRNA. Of course, an AON is not necessarily complementary to the entire sequence of a dystrophin exon or intron. AONs which are complementary to a part of such exon or intron are preferred. An AON is preferably complementary to at least part of a dystrohin exon and/or intron, said part having at least 13 nucleotides.
Splicing of a dystrophin pre-mRNA occurs via two sequential transesterification reactions. First, the 2′OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5′ splice site forming the lariat intermediate. Second, the 3′OH of the released 5′ exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3′ splice site thus joining the exons and releasing the intron lariat. The branch point and splice sites of an intron are thus involved in a splicing event. Hence, an oligonucleotide comprising a sequence which is complementary to such branch point and/or splice site is preferably used for exon skipping. Further provided is therefore a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a splice site and/or branch point of a dystrophin pre mRNA.
Since splice sites contain consensus sequences, the use of an oligonucleotide or a functional equivalent thereof (herein also called an AON) comprising a sequence which is complementary of a splice site involves the risk of promiscuous hybridization. Hybridization of AONs to other splice sites than the sites of the exon to be skipped could easily interfere with the accuracy of the splicing process. To overcome these and other potential problems related to the use of AONs which are complementary to an intron sequence, one preferred embodiment provides a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a dystrophin pre-mRNA exon. Preferably, said AON is capable of specifically inhibiting an exon inclusion signal of at least one exon in said dystrophin pre-mRNA. Interfering with an exon inclusion signal (EIS) has the advantage that such elements are located within the exon. By providing an AON for the interior of the exon to be skipped, it is possible to interfere with the exon inclusion signal thereby effectively masking the exon from the splicing apparatus. The failure of the splicing apparatus to recognize the exon to be skipped thus leads to exclusion of the exon from the final mRNA. This embodiment does not interfere directly with the enzymatic process of the splicing machinery (the joining of the exons). It is thought that this allows the method to be more specific and/or reliable. It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In this concept it is the particular spatial conformation that enables the splicing machinery to recognize the exon. However, the invention is certainly not limited to this model. It has been found that agents capable of binding to an exon are capable of inhibiting an EIS. An AON may specifically contact said exon at any point and still be able to specifically inhibit said EIS.
Using exon-internal AONs specific for an exon 46 sequence, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion11. Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also efficiently be induced in human control and series of patients with different mutations, including deletions, duplications and point mutations, for 39 different DMD exons using exon-internal AONs1,2,11-15.
Within the context of the invention, a functional equivalent of an oligonucleotide preferably means an oligonucleotide as defined herein wherein one or more nucleotides have been substituted and wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by quantifying the amount of a functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analyses. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
Hence, the use of an oligonucleotide, or a functional equivalent thereof, comprising or consisting of a sequence which is complementary to a dystrophin pre-mRNA exon provides good anti-DMD results. In one preferred embodiment an oligonucleotide, or a functional equivalent thereof, is used which comprises or consists of a sequence which is complementary to at least part of dystrophin pre-mRNA exon 2, 8, 9, 17, 19, 29, 40-46, 48-53, 55 or 59, said part having at least 13 nucleotides. However, said part may also have at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides.
Most preferably an AON is used which comprises or consists of a sequence which is complementary to at least part of dystrophin pre-mRNA exon 51, 44, 45, 53, 46, 43, 2, 8, 50 and/or 52, said part having at least 13 nucleotides. However, said part may also have at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides. Most preferred oligonucleotides are identified by each of the following sequences SEQ ID NO: 3 to SEQ ID NO: 284. Accordingly, a most preferred oligonucleotide as used herein is represented by a sequence from SEQ ID NO:3 to SEQ ID NO:284. A most preferred oligonucleotide as used herein is selected from the group consisting of SEQ ID NO:3 to NO:284.
Said exons are listed in decreasing order of patient population applicability. Hence, the use of an AON comprising a sequence which is complementary to at least part of dystrophin pre-mRNA exon 51 is suitable for use in a larger part of the DMD patient population as compared to an AON comprising a sequence which is complementary to dystrophin pre-mRNA exon 44, et cetera.
In a preferred embodiment, an oligonucleotide of the invention which comprises a sequence that is complementary to part of dystrophin pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or more. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary to part of dystrophin pre-mRNA as defined herein. For example, an oligonucleotide may comprise a sequence that is complementary to part of dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to the oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.
One preferred embodiment provides a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, which comprises:
a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is hybridized to another part of a dystrophin pre-mRNA exon (closed structure), and
a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is not hybridized in said dystrophin pre-mRNA (open structure).
For this embodiment, reference is made to our WO 2004/083432 patent application. RNA molecules exhibit strong secondary structures, mostly due to base pairing of complementary or partly complementary stretches within the same RNA. It has long since been thought that structures in the RNA play a role in the function of the RNA. Without being bound by theory, it is believed that the secondary structure of the RNA of an exon plays a role in structuring the splicing process. Through its structure, an exon is recognized as a part that needs to be included in the mRNA. Herein this signalling function is referred to as an exon inclusion signal. A complementary oligonucleotide of this embodiment is capable of interfering with the structure of the exon and thereby capable of interfering with the exon inclusion signal of the exon. It has been found that many complementary oligonucleotides indeed comprise this capacity, some more efficient than others. Oligonucleotides of this preferred embodiment, i.e. those with the said overlap directed towards open and closed structures in the native exon RNA, are a selection from all possible oligonucleotides. The selection encompasses oligonucleotides that can efficiently interfere with an exon inclusion signal. Without being bound by theory it is thought that the overlap with an open structure improves the invasion efficiency of the oligonucleotide (i.e. increases the efficiency with which the oligonucleotide can enter the structure), whereas the overlap with the closed structure subsequently increases the efficiency of interfering with the secondary structure of the RNA of the exon, and thereby interfere with the exon inclusion signal. It is found that the length of the partial complementarity to both the closed and the open structure is not extremely restricted. We have observed high efficiencies with oligonucleotides with variable lengths of complementarity in either structure. The term complementarity is used herein to refer to a stretch of nucleic acids that can hybridise to another stretch of nucleic acids under physiological conditions. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the oligonucleotide one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may to some extent be allowed, if under the circumstances in the cell, the stretch of nucleotides is capable of hybridising to the complementary part. In a preferred embodiment a complementary part (either to said open or to said closed structure) comprises at least 3, and more preferably at least 4 consecutive nucleotides. The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-)mRNA in the system. The risk that also one or more other pre-mRNA will be able to hybridise to the oligonucleotide decreases with increasing size of the oligonucleotide. It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain the capacity to hybridise to the targeted region(s) in the pre-mRNA, can be used in the present invention. However, preferably at least the complementary parts do not comprise such mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides having such mismatches in one or more complementary regions. It is thought that higher hybridisation strengths, (i.e. increasing number of interactions with the opposing strand) are favourable in increasing the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the complementarity is between 90 and 100%. In general this allows for approximately 1 or 2 mismatch(es) in an oligonucleotide of around 20 nucleotides
The secondary structure is best analysed in the context of the pre-mRNA wherein the exon resides. Such structure may be analysed in the actual RNA. However, it is currently possible to predict the secondary structure of an RNA molecule (at lowest energy costs) quite well using structure-modelling programs. A non-limiting example of a suitable program is RNA mfold version 3.1 server41. A person skilled in the art will be able to predict, with suitable reproducibility, a likely structure of the exon, given the nucleotide sequence. Best predictions are obtained when providing such modelling programs with both the exon and flanking intron sequences. It is typically not necessary to model the structure of the entire pre-mRNA.
The open and closed structure to which the oligonucleotide is directed, are preferably adjacent to one another. It is thought that in this way the annealing of the oligonucleotide to the open structure induces opening of the closed structure whereupon annealing progresses into this closed structure. Through this action the previously closed structure assumes a different conformation. The different conformation results in the disruption of the exon inclusion signal. However, when potential (cryptic) splice acceptor and/or donor sequences are present within the targeted exon, occasionally a new exon inclusion signal is generated defining a different (neo) exon, i.e. with a different 5′ end, a different 3′ end, or both. This type of activity is within the scope of the present invention as the targeted exon is excluded from the mRNA. The presence of a new exon, containing part of the targeted exon, in the mRNA does not alter the fact that the targeted exon, as such, is excluded. The inclusion of a neo-exon can be seen as a side effect which occurs only occasionally. There are two possibilities when exon skipping is used to restore (part of) an open reading frame of dystrophin that is disrupted as a result of a mutation. One is that the neo-exon is functional in the restoration of the reading frame, whereas in the other case the reading frame is not restored. When selecting oligonucleotides for restoring dystrophin reading frames by means of exon-skipping it is of course clear that under these conditions only those oligonucleotides are selected that indeed result in exon-skipping that restores the dystrophin open reading frame, with or without a neo-exon.
Further provided is a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises an oligonucleotide, or a functional equivalent thereof, which comprises a sequence that is complementary to a binding site for a serine-arginine (SR) protein in RNA of an exon of a dystrophin pre-mRNA. In our WO 2006/112705 patent application we have disclosed the presence of a correlation between the effectivity of an exon-internal antisense oligonucleotide (AON) in inducing exon skipping and the presence of a (for example by ESEfinder) predicted SR binding site in the target pre-mRNA site of said AON. Therefore, in one embodiment an oligonucleotide is generated comprising determining a (putative) binding site for an SR (Ser-Arg) protein in RNA of a dystrophin exon and producing an oligonucleotide that is complementary to said RNA and that at least partly overlaps said (putative) binding site. The term “at least partly overlaps” is defined herein as to comprise an overlap of only a single nucleotide of an SR binding site as well as multiple nucleotides of said binding site as well as a complete overlap of said binding site. This embodiment preferably further comprises determining from a secondary structure of said RNA, a region that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide that at least partly overlaps said (putative) binding site and that overlaps at least part of said closed structure and overlaps at least part of said open structure. In this way we increase the chance of obtaining an oligonucleotide that is capable of interfering with the exon inclusion from the pre-mRNA into mRNA. It is possible that a first selected SR-binding region does not have the requested open-closed structure in which case another (second) SR protein binding site is selected which is then subsequently tested for the presence of an open-closed structure. This process is continued until a sequence is identified which contains an SR protein binding site as well as a(n) (partly overlapping) open-closed structure. This sequence is then used to design an oligonucleotide which is complementary to said sequence.
Such a method for generating an oligonucleotide is also performed by reversing the described order, i.e. first generating an oligonucleotide comprising determining, from a secondary structure of RNA from a dystrophin exon, a region that assumes a structure that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide, of which at least a part of said oligonucleotide is complementary to said closed structure and of which at least another part of said oligonucleotide is complementary to said open structure. This is then followed by determining whether an SR protein binding site at least overlaps with said open/closed structure. In this way the method of WO 2004/083432 is improved. In yet another embodiment the selections are performed simultaneously.
Without wishing to be bound by any theory it is currently thought that use of an oligonucleotide directed to an SR protein binding site results in (at least partly) impairing the binding of an SR protein to the binding site of an SR protein which results in disrupted or impaired splicing.
Preferably, an open/closed structure and an SR protein binding site partly overlap and even more preferred an open/closed structure completely overlaps an SR protein binding site or an SR protein binding site completely overlaps an open/closed structure. This allows for an improved disruption of exon inclusion.
Besides consensus splice sites sequences, many (if not all) exons contain splicing regulatory sequences such as exonic splicing enhancer (ESE) sequences to facilitate the recognition of genuine splice sites by the spliceosome42,43. A subgroup of splicing factors, called the SR proteins, can bind to these ESEs and recruit other splicing factors, such as U1 and U2AF to (weakly defined) splice sites. The binding sites of the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55) have been analyzed in detail and these results are implemented in ESEfinder, a web source that predicts potential binding sites for these SR proteins42,43. There is a correlation between the effectiveness of an AON and the presence/absence of an SF2/ASF, SC35 and SRp40 binding site. In a preferred embodiment, the invention thus provides a method, combination, use or pharmaceutical preparation as described above, wherein said SR protein is SF2/ASF or SC35 or SRp40.
In one embodiment a DMD patient is provided with a functional dystrophin protein by using an oligonucleotide, or a functional equivalent thereof, which is capable of specifically binding a regulatory RNA sequence which is required for the correct splicing of a dystrophin exon in a transcript. Several cis-acting RNA sequences are required for the correct splicing of exons in a transcript. In particular, supplementary elements such as intronic or exonic splicing enhancers (ISEs and ESEs) or silencers (ISSs and ESEs) are identified to regulate specific and efficient splicing of constitutive and alternative exons. Using sequence-specific antisense oligonucleotides (AONs) that bind to the elements, their regulatory function is disturbed so that the exon is skipped, as shown for DMD. Hence, in one preferred embodiment an oligonucleotide or functional equivalent thereof is used which is complementary to an intronic splicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronic splicing silencer (ISS) and/or an exonic splicing silencer (ESS). As already described herein before, a dystrophin exon is in one preferred embodiment skipped by an agent capable of specifically inhibiting an exon inclusion signal of said exon, so that said exon is not recognized by the splicing machinery as a part that needs to be included in the mRNA. As a result, a mRNA without said exon is formed.
An AON used in a method of the invention is preferably complementary to a consecutive part of between 13 and 50 nucleotides of dystrophin exon RNA or dystrophin intron RNA. In one embodiment an AON used in a method of the invention is complementary to a consecutive part of between 16 and 50 nucleotides of a dystrophin exon RNA or dystrophin intron RNA. Preferably, said AON is complementary to a consecutive part of between 15 and 25 nucleotides of said exon RNA. More preferably, an AON is used which comprises a sequence which is complementary to a consecutive part of between 20 and 25 nucleotides of a dystrophin exon RNA or a dystrophin intron RNA.
Different types of nucleic acid may be used to generate the oligonucleotide. Preferably, said oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Since one of the aims of the exon skipping technique is to direct splicing in subjects it is preferred that the oligonucleotide RNA comprises a modification providing the RNA with an additional property, for instance resistance to endonucleases and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc. Preferably said modification comprises a 2′-O-methyl-phosphorothioate oligoribonucleotide modification. Preferably said modification comprises a 2′-O-methyl-phosphorothioate oligodeoxyribonucleotide modification. One embodiment thus provides a method, combination, use or pharmaceutical preparation according to the invention, wherein an oligonucleotide is used which comprises RNA which contains a modification, preferably a 2′-O-methyl modified ribose (RNA) or deoxyribose (DNA) modification.
In one embodiment the invention provides a hybrid oligonucleotide comprising an oligonucleotide comprising a 2′-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification and locked nucleic acid. This particular combination comprises better sequence specificity compared to an equivalent consisting of locked nucleic acid, and comprises improved effectivity when compared with an oligonucleotide consisting of 2′-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification.
With the advent of nucleic acid mimicking technology it has become possible to generate molecules that have a similar, preferably the same hybridisation characteristics in kind not necessarily in amount as nucleic acid itself. Such functional equivalents are of course also suitable for use in a method of the invention. Preferred examples of functional equivalents of an oligonucleotide are peptide nucleic acid and/or locked nucleic acid. Most preferably, a morpholino phosphorodiamidate is used. Suitable but non-limiting examples of equivalents of oligonucleotides of the invention can be found in44,50. Hybrids between one or more of the equivalents among each other and/or together with nucleic acid are of course also suitable. In a preferred embodiment locked nucleic acid is used as a functional equivalent of an oligonucleotide, as locked nucleic acid displays a higher target affinity and reduced toxicity and therefore shows a higher efficiency of exon skipping.
In one embodiment an oligonucleotide, or a functional equivalent thereof, which is capable of inhibiting inclusion of a dystrophin exon into dystrophin mRNA is combined with at least one other oligonucleotide, or functional equivalent thereof, that is capable of inhibiting inclusion of another dystrophin exon into dystrophin mRNA. This way, inclusion of two or more exons of a dystrophin pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping2,15. In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other.
Further provided is therefore a method, combination, use or pharmaceutical preparation according to the invention, wherein a nucleotide sequence is used which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first exon of a dystrophin pre-mRNA and wherein a nucleotide sequence is used which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon of said dystrophin pre-mRNA.
In one preferred embodiment said first and said second exon are separated in said dystrophin pre-mRNA by at least one exon to which said oligonucleotide is not complementary.
It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule. Further provided is therefore a method, combination, use or pharmaceutical preparation according to the invention wherein said oligonucleotide, or functional equivalent thereof, for providing said individual with a functional dystrophin protein is complementary to at least two exons in a dystrophin pre-mRNA, said oligonucleotide or functional equivalent comprising at least two parts wherein a first part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first of said at least two exons and wherein a second part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon in said dystrophin pre-mRNA. The linkage may be through any means but is preferably accomplished through a nucleotide linkage. In the latter case the number of nucleotides that do not contain an overlap between one or the other complementary exon can be zero, but is preferably between 4 to 40 nucleotides. The linking moiety can be any type of moiety capable of linking oligonucleotides. Preferably, said linking moiety comprises at least 4 uracil nucleotides. Currently, many different compounds are available that mimic hybridisation characteristics of oligonucleotides. Such a compound, called herein a functional equivalent of an oligonucleotide, is also suitable for the present invention if such equivalent comprises similar hybridisation characteristics in kind not necessarily in amount. Suitable functional equivalents are mentioned earlier in this description. As mentioned, oligonucleotides of the invention do not have to consist of only oligonucleotides that contribute to hybridisation to the targeted exon. There may be additional material and/or nucleotides added.
The DMD gene is a large gene, with many different exons. Considering that the gene is located on the X-chromosome, it is mostly boys that are affected, although girls can also be affected by the disease, as they may receive a bad copy of the gene from both parents, or are suffering from a particularly biased inactivation of the functional allele due to a particularly biased X chromosome inactivation in their muscle cells. The protein is encoded by a plurality of exons (79) over a range of at least 2.6 Mb. Defects may occur in any part of the DMD gene. Skipping of a particular exon or particular exons can, very often, result in a restructured mRNA that encodes a shorter than normal but at least partially functional dystrophin protein. A practical problem in the development of a medicament based on exon-skipping technology is the plurality of mutations that may result in a deficiency in functional dystrophin protein in the cell. Despite the fact that already multiple different mutations can be corrected for by the skipping of a single exon, this plurality of mutations, requires the generation of a large number of different pharmaceuticals as for different mutations different exons need to be skipped. An advantage of a compound capable of inducing skipping of two or more exons, is that more than one exon can be skipped with a single pharmaceutical. This property is not only practically very useful in that only a limited number of pharmaceuticals need to be generated for treating many different DMD or particular, severe BMD mutations. Another option now open to the person skilled in the art is to select particularly functional restructured dystrophin proteins and produce compounds capable of generating these preferred dystrophin proteins. Such preferred end results are further referred to as mild phenotype dystrophins.
Each compound, an oligonucleotide and/or an adjunct compound as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro.
Alternatively, suitable means for providing cells with an oligonucleotide or equivalent thereof are present in the art. An oligonucleotide or functional equivalent thereof may for example be provided to a cell in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into the cell via a gene delivery vehicle. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector4,51,52 and the like. Also plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from PolIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are PolIII driven transcripts. Preferably in the form of a fusion transcript with an U1 or U7 transcript4,51,52. Such fusions may be generated as described53,54. The oligonucleotide may be delivered as is. However, the oligonucleotide may also be encoded by the viral vector. Typically this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.
Improvements in means for providing cells with an oligonucleotide or equivalent thereof, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. The oligonucleotide or equivalent thereof can be delivered as is to the cells. When administering the oligonucleotide or equivalent thereof to an individual, it is preferred that the oligonucleotide is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will aid in delivery of a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound to a cell and into a cell, preferably a muscle cell. Preferred are excipients capable of forming complexes, vesicles and/or liposomes that deliver such a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, preferably an oligonucleotide and optionally together with an adjunct compound as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
In addition, a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide (-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and optionally together with an adjunct compound and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
It is to be understood that an oligonucleotide and an adjunct compound may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
In a preferred embodiment the invention provides a kit of parts comprising a compound for providing an individual with a functional dystrophin protein and an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival.
In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged between about 0.1 nM and about 1 μM is used. More preferably, the concentration used is ranged between about 0.3 to about 400 nM, even more preferably between about 1 to about 200 nM. If several oligonucleotides are used, this concentration may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, a compound preferably an oligonucleotide and an adjunct compound to be used in the invention to prevent, treat DMD or BMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
Besides exon skipping, it is also possible to provide a DMD patient with a functional dystrophin protein with a therapy based on read-through of stopcodons. Compounds capable of suppressing stopcodons are particularly suitable for a subgroup of DMD patients which is affected by nonsense mutations (˜7%) resulting in the formation of a stop codon within their dystrophin gene. In one embodiment said compound capable of suppressing stopcodons comprises the antibiotic gentamicin. In a recent study in mdx mice, gentamicin treatment induced novel dystrophin expression up to 20% of normal level, albeit with variability among animals. Human trials with gentamicin have however been inconclusive55. PTC124 belongs to a new class of small molecules that mimics at lower concentrations the readthrough activity of gentamicin. Administration of PTC124 resulted in the production of full-length and functionally active dystrophin both in vitro and in mdx mice16. Phase I/II trials with PTC124 are currently ongoing, not only for application in DMD but also for cystic fibrosis16,17. The references 16 and 17 also describe preferred dosages of the PCT124 compound for use in the present invention. Further provided is therefore a method, combination, use or pharmaceutical preparation according to the invention, wherein said compound for providing said individual with a functional dystrophin protein comprises a compound for suppressing stop codons. Said compound for suppressing stop codons preferably comprises gentamicin, PTC124 or a functional equivalent thereof. Most preferably, said compound comprises PTC124.
In one embodiment an individual is provided with a functional dystrophin protein using a vector, preferably a viral vector, comprising a micro-mini-dystrophin gene. Most preferably, a recombinant adeno-associated viral (rAAV) vector is used. AAV is a single-stranded DNA parvovirus that is non-pathogenic and shows a helper-dependent life cycle. In contrast to other viruses (adenovirus, retrovirus, and herpes simplex virus), rAAV vectors have demonstrated to be very efficient in transducing mature skeletal muscle. Application of rAAV in classical DMD “gene addition” studies has been hindered by its restricted packaging limits (<5 kb). Therefore, rAAV is preferably applied for the efficient delivery of a much smaller micro- or mini-dystrophin gene. Administration of such micro- or mini-dystrophin gene results in the presence of a at least partially functional dystrophin protein. Reference is made to18-20.
A compound for providing an individual with a functional dystrophin protein and at least one adjunct compound according to the invention can be administered to an individual in any order. In one embodiment, said compound for providing an individual with a functional dystrophin protein and said at least one adjunct compound are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In one embodiment at least one adjunct compound is administered to an individual in need thereof before administration of a compound for providing an individual with a functional dystrophin protein. Further provided is therefore a method according to the invention, comprising:
administering to an individual in need thereof an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or administering to said individual an adjunct compound for improving muscle fiber function, integrity and/or survival, and, subsequently,
administering to said individual a compound for providing said individual with a functional dystrophin protein.
In yet another embodiment, said compound for providing an individual with a functional dystrophin protein is administered before administration of said at least one adjunct compound.
Further provided is a method for at least in part increasing the production of a functional dystrophin protein in a cell, said cell comprising pre-mRNA of a dystrophin gene encoding aberrant dystrophin protein, the method comprising:
providing said cell with a compound for inhibiting inclusion of an exon into mRNA produced from splicing of said dystrophin pre-mRNA, and
providing said cell with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or providing said cell with an adjunct compound for improving muscle fiber function, integrity and/or survival, the method further comprising allowing translation of mRNA produced from splicing of said pre-mRNA. In one embodiment said method is performed in vitro, for instance using a cell culture.
In this context, increasing the production of a functional dystrophin protein has been earlier defined herein.
Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B. Schematic Representation of Exon Skipping.
In a patient with Duchenne's muscular dystrophy who has a deletion of exon 50, an out-of-frame transcript is generated in which exon 49 is spliced to exon 51 (A). As a result, a stop codon is generated in exon 51, which prematurely aborts dystrophin synthesis. The sequence-specific binding of the exon-internal antisense oligonucleotide PRO051 interferes with the correct inclusion of exon 51 during splicing so that the exon is actually skipped (B). This restores the open reading frame of the transcript and allows the synthesis of a dystrophin similar to that in patients with Becker's muscular dystrophy (BMD).
FIGS. 2A-2E. Prescreening Studies of the Four Patients.
Magnetic resonance images of the lower legs of the four patients (the left leg of Patient 3 and right legs of the other three patients) show the adequate condition of the tibialis anterior muscle (less than 50% fat infiltration and fibrosis) (A). The diagnosis of Duchenne's muscular dystrophy in these patients was confirmed by diaminobenzidine tetrahydrochloride staining of cross sections of biopsy specimens obtained previously from the quadriceps muscle (B). No dystrophin expression was observed, with the exception of one dystrophin-positive, or revertant, fiber in Patient 2 (arrow). Reverse-transcriptase-polymerase chain-reaction (RT-PCR) analysis of the transcript region flanking the patients' mutations and exon 51 confirmed both the individual mutations in nontreated myotubes (NT) and the positive response to PRO051 (i.e., exon 51 skipping) in treated myotubes (T) on the RNA level (C). The efficiencies of exon skipping were 49% for Patient 1, 84% for Patient 2, 58% for Patient 3, and 90% for Patient 4. A cryptic splice site within exon 51 is sometimes activated by PRO051 in cell culture, resulting in an extra aberrant splicing product, as seen in the treated sample from Patient 4. Lane M shows a 100-bp size marker, and lane C RNA from healthy control muscle. Sequence analysis of the RT-PCR fragments from treated and untreated myotubes identified the precise skipping of exon 51 for each patient (D). The new in-frame transcripts led to substantial dystrophin synthesis, as detected by immunofluorescence analysis of treated myotubes with the use of monoclonal antibody NCL-DYS2 (E).
No dystrophin was detected before treatment.
FIG. 3. RT-PCR Analysis of RNA Isolated from Serial Sections of Biopsy Specimens from the Patients.
After treatment with PRO051, reverse-transcriptase-polymerase-chain-reaction (RT-PCR) analysis shows novel, shorter transcript fragments for each patient. Both the size and sequence of these fragments confirm the precise skipping of exon 51. No additional splice variants were observed. At 28 days, still significant in-frame RNA transcripts were detected, suggesting prolonged persistence of PRO051 in muscle. Owing to the small amount of section material, high-sensitivity PCR conditions were used; this process precluded the accurate quantification of skipping efficiencies and the meaningful correlation between levels of RNA and protein. M denotes size marker, and C control.
FIGS. 4A-4B. Dystrophin-Restoring Effect of a Single Intramuscular Dose of PRO051. Immuno fluorescence analysis with the use of the dystrophin antibody MANDYS106 clearly shows dystrophin expression at the membranes of the majority of fibers throughout the biopsy specimen obtained from each patient (B). Western blot analysis of total protein extracts isolated from the patients' biopsy specimens with the use of NCL-DYS1 antibody show restored dystrophin expression in all patients (A).
FIG. 5. Exon 23 skipping levels on RNA level in different muscle groups (Q: quadriceps muscle; TA: tibialis anterior muscle; DIA: diaphragm muscle) in mdx mice (two mice per group) treated with PS49 alone (group 3) or with PS49 and prednisolone (group 4).
FIGS. 6A-6B. In muscle cells, DMD gene exon 44 (A) or exon 45 (B) skipping levels are enhanced with increasing concentrations of pentoxyfilline (from 0 to 0.5 mg/ml). FIG. 6C Exon 23 skipping levels on RNA level in different muscle groups (Q: quadriceps muscle; TA: tibialis anterior muscle; Tri: triceps muscle; HRT: heart muscle) in mdx mice (two mice per group) treated with PS49 alone (group 3) or with PS49 and pentoxyfilline (group 4).
FIGS. 7A-7B. Dystrophin (DMD) gene amino acid sequence
FIG. 8. Human IGF-1 Isoform 4 amino acid sequence.
FIGS. 9A-9M. Various oligonucleotides directed against the indicated exons of the dystrophin 20 (DMD)
EXAMPLES
Example 1
In a recent clinical study the local safety, tolerability, and dystrophin-restoring effect of antisense compound PRO051 was assessed. The clinical study was recently published. The content of the publication is reproduced herein under example 1A. In brief, PRO051 is a synthetic, modified RNA molecule with sequence 5′-UCA AGG AAG AUG GCA UUU CU-3′, and designed to specifically induce exon 51 skipping59. It carries full-length 2′-O-methyl substituted ribose moieties and phosphorothioate internucleotide linkages. Four DMD patients with different specific DMD gene deletions correctable by exon 51 skipping were included. At day 0, a series of safety parameters was assessed. The patient's leg (i.e. tibialis anterior muscle) was fixed with a tailor-made plastic mould and its position was carefully recorded. A topical anesthetic (EMLA) was used to numb the skin Four injections of PRO051 were given along a line of 1.5 cm between two small skin tattoos, using a 2.5 cm electromyographic needle (MyoJect Disposable Hypodermic Needle Electrode, TECA Accessories) to ensure intramuscular delivery. Each injection volume was 200 μl, containing 200 μg PRO051, dispersed in equal portions at angles of approximately 30 degrees. At day 28, the same series of safety parameters was assessed again. The leg was positioned using the patient's own mould, and a semi-open muscle biopsy was taken between the tattoos under local anesthesia using a forceps with two sharp-edged jaws (Blakesley Conchotoma, DK Instruments). The biopsy was snap-frozen in liquid nitrogen-cooled 2-methylbutane. Patients were treated sequentially. At the time of study, two patients (nr. 1 and 2) were also on corticosteroids (prednisone or deflazacort), one had just stopped steroid treatment (nr. 4) and one patient never used steroids (nr. 3) (see Table 1). This latter patient was also the one who lost ambulance at the youngest age when compared to the other three patients. The biopsy was analysed, for detection of specific exon skipping on RNA level (RT-PCR analysis, not shown) and novel expression of dystrophin on protein level (immunofluorescence and western blot analyses, summarized in Table 1). Assessment of the series of safety parameters (routine plasma and urine parameters for renal and liver function, electrolyte levels, blood cell counts, hemoglobin, aPTT, AP50 and CH50 values) before and after treatment, indicated that the PRO051 compound was locally safe and well tolerated. For immunofluorescence analysis, acetone-fixed cross-sections of the biopsy were incubated for 90 minutes with monoclonal antibodies against the central rod domain (MANDYS106, Dr. G. Morris, UK, 1:60), the C-terminal domain (NCL-DYS2, Novocastra Laboratories Ltd., 1:30) or, as reference, laminin-α2 (Chemicon International, Inc, 1:150), followed by Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular Probes, Inc, 1:250) antibody for one hour. Sections were mounted with Vectashield Mounting Medium (Vector Laboratories Inc.). For quantitative image analysis the ImageJ software (W. Rasband, NIH, USA; http://rsb.info.nih.gov/ij) was used as described60,61. Entire cross-sections were subdivided into series of 6-10 adjacent images, depending on section size. To ensure reliable measurements, staining of the sections and recording of all images was performed in one session, using fixed exposure settings, and avoiding pixel saturation. The lower intensity threshold was set at Duchenne muscular dystrophy background, and positive fluorescence was quantified for each section (area percentage), both for dystrophin and laminin-α2. Western blot analysis was performed as described1, using pooled homogenates from sets of four serial 50 μm sections throughout the biopsy. For the patients 30 and 60 μg total protein was applied and for the control sample 3 μg. The blot was incubated overnight with dystrophin monoclonal antibody NCL-DYS1 (Novocastra Laboratories, 1:125), followed by goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, 1:10.000) for one hour Immuno-reactive bands were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare) and Hyperfilm ECL (Amersham, Biosciences). Signal intensities were measured using ImageJ. Novel dystrophin protein expression at the sarcolemma was detected in the majority of muscle fibers in the treated area in all four patients. The fibers in each section were manually counted after staining for laminin-α2, a basal lamina protein unaffected by dystrophin deficiency. The individual numbers varied, consistent with the biopsy size and the quality of the patients' muscles. In the largest sections, patient 2 had 726 fibers, of which 620 were dystrophin-positive, while patient 3 had 120 fibers, of which 117 were dystrophin-positive. The dystrophin intensities were typically lower than those in a healthy muscle biopsy. Western blot analysis confirmed the presence of dystrophin in varying amounts. The dystrophin signals were scanned and correlated to the control (per μg total protein). The amounts varied from 3% in patient 3 with the most dystrophic muscle, to 12% in patient 2 with the best preserved muscle. Since such comparison based on total protein does not correct for the varying amounts of fibrotic and adipose tissue in Duchenne muscular dystrophy patients, we also quantified the dystrophin fluorescence signal relative to that of the similarly-located laminin-α2 in each section, by ImageJ analysis. When this dystrophin/laminin-α2 ratio was set at 100% for the control section, the two patients that were co-treated with corticosteroids showed the highest percentages of dystrophin, 32% in patient 1 and 35% in patient 2 (Table 1). The lowest percentage of dystrophin was detected in patient 3, 17%. In patient 4 an intermediate percentage of 25% was observed. These percentages correlated to the relative quality of the target muscle, which was best in patients nr. 1 and 2, and worst in patient nr. 3.
TABLE 1
Patient 1
Patient 2
Patient 3
Patient 4
Age (yrs)
10
13
13
11
Age at Loss of
9
11
7
10
Ambulation (yrs)
Steroid Treatment
Yes
Yes
Never
Until
January
2006
Ratio Dystrophin/
32%
35%
17%
25%
laminin-alpha2
Conclusion: the effect of the PRO051 antisense compound was more prominent in those patients that were also subjected to corticosteroids.
Example 1A
Reproduced from Van Deutekom J C et al, (2007) Antisense Oligonucleotide PRO051 Restores Local Dystrophin in DMD Patients. N Engl J. Med., 357(26): 2677-86.
Methods
Patients and Study Design
Patients with Duchenne's muscular dystrophy who were between the ages of 8 and 16 years were eligible to participate in the study. All patients had deletions that were correctable by exon-51 skipping and had no evidence of dystrophin on previous diagnostic muscle biopsy. Concurrent glucocorticoid treatment was allowed. Written informed consent was obtained from the patients or their parents, as appropriate. During the prescreening period (up to 60 days), each patient's mutational status and positive exon-skipping response to PRO051 in vitro were confirmed, and the condition of the tibialis anterior muscle was determined by T1-weighted magnetic resonance imaging (MRI).62 For patients to be included in the study, fibrotic and adipose tissue could make up no more than 50% of their target muscle.
During the baseline visit, safety measures were assessed. In each patient, the leg that was to be injected was fixed with a tailor-made plastic mold and its position was recorded. A topical eutectic mixture of local anesthetics (EMLA) was used to numb the skin. Four injections of PRO051 were given along a line measuring 1.5 cm running between two small skin tattoos with the use of a 2.5-cm electromyographic needle (MyoJect Disposable Hypodermic Needle Electrode, TECA Accessories) to ensure intramuscular delivery. The volume of each injection was 200 μl containing 200 μg of PRO051, which was dispersed in equal portions at angles of approximately 30 degrees.
At day 28, safety measures were assessed again. The leg that had been injected was positioned with the use of the patient's own mold, and a semiopen muscle biopsy was performed between the tattoos under local anesthesia with a forceps with two sharp-edged jaws (Blakesley Conchotoma, DK Instruments).63 The biopsy specimen was snap-frozen in 2-methylbutane cooled in liquid nitrogen.
Patients were treated sequentially from May 2006 through March 2007 and in compliance with Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki. The study was approved by the Dutch Central Committee on Research Involving Human Subjects and by the local institutional review board at Leiden University Medical Center. All authors contributed to the study design, participated in the collection and analysis of the data, had complete and free access to the data, jointly wrote the manuscript, and vouch for the completeness and accuracy of the data and analyses presented.
Description of PRO051
PRO051 is a synthetic, modified RNA molecule with sequence 5′-UCAAGGAAGAUGGCAUUUCU-3′.12 It carries full-length 2′-O-methyl-substituted ribose molecules and phosphorothioate internucleotide linkages. The drug was provided by Prosensa B.V. in vials of 1 mg of freeze-dried material with no excipient. It was dissolved and administered in sterile, unpreserved saline (0.9% sodium chloride). PRO051 was not found to be mutagenic by bacterial Ames testing. In regulatory Good Laboratory Practice safety studies, rats that received a single administration of up to 8 mg per kilogram of body weight intramuscularly and 50 mg per kilogram intravenously showed no adverse effects; monkeys receiving PRO051 for 1 month appeared to tolerate doses up to 16 mg per kilogram per week when the drug was administered by intravenous 1-hour infusion or by subcutaneous injection, without clinically relevant adverse effects.
In Vitro Prescreening
A preexisting primary myoblast culture1 was used for the prescreening of Patient 4. For the other three patients, fibroblasts were converted into myogenic cells after infection with an adenoviral vector containing the gene for the myogenic transcription factor (MyoD) as described previously.1,64,65 Myotube cultures were transfected with PRO051 (100 nM) and polyethylenimine (2 μl per microgram of PRO051), according to the manufacturer's instructions for ExGen500 (MBI Fermentas). RNA was isolated after 48 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analyses were performed as reported previously1,12 PCR fragments were analyzed with the use of the 2100 Bioanalyzer (Agilent) and isolated for sequencing by the Leiden Genome Technology Center.
Safety Assessment
At baseline and at 2 hours, 1 day, and 28 days after injection, all patients received a full physical examination (including the measurement of vital signs) and underwent electrocardiography. In addition, plasma and urine were obtained to determine renal and liver function, electrolyte levels, complete cell counts, the activated partial-thromboplastin time, and complement activity values in the classical (CH50) and alternative (AP50) routes. The use of concomitant medications was recorded. At baseline and on day 28, the strength of the tibialis anterior muscle was assessed with the use of the Medical Research Council scale66 to evaluate whether the procedures had affected muscle performance. (On this scale, a score of 0 indicates no movement and a score of 5 indicates normal muscle strength.) Since only a small area of the muscle was treated, clinical benefit in terms of increased muscle strength was not expected. At each visit, adverse events were recorded.
RNA Assessment
Serial sections (50 μm) of the frozen muscle-biopsy specimen were homogenized in RNA-Bee solution (Campro Scientific) and MagNA Lyser Green Beads (Roche Diagnostics). Total RNA was isolated and purified according to the manufacturer's instructions. For complementary DNA, synthesis was accomplished with Transcriptor reverse transcriptase (Roche Diagnostics) with the use of 500 ng of RNA in a 20-μl reaction at 55° C. for 30 minutes with human exon 53 or 54 specific reverse primers. PCR analyses were performed as described previously.1,12 Products were analyzed on 2% agarose gels and sequenced. In addition, RT-PCR with the use of a primer set for the protein-truncation test67 was used to rapidly screen for aspecific aberrant splicing events throughout the DMD gene.
Assessment of Protein Level
For immunofluorescence analysis, acetone-fixed sections were incubated for 90 minutes with monoclonal antibodies against the central rod domain (MANDYS106, Dr. G. Morris, United Kingdom) at a dilution of 1:60, the C-terminal domain (NCL-DYS2, Novocastra Laboratories) at a dilution of 1:30, or (as a reference) laminin (Chemicon International), a basal lamina protein that is unaffected by dystrophin deficiency, at a dilution of 1:150, followed by Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody (Molecular Probes) at a dilution of 1:250 for 1 hour. Sections were mounted with Vectashield Mounting Medium (Vector Laboratories). ImageJ software (W. Rasband, National Institutes of Health, http://rsb.info.nih.gov/ij) was used for quantitative image analysis as described previously.60,61 Entire cross sections were subdivided into series of 6 to 10 adjacent images, depending on the size of the section. To ensure reliable measurements, staining of the sections and recording of all images were performed during one session with the use of fixed exposure settings and the avoidance of pixel saturation. The lower-intensity threshold was set at background for Duchenne's muscular dystrophy, and positive fluorescence was quantified for each section (area percentage), both for dystrophin and laminin α2.
Western blot analysis was performed as described previously1 with the use of pooled homogenates from sets of four serial 50-μm sections throughout the biopsy specimen. For each patient, two amounts of total protein—30 μg and 60 μg—were applied, and for the control sample, 3 μg. The Western blot was incubated overnight with dystrophin monoclonal antibody NCL-DYS1 (Novocastra Laboratories) at a dilution of 1:125, followed by horseradish-peroxidase-labeled goat antimouse IgG (Santa Cruz Biotechnology) at a dilution of 1:10,000 for 1 hour. Immunoreactive bands were visualized with the use of the ECL Plus Western blotting detection system (GE Healthcare) and Hyperfilm ECL (Amersham Biosciences). Signal intensities were measured with the use of ImageJ software.
Results
Prescreening of Patients
The study was planned to include four to six patients. Six patients were invited to participate, and one declined. The remaining five patients were prescreened. First, the condition of the tibialis anterior muscle was evaluated on MRI. The muscle condition of four patients was deemed to be adequate for the study (FIG. 2B), and the absence of dystrophin was confirmed in the patients' original biopsy specimens (FIG. 2B). Second, the mutational status and positive exon-skipping response to PRO051 of these four patients were confirmed in fibroblast cultures. PRO051 treatment generated a novel, shorter fragment of messenger RNA for each patient, representing 46% (in Patient 4) to 90% (in Patient 1) of the total RT-PCR product (FIG. 2C). Precise exon-51 skipping was confirmed by sequencing (FIG. 2D). No other transcript regions were found to be altered. Immunofluorescence analyses showed a preponderance of dystrophin-positive myotubes (FIG. 2E), a finding that was confirmed by Western blot analysis (not shown). Thus, the four patients were judged to be eligible for PRO051 treatment. Their baseline characteristics are shown in Table 2.
Safety and Adverse Events
All patients had one or more adverse events. However, only one patient reported mild local pain at the injection site, which was considered to be an adverse event related to the study drug. Other events included mild-to-moderate pain after the muscle biopsy. Two patients had blistering under the bandages used for wound closure. In the period between injection and biopsy, two patients reported a few days of flulike symptoms, and one patient had mild diarrhea for 1 day. At baseline, the muscle-strength scores of the treated tibialis anterior muscle in Patients 1, 2, 3, and 4 were 4, 2, 3, and 4, respectively, on the Medical Research Council scale. None of the patients showed changes in the strength of this muscle during the study or significant alterations in standard laboratory measures or increased measures of complement split products or activated partial-thromboplastin time. No local inflammatory or toxic response was detected in the muscle sections of the patients (data not shown). Patient 3 successfully underwent preplanned surgery for scoliosis in the month after the study was completed.
RNA and Protein Level
At day 28, a biopsy of the treated area was performed in each patient. Total muscle RNA was isolated from serial sections throughout the biopsy specimen. In all patients, RT-PCR identified a novel, shorter fragment caused by exon-51 skipping, as confirmed by sequencing (FIG. 3). Further transcript analysis showed no other alterations (data not shown). Immunofluorescence analyses of sections throughout the biopsy specimen of each patient showed clear sarcolemmal dystrophin signals in the majority of muscle fibers (FIGS. 4A and 4B). Dystrophin antibodies proximal and distal to the deletions that were used included MANDYS106 (FIGS. 4A and 4B) and NCL-DYS2 (similar to MANDYS106, not shown). The fibers in each section were manually counted after staining for laminin α2.68 The individual numbers varied, consistent with the size of the biopsy specimen and the quality of the muscle. In the largest sections, Patient 2 had 726 fibers, of which 620 were dystrophin-positive, whereas Patient 3 had 120 fibers, of which 117 were dystrophin-positive (Data not shown). The dystrophin intensities were typically lower than those in a healthy muscle biopsy specimen (Data not shown). The single fibers with a more intense dystrophin signal in Patients 2 and 3 could well be revertant fibers (Data not shown).
Western blot analysis confirmed the presence of dystrophin in varying amounts (FIG. 4A). The dystrophin signals were scanned and correlated to the control (per microgram of total protein). The amounts varied from 3% in Patient 3, who had the most-dystrophic muscle, to 12% in Patient 2, who had the best-preserved muscle. Since such comparison on the basis of total protein does not correct for the varying amounts of fibrotic and adipose tissue in patients with Duchenne's muscular dystrophy, we also quantified the dystrophin fluorescence signal (Data not shown) relative to that of the similarly located laminin α2 in each section by ImageJ analysis. When the ratio of dystrophin to laminin α2 was set at 100 for the control section, Patients 1, 2, 3, and 4 had ratios of 33, 35, 17, and 25, respectively (Table 1).
Discussion
Our study showed that local intramuscular injection of PRO051, a 2OMePS antisense oligoribonucleotide complementary to a 20-nucleotide sequence within exon 51, induced exon-51 skipping, corrected the reading frame, and thus introduced dystrophin in the muscle in all four patients with Duchenne's muscular dystrophy who received therapy. Dystrophin-positive fibers were found throughout the patients' biopsy specimens, indicating dispersion of the compound in the injected area. Since no delivery-enhancing excipient was used, PRO051 uptake did not seem to be a major potentially limiting factor. We cannot rule out that increased permeability of the dystrophic fiber membrane had a favorable effect. The patients produced levels of dystrophin that were 3 to 12% of the level in healthy control muscle, as shown on Western blot analysis of total protein. Since the presence of fibrosis and fat may lead to some underestimation of dystrophin in total protein extracts, we determined the ratio of dystrophin to laminin α2 in the cross sections, which ranged from 17 to 35, as compared with 100 in control muscle. The dystrophin-restoring effect of PRO051 was limited to the treated area, and no strength improvement of the entire muscle was observed. Future systemic treatment will require repeated administration to increase and maintain dystrophin expression at a higher level and to obtain clinical efficacy.
Because of medical-ethics regulations regarding interventions in minors, we could not obtain a biopsy specimen from the patients' contralateral muscles that had not been injected. However, the patients showed less than 1% of revertant fibers in the original diagnostic biopsy specimens obtained 5 to 9 years before the initiation of the study (Table 2 and FIG. 2B). We consider it very likely that the effects we observed were related to the nature and sequence of the PRO051 reagent rather than to a marked increase in revertant fibers. Indeed, a single, possibly revertant fiber that had an increased dystrophin signal was observed in both Patient 2 and Patient 3 (FIG. 4B).
In summary, our study showed that local administration of PRO051 to muscle in four patients with Duchenne' s muscular dystrophy restored dystrophin to levels ranging from 3 to 12% or 17 to 35%, depending on quantification relative to total protein or myofiber content. Consistent with the distinctly localized nature of the treatment, functional improvement was not observed. The consistently poorer result in Patient 3, who had the most advanced disease, suggests the importance of performing clinical trials in patients at a relatively young age, when relatively little muscle tissue has been replaced by fibrotic and adipose tissue. Our findings provide an indication that antisense-mediated exon skipping may be a potential approach to restoring dystrophin synthesis in the muscles of patients with Duchenne's muscular dystrophy.
Example 2
In a pre-clinical study in mdx mice (animal model for DMD) the effect of adjunct compound prednisone on AON-induced exon skipping was assessed.
Mdx mice (C57Bl/10ScSn-mdx/J) were obtained from Charles River Laboratories (The Netherlands). These mice are dystrophin-deficient due to a nonsense mutation in exon 23. AON-induced exon 23 skipping is therapeutic in mdx mice by removing the nonsense mutation and correction of the open reading frame. Two mdx mice per group were injected subcutaneously with: Group 1) physiologic salt (wk 1-8), Group 2) prednisolone (1 mg/kg, wk 1-8), Group 3) mouse-specific antisense oligonucleotide PS49 designed to specifically induce exon 23 skipping (100 mg/kg, wk 4 (5 times), week 5-8 (2 times), Group 4) prednisolone (1 mg/kg, wk 1-8)+PS49 (100 mg/kg, wk 4 (5 times), week 5-8 (2 times). PS49 (5′ GGCCAAACCUCGGCUUACCU 3′) has a full-length phosphorothioate backbone and 2′O-methyl modified ribose molecules.
All mice were sacrificed at 1 week post-last-injection. Different muscles groups, including quadriceps, tibialis anterior, and diaphragm muscles were isolated and frozen in liquid nitrogen-cooled 2-methylbutane. For RT-PCR analysis, the muscle samples were homogenized in the RNA-Bee solution (Campro Scientific, The Netherlands). Total RNA was isolated and purified according to the manufacturer's instructions. For cDNA synthesis with reverse transcriptase (Roche Diagnostics, The Netherlands), 300 ng of RNA was used in a 20 μl reaction at 55° C. for 30 min, reverse primed with mouse DMD gene-specific primers. First PCRs were performed with outer primer sets, for 20 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). One μl of this reaction (diluted 1:10) was then re-amplified using nested primer combinations in the exons directly flanking exon 23, with 30 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). PCR products were analysed on 2% agarose gels. Skipping efficiencies were determined by quantification of PCR products using the DNA 1000 LabChip® Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, The Netherlands). No exon 23 skipping was observed in the muscles from mice treated with physiologic salt or prenisolone only (groups 1 and 2). Levels of exon 23 skipping were detected and per muscle group compared between mice treated with PS49 only (group 3) and mice treated with PS49 and adjunct compound prednisolone (group 4). In the quadriceps (Q), tibialis anterior (TA), and diaphragm (DIA) muscles, exon 23 skipping levels were typically higher in group 4 when compared to group 3 (FIG. 5). This indicates that adjunct compound prednisolone indeed enhances exon 23 skipping levels in mdx mice treated with PS49.
Example 3
A., B. Differentiated muscle cell cultures (myotubes) derived from a healthy control individual were transfected with 250 nM PS188 ([5′ UCAGCUUCUGUUAGCCACUG 3′; SEQ ID NO:10] an AON optimized to specifically skip exon 44) or 250 nM PS221 ([5′ AUUCAAUGUUCUGACAACAGUUUGC 3′; SEQ ID NO: 60] an AON optimized to specifically skip exon 45) in the presence of 0 to 0.5 mg/ml pentoxifylline, using the transfection reagent polymer UNIFectylin (2.0 μl UNIFectylin per μg AON in 0.15M NaCl). UNIFectylin interacts electrostatically with nucleic acids, provided that the nucleic acid is negatively charged (such as 2′-O-methyl phosphorothioate AONs). Pentoxyfillin (Sigma Aldrich) was dissolved in water. Total RNA was isolated 24 hrs after transfection in RNA-Bee solution (Campro Scientific, The Netherlands) according to the manufacturer's instructions. For cDNA synthesis with reverse transcriptase (Roche Diagnostics, The Netherlands), 500 ng of RNA was used in a 20 μl reaction at 55° C. for 30 min, reverse primed with DMD gene-specific primers. First PCRs were performed with outer primer sets, for 20 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). One μl of this reaction (diluted 1:10) was then re-amplified using nested primer combinations in the exons directly flanking exon 44 or 45, with 30 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). PCR products were analysed on 2% agarose gels. Skipping efficiencies were determined by quantification of PCR products using the DNA 1000 LabChip® Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, The Netherlands).
Both with PS188 and PS221, increasing levels of exon 44 or 45 skipping were obtained with increasing concentrations of the adjunct compound pentoxifylline when compared to those obtained in cells that were not co-treated with pentoxyfilline (see FIG. 6). These results indicate that pentoxifylline enhances exon skipping levels in the muscle cells.
C.
In a pre-clinical study in mdx mice (animal model for DMD) the effect of adjunct compound pentoxyfilline on AON-induced exon skipping was assessed. Mdx mice (C57Bl/10ScSn-mdx/J) were obtained from Charles River Laboratories (The Netherlands). These mice are dystrophin-deficient due to a nonsense mutation in exon 23. AON-induced exon 23 skipping is therapeutic in mdx mice by removing the nonsense mutation and correction of the open reading frame. Two mdx mice per group were injected subcutaneously with: Group 1) pentoxyfilline (50 mg/kg, wk 1-2), Group 2) mouse-specific antisense oligonucleotide PS49 designed to specifically induce exon 23 skipping (100 mg/kg, wk 2 (2 times), Group 3) pentoxyfilline (50 mg/kg, wk 1-2)+PS49 (100 mg/kg, wk 2 (2 times). PS49 (5′ GGCCAAACCUCGGCUUACCU 3′) has a full-length phosphorothioate backbone and 2′O-methyl modified ribose molecules.
All mice were sacrificed at 1 week post-last-injection. Different muscles groups, including quadriceps, tibialis anterior, triceps and heart muscles were isolated and frozen in liquid nitrogen-cooled 2-methylbutane. For RT-PCR analysis, the muscle samples were homogenized in the RNA-Bee solution (Campro Scientific, The Netherlands). Total RNA was isolated and purified according to the manufacturer's instructions. For cDNA synthesis with reverse transcriptase (Roche Diagnostics, The Netherlands), 300 ng of RNA was used in a 20 μl reaction at 55° C. for 30 min, reverse primed with mouse DMD gene-specific primers. First PCRs were performed with outer primer sets, for 20 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). One μl of this reaction (diluted 1:10) was then re-amplified using nested primer combinations in the exons directly flanking exon 23, with 30 cycles of 94° C. (40 sec), 60° C. (40 sec), and 72° C. (60 sec). PCR products were analysed on 2% agarose gels. Skipping efficiencies were determined by quantification of PCR products using the DNA 1000 LabChip® Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, The Netherlands). No exon 23 skipping was observed in the muscles from mice treated with pentoxyfilline only (groups 1). Levels of exon 23 skipping were detected and per muscle group compared between mice treated with PS49 only (group 2) and mice treated with PS49 and adjunct compound pentoxyfilline (group 3). In the quadriceps (Q), tibialis anterior (TA), triceps (Tri) and heart (HRT) muscles, exon 23 skipping levels were typically higher in group 3 when compared to group 2 (FIG. 6c). This indicates that adjunct compound pentoxyfilline indeed enhances exon 23 skipping levels in mdx mice treated with PS49.
TABLE 2
Baseline characteristics of the DMD patients
Patient 1
Patient 2
Patient 3
Patient 4
Age (yrs)
10
13
13
11
Deletion
Exon 50
Exons
Exons
Exon 52
48-50
49-50
Age at Loss of
9
11
7
10
Ambulation (yrs)
Scoliosis
No
No
Yes
Yes
Creatine Kinase
5823
2531
717
4711
Levels (U/I)1
Steroid treatment
Yes
Yes
Never
Until
January
2006
Strength TA
4
2
3
4
muscle (MRC
scale)
MRI status TA
Moderate2
Moderate2
Moderate2
Moderate2
muscle
% Revertant
N.D.
<1%
N.D.
fibers
1normal level: <200 U/I
2less than 50% fat infiltration and/or fibrosis [Mercuri et al., 2005)
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26. Pierno S, Nico B, Burdi R, et al. Role of tumour necrosis factor alpha, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice: a pharmacological approach. Neuropathology and applied neurobiology 2007; 33(3):344-59.
27. Musaro A, McCullagh K, Paul A, et al. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet. 2001; 27(2):195-200.
28. Barton E R, Morris L, Musaro A, Rosenthal N, Sweeney H L. Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J Cell Biol 2002; 157(1):137-48.
29. Disatnik M H, Dhawan J, Yu Y, et al. Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state. J Neurol Sci 1998; 161(1):77-84.
30. Nelson S K, Bose S K, Grunwald G K, Myhill P, McCord J M. The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy. Free radical biology & medicine 2006; 40(2):341-7.
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32. Rolland J F, De Luca A, Burdi R, Andreetta F, Confalonieri P, Conte Camerino D. Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline. Neurobiol Dis 2006; 24(3):466-74.
33. Whitehead N P, Streamer M, Lusambili L I, Sachs F, Allen D G. Streptomycin reduces stretch-induced membrane permeability in muscles from mdx mice. Neuromuscul Disord 2006; 16(12):845-54.
34. Badalamente M A, Stracher A. Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition. Muscle Nerve 2000; 23(1):106-11.
35. Burdi R, Didonna M P, Pignol B, et al. First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant. Neuromuscul Disord 2006; 16(4):237-48.
36. Bonuccelli G, Sotgia F, Schubert W, et al. Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. Am J Pathol 2003; 163(4):1663-75.
37. Voisin V, Sebrie C, Matecki S, et al. L-arginine improves dystrophic phenotype in mdx mice. Neurobiol Dis 2005; 20(1):123-30.
38. Soret J, Bakkour N, Maire S, et al. Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors. Proc Natl Acad Sci USA 2005; 102(24):8764-9.
39. Mann C J, Honeyman K, McClorey G, Fletcher S, Wilton S D. Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. J Gene Med 2002; 4(6):644-54.
40. Graham I R, Hill V J, Manoharan M, Inamati G B, Dickson G. Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays. J Gene Med 2004; 6(10):1149-58.
41. Mathews D H, Sabina J, Zuker M, Turner D H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 1999; 288(5):911-40.
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43. Cartegni L, Wang J, Zhu Z, Zhang M Q, Krainer A R. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 2003; 31(13):3568-71.
44. Braasch D A, Corey D R. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 2001; 8(1):1-7.
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46. Elayadi A N, Corey D R. Application of PNA and LNA oligomers to chemotherapy. Curr Opin Investig Drugs 2001; 2(4):558-61.
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48. Summerton J. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1999; 1489(1):141-58.
49. Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997; 7(3):187-95.
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51. De Angelis F G, Sthandier O, Berarducci B, et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells. Proc Natl Acad Sci USA 2002; 99(14):9456-61.
52. Denti M A, Rosa A, D'Antona G, et al. Chimeric adeno-associated virus/antisense U1 small nuclear RNA effectively rescues dystrophin synthesis and muscle function by local treatment of mdx mice. Hum Gene Ther 2006; 17(5):565-74.
53. Gorman L, Suter D, Emerick V, Schumperli D, Kole R. Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proc Natl Acad Sci USA 1998; 95(9):4929-34.
54. Suter D, Tomasini R, Reber U, Gorman L, Kole R, Schumperli D. Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations. Hum Mol Genet. 1999; 8(13):2415-23.
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67. Roest P A, Roberts R G, van der Tuijn A C, Heikoop J C, van Ommen G J, den Dunnen J T. Protein truncation test (PTT) to rapidly screen the DMD gene for translation terminating mutations. Neuromuscul Disord 1993; 3:391-4.
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15881574
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biomarin technologies b.v.
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USA
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E1
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Reissue Patent
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Dec 27th, 2016 12:00AM
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Nov 14th, 2014 12:00AM
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https://www.uspto.gov?id=US09528109-20161227
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Methods and means for efficient skipping of exon 45 in duchenne muscular dystrophy pre-mRNA
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The invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing an isolated muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within the exon. The invention further relates to such molecule used in the method.
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9528109
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1. An antisense oligonucleotide whose base sequence consists of the base sequence of 5 ′UUUGCCGCUGCCCAAUGCCAUCCUG-3′ (SEQ ID: NO: 3), said oligonucleotide comprising a modification.
2. The oligonucleotide of claim 1, comprising a phosphorodiamidate morpholino oligomer (PMO).
3. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a locked nucleic acid (LNA).
4. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified base.
5. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified sugar moiety.
6. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified internucleoside linkage.
7. The oligonucleotide of claim 1 wherein said oligonucleotide comprises a phosphorothioate internucleoside linkage and a 2′-O-alkyl substituted ribose moiety.
8. The oligonucleotide of claim 5, wherein the modified sugar moiety is selected from the group consisting of: a ribose that is mono- or di-substituted at the 2′, 3′, and/or 5′ position.
9. The oligonucleotide of claim 8, wherein the ribose is a 2′-O-substituted ribose.
10. The oligonucleotide of claim 9, wherein the ribose is a 2′-O-methyl ribose.
11. The oligonucleotide of claim 6, said oligonucleotide comprising a modified backbone such that all of internucleoside linkages of said oligonucleotide are modified.
12. The oligonucleotide of claim 11, said internucleoside linkages comprising phosphorothioate.
13. The oligonucleotide of claim 11, said internucleoside linkages comprising a phosphorodiamidate morpholino oligomer (PMO).
14. The oligonucleotide of claim 1 said modification comprising a peptide nucleic acid, and/or locked nucleic acid.
15. The oligonucleotide of claim 11, wherein said backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.
16. The antisense oligonucleotide of claim 1 wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 50%.
17. The antisense oligonucleotide of claim 16, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 60%.
18. The antisense oligonucleotide of claim 17, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 70%.
19. The antisense oligonucleotide of claim 18, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 80%.
20. The antisense oligonucleotide of claim 19, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 90%.
21. A viral-based vector comprising an expression cassette comprising a nucleotide sequence encoding the oligonucleotide of claim 1.
22. A pharmaceutical composition comprising the oligonucleotide of claim 1, and a pharmaceutically acceptable carrier.
23. The pharmaceutical composition of claim 1, further comprising an antisense oligonucleotide which induces or promotes skipping of exon 7, 44, 46, 51, 53, 59, or 67 of dystrophin pre-mRNA of a patient.
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PRIORITY
This application is a U.S. continuation patent application Ser. No. 14/200,251 filed Mar. 7, 2014, which is a U.S. continuation patent application of U.S. patent application Ser. No. 14/134,971 filed Dec. 19, 2013 which is a U.S. continuation patent application of U.S. patent application Ser. No. 14/097,210 filed Dec. 4, 2013, which is a U.S. continuation patent application of U.S. patent application Ser. No. 13/094,548 filed Apr. 26, 2011, which is a U.S. continuation patent application of PCT/NL2009/050006, filed on Jan. 13, 2009, which claims priority to PCT/NL2008/050673, filed on Oct. 27, 2008, the entirety of which is incorporated herein by reference.
SEQUENCE LISTING
The attached sequence listing is hereby incorporated by reference.
FIELD
The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to human Duchenne Muscular Dystrophy.
BACKGROUND OF THE INVENTION
Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are mainly characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the DMD transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008; 10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, van Deutekom et al., N Engl J Med. 2007; 357(26):2677-86. Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol Chem. 2008; 283: 5899-907).
As the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively large numbers of patients. The actual applicability of exon skipping can be determined for deletions, duplications and point mutations reported in DMD mutation databases such as the Leiden DMD mutation database available at www.dmd.nl. Therapeutic skipping of exon 45 of the DMD pre-mRNA would restore the open reading frame of DMD patients having deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA, occurring in a total of 16% of all DMD patients with a deletion (Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics) Research Focus, 2007 Nova Science Publishers, Inc). Furthermore, for some DMD patients the simultaneous skipping of one of more exons in addition to exon 45, such as exons 51 or 53 is required to restore the correct reading frame. None-limiting examples include patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.
Recently, a first-in-man study was successfully completed where an AON inducing the skipping of exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J Med. 2007; 357: 2677-86).
Most AONs studied contain up to 20 nucleotides, and it has been argued that this relatively short size improves the tissue distribution and/or cell penetration of an AON. However, such short AONs will result in a limited specificity due to an increased risk for the presence of identical sequences elsewhere in the genome, and a limited target binding or target affinity due to a low free energy of the AON-target complex. Therefore the inventors decided to design new and optionally improved oligonucleotides that would not exhibit all of these drawbacks.
DESCRIPTION OF THE INVENTION
Method
In a first aspect, the invention provides a method for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.
Accordingly, a method is herewith provided for inducing and/or promoting skipping of exon 45 of DMD pre-mRNA, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon.
It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method.
As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. The DMD gene or protein corresponds to the dystrophin gene or protein.
A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background.
Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with sequences such as, for example, the splice donor or splice acceptor sequence that are both required for allowing the enzymatic process of splicing, or a molecule that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
Within the context of the invention inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.
Preferably, a method of the invention by inducing or promoting skipping of exon 45 of the DMD pre-mRNA in one or more cells of a patient provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient. Therefore a preferred method is a method, wherein a patient or a cell of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient or in a cell of said patient is decreased
Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.
Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the DMD gene that prevent synthesis of the complete dystrophin protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared to the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon-skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of the remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
A method of the invention may alleviate one or more characteristics of a muscle cell from a DMD patient comprising deletions including but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA of said patient (Aartsma-Rus and van Deutekom, 2007, Antisense Elements (Genetics) Research Focus, 2007 Nova Science Publishers, Inc) as well as from DMD patients requiring the simultaneous skipping of one of more exons in addition to exon 45 including but not limited to patients with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, or with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53.
In a preferred method, one or more symptom(s) or characteristic(s) of a myogenic cell or muscle cell from a DMD patient is/are alleviated. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.
An alleviation of one or more symptoms or characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
The improvement of muscle fiber function, integrity and/or survival may also be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD patient before treatment.
A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD patient before treatment.
A detectable increase of the homogeneity of the diameter of muscle fibers is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of muscle fibers in a muscle biopsy cross-section of a same DMD patient before treatment.
An alleviation of one or more symptoms or characteristics may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
A treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. The frequency of administration of an oligonucleotide, composition, compound of the invention may depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.
Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD and may be administered directly in vivo, ex vivo or in vitro. An oligonucleotide as used herein may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne muscular dystrophy has a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably said cells comprise a gene encoding a mutant dystrophin protein. Preferably said cells are cells of an individual suffering from DMD.
A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipient are defined in the section entitled “pharmaceutical composition”. In vitro, we obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example.
In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of a distinct exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 7, 44, 46, 51, 53, 59, 67 of the dystrophin pre-mRNA of a patient. Molecules which can be used are depicted in table 2. Preferred molecules comprise or consist of any of the oligonucleotides as disclosed in table 2. Several oligonucleotides may also be used in combination. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006; 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other.
It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.
In case, more than one compounds are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.
Molecule
In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled “Method”. This molecule preferably comprises or consists of an oligonucleotide, Said oligonucleotide is preferably an antisense oligonucleotide (AON) or antisense oligoribonucleotide.
It was found by the present investigators that especially exon 45 is specifically skipped at a high frequency using a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. Although this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 21 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex. In a preferred embodiment, a molecule that binds to a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within said exon is used.
In a preferred embodiment, a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of exon 45 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100%. “A part of exon 45” preferably means a stretch of at least 21 nucleotides. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of exon 45 dystrophin pre-mRNA as defined herein. Alternatively, an oligonucleotide may comprise a sequence that is complementary to part of exon 45 dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Several types of flanking sequences may be used. Preferably, additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity. In another preferred embodiment, additional flanking sequences are complementary to sequences of the DMD pre-mRNA which are not present in exon 45. Such flanking sequences are preferably complementary to sequences comprising or consisting of the splice site acceptor or donor consensus sequences of exon 45. In a preferred embodiment, such flanking sequences are complementary to sequences comprising or consisting of sequences of an intron of the DMD pre-mRNA which is adjacent to exon 45; i.e. intron 44 or 45.
A continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides within exon 45 is preferably selected from the sequence:
(SEQ ID NO 2)
5′-CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGA
ACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUC-3′.
It was found that a molecule that binds to a nucleotide sequence comprising or consisting of a continuous stretch of at least 21, 25, 30, 35, 40, 45, 50 nucleotides of SEQ ID NO. 2 results in highly efficient skipping of exon 45 in a cell provided with this molecule. Molecules that bind to a nucleotide sequence comprising a continuous stretch of less than 21 nucleotides of SEQ ID NO:2 were found to induce exon skipping in a less efficient way than the molecules of the invention. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 21, 25, 30, 35 nucleotides within SEQ ID NO:2. Contrary to what was generally thought, the inventors surprisingly found that a higher specificity and efficiency of exon skipping may be reached using an oligonucleotides having a length of at least 21 nucleotides. None of the indicated sequences is derived from conserved parts of splice-junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.
In one embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the indicated nucleotide sequence on a RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are for example disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein enclosed by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein enclosed by reference.
In a further embodiment, a molecule of the invention capable of interfering with the inclusion of exon 45 of the DMD pre-mRNA comprises an antisense oligonucleotide that is complementary to and can base-pair with the coding strand of the pre-mRNA of the DMD gene. Said antisense oligonucleotide preferably contains a RNA residue, a DNA residue, and/or a nucleotide analogue or equivalent, as will be further detailed herein below.
A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 21 and 50 nucleotides or bases, more preferred between 21 and 40 nucleotides, more preferred between 21 and 30 nucleotides, such as 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides.
A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.
In a preferred embodiment, a molecule of the invention binds to a continuous stretch of or is complementary to or is antisense to at least a continuous stretch of at least 21 nucleotides within the nucleotide sequence SEQ ID NO:2.
In a certain embodiment, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences as depicted in Table 1, except SEQ ID NO:68. A molecule of the invention that is antisense to the sequence of SEQ ID NO 2, which is present in exon 45 of the DMD gene preferably comprises or consists of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66 and/or SEQ ID NO:67.
In a more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and/or SEQ ID NO 8.
In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 3. It was found that this molecule is very efficient in modulating splicing of exon 45 of the DMD pre-mRNA in a muscle cell.
A nucleotide sequence of a molecule of the invention may contain a RNA residue, a DNA residue, a nucleotide analogue or equivalent as will be further detailed herein below. In addition, a molecule of the invention may encompass a functional equivalent of a molecule of the invention as defined herein.
It is preferred that a molecule of the invention comprises a or at least one residue that is modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, an antisense nucleotide sequence comprises a or at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
In a preferred embodiment, a nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
It is further preferred that the linkage between a residue in a backbone does not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of at least one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; aminoxy, methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or deoxyribose or derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
A preferred antisense oligonucleotide according to the invention comprises a 2′-O-alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
A most preferred antisense oligonucleotide according to the invention comprises a 2′-O-methyl phosphorothioate ribose.
A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 45 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 45 skipping and quantifying the amount of a functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analysis. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
It will also be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping of exon 45 of the human DMD pre-mRNA. In a preferred embodiment, a combination of at least two antisense oligonucleotides are used in a method of the invention, such as two distinct antisense oligonucleotides, three distinct antisense oligonucleotides, four distinct antisense oligonucleotides, or five distinct antisense oligonucleotides or even more. It is also encompassed by the present invention to combine several oligonucleotides or molecules as depicted in table 1 except SEQ ID NO:68.
An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably myogenic cells or muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
A preferred antisense oligonucleotide comprises a peptide-linked PMO.
A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for modulating skipping of exon 45 of the human DMD pre-mRNA.
A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 45 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by viral-based vector. Such a viral-based vector comprises an expression cassette that drives expression of an antisense molecule as defined herein. Preferred virus-based vectors include adenovirus- or adeno-associated virus-based vectors. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.
One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 45 of the DMD pre-mRNA.
A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 45 of the human DMD pre-mRNA.
Pharmaceutical Composition
If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.
Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.
A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector which is able to modulate skipping of exon 7, 44, 46, 51, 53, 59, 67 of the DMD pre-mRNA.
Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils, Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. We obtained very good results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the example. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy in humans.
In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 □M is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, a compound preferably an oligonucleotide and an adjunct compound to be used in the invention to prevent, treat DMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
Use
In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for inducing and/or promoting splicing of the DMD pre-mRNA. The splicing is preferably modulated in a human myogenic cell or a muscle cell in vitro. More preferred is that splicing is modulated in human a myogenic cell or muscle cell in vivo.
Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for inducing and/or promoting splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD patient.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. In human control myotubes, a series of AONs (PS220 to PS225; SEQ ID NO: 3 to 8), all binding to a continuous stretch of at least 21 nucleotides within a specific sequence of exon 45 (i.e. SEQ ID NO:2), were tested at two different concentrations (200 and 500 nM). All six AONs were effective in inducing specific exon 45 skipping, as confirmed by sequence analysis (not shown). PS220 (SEQ ID NO:3) however, reproducibly induced highest levels of exon 45 skipping (see FIG. 2). (NT: non-treated cells, M: size marker).
FIG. 2. In human control myotubes, 25-mer PS220 (SEQ ID NO: 3) was tested at increasing concentration. Levels of exon 45 skipping of up to 75% (at 400 nM) were observed reproducibly, as assessed by Agilent LabChip Analysis.
FIG. 3. In human control myotubes, the efficiencies of a “short” 17-mer AON45-5 (SEQ ID NO:68) and its overlapping “long” 25-mer counterpart PS220 were directly compared at 200 nM and 500 nM. PS220 was markedly more efficient at both concentrations: 63% when compared to 3% obtained with 45-5. (NT: non-treated cells, M: size marker).
EXAMPLES
Examples 1 and 2
Materials and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program (Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 31, 3406-3415), and on (partly) overlapping putative SR-protein binding sites as predicted by numerous software programs such as ESEfinder (Cartegni, L. et al. (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res, 31, 3568-71; Smith, P. J. et al. (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum. Mol. Genet., 15, 2490-2508) that predicts binding sites for the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55). AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) were obtained as described previously (Aartsma-Rus et al. Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 0 to 500 nM of each AON. The transfection reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) was used according to manufacturer's instructions, with 2 μl PEI per μg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the Agilent DNA 1000 LabChip Kit and the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
A series of AONs targeting sequences within SEQ ID NO:2 within exon 45 were designed and tested in normal myotube cultures, by transfection and subsequent RT-PCR and sequence analysis of isolated RNA. PS220 (SEQ ID NO: 3) reproducibly induced highest levels of exon 45 skipping, when compared to PS221-PS225 (FIG. 1). High levels of exon 45 skipping of up to 75% were already obtained at 400 nM PS220 (FIG. 2). In a direct comparison, PS220 (a 25-mer) was reproducibly more efficient in inducing exon 45 skipping than its shorter 17-mer counterpart AON 45-5 (SEQ ID NO: 68; previously published as h45AON5 (Aartsma-Rus et al. Am J Hum Genet 2004; 74: 83-92)), at both AON concentrations of 200 nM and 500 nM and with 63% versus 3% respectively at 500 nM (FIG. 3). This result is probably due to the fact that the extended length of PS220, in fact completely overlapping AON 45-5, increases the free energy of the AON-target complex such that the efficiency of inducing exon 45 skipping is also increased.
TABLE 1
AONs in exon 45
SEQ ID NO 3
UUUGCCGCUGCCCAAUGCCAUCCUG
(PS220)
SEQ ID NO 4
AUUCAAUGUUCUGACAACAGUUUGC
(PS221)
SEQ ID NO 5
CCAGUUGCAUUCAAUGUUCUGACAA
(PS222)
SEQ ID NO 6
CAGUUGCAUUCAAUGUUCUGAC
(PS223)
SEQ ID NO 7
AGUUGCAUUCAAUGUUCUGA
(PS224)
SEQ ID NO 8
GAUUGCUGAAUUAUUUCUUCC
(PS225)
SEQ ID NO 9
GAUUGCUGAAUUAUUUCUUCCCCAG
SEQ ID NO 10
AUUGCUGAAUUAUUUCUUCCCCAGU
SEQ ID NO 11
UUGCUGAAUUAUUUCUUCCCCAGUU
SEQ ID NO 12
UGCUGAAUUAUUUCUUCCCCAGUUG
SEQ ID NO 13
GCUGAAUUAUUUCUUCCCCAGUUGC
SEQ ID NO 14
CUGAAUUAUUUCUUCCCCAGUUGCA
SEQ ID NO 15
UGAAUUAUUUCUUCCCCAGUUGCAU
SEQ ID NO 16
GAAUUAUUUCUUCCCCAGUUGCAUU
SEQ ID NO 17
AAUUAUUUCUUCCCCAGUUGCAUUC
SEQ ID NO 18
AUUAUUUCUUCCCCAGUUGCAUUCA
SEQ ID NO 19
UUAUUUCUUCCCCAGUUGCAUUCAA
SEQ ID NO 20
UAUUUCUUCCCCAGUUGCAUUCAAU
SEQ ID NO 21
AUUUCUUCCCCAGUUGCAUUCAAUG
SEQ ID NO 22
UUUCUUCCCCAGUUGCAUUCAAUGU
SEQ ID NO 23
UUCUUCCCCAGUUGCAUUCAAUGUU
SEQ ID NO 24
UCUUCCCCAGUUGCAUUCAAUGUUC
SEQ ID NO 25
CUUCCCCAGUUGCAUUCAAUGUUCU
SEQ ID NO 26
UUCCCCAGUUGCAUUCAAUGUUCUG
SEQ ID NO 27
UCCCCAGUUGCAUUCAAUGUUCUGA
SEQ ID NO 28
CCCCAGUUGCAUUCAAUGUUCUGAC
SEQ ID NO 29
CCCAGUUGCAUUCAAUGUUCUGACA
SEQ ID NO 30
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID NO 31
CAGUUGCAUUCAAUGUUCUGACAAC
SEQ ID NO 32
AGUUGCAUUCAAUGUUCUGACAACA
SEQ ID NO 33
UCC UGU AGA AUA CUG GCA UC
SEQ ID NO 34
UGC AGA CCU CCU GCC ACC GCA GAU UCA
SEQ ID NO 35
UUGCAGACCUCCUGCCACCGCAGAUUCAG
GCUUC
SEQ ID NO 36
GUUGCAUUCAAUGUUCUGACAACAG
SEQ ID NO 37
UUGCAUUCAAUGUUCUGACAACAGU
SEQ ID NO 38
UGCAUUCAAUGUUCUGACAACAGUU
SEQ ID NO 39
GCAUUCAAUGUUCUGACAACAGUUU
SEQ ID NO 40
CAUUCAAUGUUCUGACAACAGUUUG
SEQ ID NO 41
AUUCAAUGUUCUGACAACAGUUUGC
SEQ ID NO 42
UCAAUGUUCUGACAACAGUUUGCCG
SEQ ID NO 43
CAAUGUUCUGACAACAGUUUGCCGC
SEQ ID NO 44
AAUGUUCUGACAACAGUUUGCCGCU
SEQ ID NO 45
AUGUUCUGACAACAGUUUGCCGCUG
SEQ ID NO 46
UGUUCUGACAACAGUUUGCCGCUGC
SEQ ID NO 47
GUUCUGACAACAGUUUGCCGCUGCC
SEQ ID NO 48
UUCUGACAACAGUUUGCCGCUGCCC
SEQ ID NO 49
UCUGACAACAGUUUGCCGCUGCCCA
SEQ ID NO 50
CUGACAACAGUUUGCCGCUGCCCAA
SEQ ID NO 51
UGACAACAGUUUGCCGCUGCCCAAU
SEQ ID NO 52
GACAACAGUUUGCCGCUGCCCAAUG
SEQ ID NO 53
ACAACAGUUUGCCGCUGCCCAAUGC
SEQ ID NO 54
CAACAGUUUGCCGCUGCCCAAUGCC
SEQ ID NO 55
AACAGUUUGCCGCUGCCCAAUGCCA
SEQ ID NO 56
ACAGUUUGCCGCUGCCCAAUGCCAU
SEQ ID NO 57
CAGUUUGCCGCUGCCCAAUGCCAUC
SEQ ID NO 58
AGUUUGCCGCUGCCCAAUGCCAUCC
SEQ ID NO 59
GUUUGCCGCUGCCCAAUGCCAUCCU
SEQ ID NO 60
UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID NO 61
UUGCCGCUGCCCAAUGCCAUCCUGG
SEQ ID NO 62
UGCCGCUGCCCAAUGCCAUCCUGGA
SEQ ID NO 63
GCCGCUGCCCAAUGCCAUCCUGGAG
SEQ ID NO 64
CCGCUGCCCAAUGCCAUCCUGGAGU
SEQ ID NO 65
CGCUGCCCAAUGCCAUCCUGGAGUU
SEQ ID NO 66
UGU UUU UGA GGA UUG CUG AA
SEQ ID NO 67
UGUUCUGACAACAGUUUGCCGCUGCCCAAUGC
CAUCCUGG
SEQ ID NO 68
GCCCAAUGCCAUCCUGG
(45-5)
TABLE 2
AONs in exons 51, 53, 7, 44, 46, 59, and 67
DMD Gene Exon 51
SEQ ID NO 69
AGAGCAGGUACCUCCAACAUCAAGG
SEQ ID NO 70
GAGCAGGUACCUCCAACAUCAAGGA
SEQ ID NO 71
AGCAGGUACCUCCAACAUCAAGGAA
SEQ ID NO 72
GCAGGUACCUCCAACAUCAAGGAAG
SEQ ID NO 73
CAGGUACCUCCAACAUCAAGGAAGA
SEQ ID NO 74
AGGUACCUCCAACAUCAAGGAAGAU
SEQ ID NO 75
GGUACCUCCAACAUCAAGGAAGAUG
SEQ ID NO 76
GUACCUCCAACAUCAAGGAAGAUGG
SEQ ID NO 77
UACCUCCAACAUCAAGGAAGAUGGC
SEQ ID NO 78
ACCUCCAACAUCAAGGAAGAUGGCA
SEQ ID NO 79
CCUCCAACAUCAAGGAAGAUGGCAU
SEQ ID NO 80
CUCCAACAUCAAGGAAGAUGGCAUU
SEQ ID NO 81
CUCCAACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 82
UCCAACAUCAAGGAAGAUGGCAUUU
SEQ ID NO 83
CCAACAUCAAGGAAGAUGGCAUUUC
SEQ ID NO 84
CAACAUCAAGGAAGAUGGCAUUUCU
SEQ ID NO 85
AACAUCAAGGAAGAUGGCAUUUCUA
SEQ ID NO 86
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 87
ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID NO 88
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO 89
CAUCAAGGAAGAUGGCAUUUCUAGU
SEQ ID NO 90
AUCAAGGAAGAUGGCAUUUCUAGUU
SEQ ID NO 91
UCAAGGAAGAUGGCAUUUCUAGUUU
SEQ ID NO 92
UCAAGGAAGAUGGCAUUUCU
SEQ ID NO 93
CAAGGAAGAUGGCAUUUCUAGUUUG
SEQ ID NO 94
AAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID NO 95
AGGAAGAUGGCAUUUCUAGUUUGGA
SEQ ID NO 96
GGAAGAUGGCAUUUCUAGUUUGGAG
SEQ ID NO 97
GAAGAUGGCAUUUCUAGUUUGGAGA
SEQ ID NO 98
AAGAUGGCAUUUCUAGUUUGGAGAU
SEQ ID NO 99
AGAUGGCAUUUCUAGUUUGGAGAUG
SEQ ID NO 100
GAUGGCAUUUCUAGUUUGGAGAUGG
SEQ ID NO 101
AUGGCAUUUCUAGUUUGGAGAUGGC
SEQ ID NO 102
UGGCAUUUCUAGUUUGGAGAUGGCA
SEQ ID NO 103
GGCAUUUCUAGUUUGGAGAUGGCAG
SEQ ID NO 104
GCAUUUCUAGUUUGGAGAUGGCAGU
SEQ ID NO 105
CAUUUCUAGUUUGGAGAUGGCAGUU
SEQ ID NO 106
AUUUCUAGUUUGGAGAUGGCAGUUU
SEQ ID NO 107
UUUCUAGUUUGGAGAUGGCAGUUUC
SEQ ID NO 108
UUCUAGUUUGGAGAUGGCAGUUUCC
DMD Gene Exon 53
SEQ ID NO 109
CCAUUGUGUUGAAUCCUUUAACAUU
SEQ ID NO 110
CCAUUGUGUUGAAUCCUUUAAC
SEQ ID NO 111
AUUGUGUUGAAUCCUUUAAC
SEQ ID NO 112
CCUGUCCUAAGACCUGCUCA
SEQ ID NO 113
CUUUUGGAUUGCAUCUACUGUAUAG
SEQ ID NO 114
CAUUCAACUGUUGCCUCCGGUUCUG
SEQ ID NO 115
CUGUUGCCUCCGGUUCUGAAGGUG
SEQ ID NO 116
CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG
SEQ ID NO 117
CUGAAGGUGUUCUUGUACUUCAUCC
SEQ ID NO 118
UGUAUAGGGACCCUCCUUCCAUGACUC
SEQ ID NO 119
AUCCCACUGAUUCUGAAUUC
SEQ ID NO 120
UUGGCUCUGGCCUGUCCUAAGA
SEQ ID NO 121
AAGACCUGCUCAGCUUCUUCCUUAGCUUCCAGCCA
DMD Gene Exon 7
SEQ ID NO 122
UGCAUGUUCCAGUCGUUGUGUGG
SEQ ID NO 123
CACUAUUCCAGUCAAAUAGGUCUGG
SEQ ID NO 124
AUUUACCAACCUUCAGGAUCGAGUA
SEQ ID NO 125
GGCCUAAAACACAUACACAUA
DMD Gene Exon 44
SEQ ID NO 126
UCAGCUUCUGUUAGCCACUG
SEQ ID NO 127
UUCAGCUUCUGUUAGCCACU
SEQ ID NO 128
UUCAGCUUCUGUUAGCCACUG
SEQ ID NO 129
UCAGCUUCUGUUAGCCACUGA
SEQ ID NO 130
UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 131
UCAGCUUCUGUUAGCCACUGA
SEQ ID NO 132
UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 133
UCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 134
UUCAGCUUCUGUUAGCCACUGAU
SEQ ID NO 135
UCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 136
UUCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 137
UCAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 138
UUCAGCUUCUGUUAGCCACUGAUA
SEQ ID NO 139
UCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 140
UUCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 141
UCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 142
UUCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 143
CAGCUUCUGUUAGCCACUG
SEQ ID NO 144
CAGCUUCUGUUAGCCACUGAU
SEQ ID NO 145
AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 146
CAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 147
AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 148
CAGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 149
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 150
CAGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 151
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 152
CAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 153
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 154
AGCUUCUGUUAGCCACUGAU
SEQ ID NO 155
GCUUCUGUUAGCCACUGAUU
SEQ ID NO 156
AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 157
GCUUCUGUUAGCCACUGAUUA
SEQ ID NO 158
AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 159
GCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 160
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 161
GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 162
AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 163
GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 164
CCAUUUGUAUUUAGCAUGUUCCC
SEQ ID NO 165
AGAUACCAUUUGUAUUUAGC
SEQ ID NO 166
GCCAUUUCUCAACAGAUCU
SEQ ID NO 167
GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 168
AUUCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 169
UCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 170
GUUCAGCUUCUGUUAGCC
SEQ ID NO 171
CUGAUUAAAUAUCUUUAUAU C
SEQ ID NO 172
GCCGCCAUUUCUCAACAG
SEQ ID NO 173
GUAUUUAGCAUGUUCCCA
SEQ ID NO 174
CAGGAAUUUGUGUCUUUC
DMD Gene Exon 46
SEQ ID NO 175
GCUUUUCUUUUAGUUGCUGCUCUUU
SEQ ID NO 176
CUUUUCUUUUAGUUGCUGCUCUUUU
SEQ ID NO 177
UUUUCUUUUAGUUGCUGCUCUUUUC
SEQ ID NO 178
UUUCUUUUAGUUGCUGCUCUUUUCC
SEQ ID NO 179
UUCUUUUAGUUGCUGCUCUUUUCCA
SEQ ID NO 180
UCUUUUAGUUGCUGCUCUUUUCCAG
SEQ ID NO 181
CUUUUAGUUGCUGCUCUUUUCCAGG
SEQ ID NO 182
UUUUAGUUGCUGCUCUUUUCCAGGU
SEQ ID NO 183
UUUAGUUGCUGCUCUUUUCCAGGUU
SEQ ID NO 184
UUAGUUGCUGCUCUUUUCCAGGUUC
SEQ ID NO 185
UAGUUGCUGCUCUUUUCCAGGUUCA
SEQ ID NO 186
AGUUGCUGCUCUUUUCCAGGUUCAA
SEQ ID NO 187
GUUGCUGCUCUUUUCCAGGUUCAAG
SEQ ID NO 188
UUGCUGCUCUUUUCCAGGUUCAAGU
SEQ ID NO 189
UGCUGCUCUUUUCCAGGUUCAAGUG
SEQ ID NO 190
GCUGCUCUUUUCCAGGUUCAAGUGG
SEQ ID NO 191
CUGCUCUUUUCCAGGUUCAAGUGGG
SEQ ID NO 192
UGCUCUUUUCCAGGUUCAAGUGGGA
SEQ ID NO 193
GCUCUUUUCCAGGUUCAAGUGGGAC
SEQ ID NO 194
CUCUUUUCCAGGUUCAAGUGGGAUA
SEQ ID NO 195
UCUUUUCCAGGUUCAAGUGGGAUAC
SEQ ID NO 196
CUUUUCCAGGUUCAAGUGGGAUACU
SEQ ID NO 197
UUUUCCAGGUUCAAGUGGGAUACUA
SEQ ID NO 198
UUUCCAGGUUCAAGUGGGAUACUAG
SEQ ID NO 199
UUCCAGGUUCAAGUGGGAUACUAGC
SEQ ID NO 200
UCCAGGUUCAAGUGGGAUACUAGCA
SEQ ID NO 201
CCAGGUUCAAGUGGGAUACUAGCAA
SEQ ID NO 202
CAGGUUCAAGUGGGAUACUAGCAAU
SEQ ID NO 203
AGGUUCAAGUGGGAUACUAGCAAUG
SEQ ID NO 204
GGUUCAAGUGGGAUACUAGCAAUGU
SEQ ID NO 205
GUUCAAGUGGGAUACUAGCAAUGUU
SEQ ID NO 206
UUCAAGUGGGAUACUAGCAAUGUUA
SEQ ID NO 207
UCAAGUGGGAUACUAGCAAUGUUAU
SEQ ID NO 208
CAAGUGGGAUACUAGCAAUGUUAUC
SEQ ID NO 209
AAGUGGGAUACUAGCAAUGUUAUCU
SEQ ID NO 210
AGUGGGAUACUAGCAAUGUUAUCUG
SEQ ID NO 211
GUGGGAUACUAGCAAUGUUAUCUGC
SEQ ID NO 212
UGGGAUACUAGCAAUGUUAUCUGCU
SEQ ID NO 213
GGGAUACUAGCAAUGUUAUCUGCUU
SEQ ID NO 214
GGAUACUAGCAAUGUUAUCUGCUUC
SEQ ID NO 215
GAUACUAGCAAUGUUAUCUGCUUCC
SEQ ID NO 216
AUACUAGCAAUGUUAUCUGCUUCCU
SEQ ID NO 217
UACUAGCAAUGUUAUCUGCUUCCUC
SEQ ID NO 218
ACUAGCAAUGUUAUCUGCUUCCUCC
SEQ ID NO 219
CUAGCAAUGUUAUCUGCUUCCUCCA
SEQ ID NO 220
UAGCAAUGUUAUCUGCUUCCUCCAA
SEQ ID NO 221
AGCAAUGUUAUCUGCUUCCUCCAAC
SEQ ID NO 222
GCAAUGUUAUCUGCUUCCUCCAACC
SEQ ID NO 223
CAAUGUUAUCUGCUUCCUCCAACCA
SEQ ID NO 224
AAUGUUAUCUGCUUCCUCCAACCAU
SEQ ID NO 225
AUGUUAUCUGCUUCCUCCAACCAUA
SEQ ID NO 226
UGUUAUCUGCUUCCUCCAACCAUAA
SEQ ID NO 227
GUUAUCUGCUUCCUCCAACCAUAAA
SEQ ID NO 228
GCUGCUCUUUUCCAGGUUC
SEQ ID NO 229
UCUUUUCCAGGUUCAAGUGG
SEQ ID NO 230
AGGUUCAAGUGGGAUACUA
DMD Gene Exon 59
SEQ ID NO 231
CAAUUUUUCCCACUCAGUAUU
SEQ ID NO 232
UUGAAGUUCCUGGAGUCUU
SEQ ID NO 233
UCCUCAGGAGGCAGCUCUAAAU
DMD Gene Exon 67
SEQ ID NO 234
GCGCUGGUCACAAAAUCCUGUUGAAC
SEQ ID NO 235
CACUUGCUUGAAAAGGUCUACAAAGGA
SEQ ID NO 236
GGUGAAUAACUUACAAAUUUGGAAGC
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14542183
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biomarin technologies b.v.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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BioMarin Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:bmrn
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BioMarin Pharmaceutical
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Dec 29th, 2020 12:00AM
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Jun 29th, 2018 12:00AM
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https://www.uspto.gov?id=US10876114-20201229
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Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53
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The invention relates a method wherein a molecule is used for inducing and/or promoting skipping of at least one of exon 43, exon 46, exons 50-53 of the DMD pre-mRNA in a patient, preferably in an isolated cell of a patient, the method comprising providing the cell and/or the patient with a molecule. The invention also relates to the molecule as such.
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10876114
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1. An isolated antisense oligonucleotide whose base sequence consists of 5′-UUCCAACUGGGGACGCCUCUGUUCC-3′ (SEQ ID NO: 299), wherein the oligonucleotide comprises a modification.
2. The isolated antisense oligonucleotide of claim 1, wherein the modification comprises at least one nucleotide analogue, wherein the nucleotide analogue comprises a modified sugar moiety, a modified backbone, a modified internucleoside linkage, or a modified base, or a combination thereof.
3. The isolated antisense oligonucleotide of claim 1, wherein the modification comprises a modified sugar moiety.
4. The isolated antisense oligonucleotide of claim 3, wherein the modified sugar moiety is mono- or di-substituted at the 2′, 3′ and/or 5′ position.
5. The isolated antisense oligonucleotide of claim 4, wherein the modified sugar moiety comprises a 2′-O-methyl ribose.
6. The isolated antisense oligonucleotide of claim 1, wherein the modification comprises a modified backbone.
7. The isolated antisense oligonucleotide of claim 6, wherein the modified backbone comprises a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone or an amide backbone, or a combination thereof.
8. The isolated antisense oligonucleotide of claim 7, wherein the modified backbone comprises a morpholino backbone.
9. The isolated antisense oligonucleotide of claim 1, wherein the modification comprises a modified internucleoside linkage.
10. The isolated antisense oligonucleotide of claim 9, wherein the modified internucleoside linkage comprises a phosphorothioate linkage.
11. The isolated antisense oligonucleotide of claim 1, wherein the modification comprises a modified base.
12. The isolated antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a morpholino ring, a phosphorodiamidate internucleoside linkage, a peptide nucleic acid, a locked nucleic acid (LNA), or a combination thereof.
13. The isolated antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a 2′-O-methyl phosphorothioate ribose.
14. The isolated antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).
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14
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 15/289,053 filed on Oct. 7, 2016 which is a continuation of U.S. application Ser. No. 14/631,686 filed on Feb. 25, 2015, now U.S. Pat. No. 9,499,818, issued Nov. 22, 2016, which is a continuation of U.S. application Ser. No. 13/094,571 filed Apr. 26, 2011, which is a continuation of International Application No. PCT/NL2009/050113, filed on Mar. 11, 2009, which is a continuation of PCT/NL2008/050673, filed on Oct. 27, 2008, the contents of each of which are herein incorporated by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING
The present specification is being filed with a Sequence Listing in Computer Readable Form (CFR), which is entitled 11808-364-999_SEQLIST.txt of 128,829 bytes in size and was created Nov. 15, 2018; the content of which is incorporated herein by reference in its entirety.
FIELD
The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to modulation of splicing of the human Duchenne Muscular Dystrophy pre-mRNA.
BACKGROUND
Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the dystrophin transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther. 2008; 10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, van Deutekom et al., N Engl J Med. 2007; 357(26):2677-86).
Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; ′t Hoen et al. J Biol Chem. 2008; 283: 5899-907).
Recently, a first-in-man study was successfully completed where an AON inducing the skipping of exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J Med. 2007; 357: 2677-86).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. In human control myotubes, a series of AONs (PS237, PS238, and PS240; SEQ ID NO 65, 66, 16 respectively) targeting exon 43 was tested at 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels of exon 43 skipping. (M: DNA size marker; NT: non-treated cells)
FIG. 2. In myotubes from a DMD patient with an exon 45 deletion, a series of AONs (PS177, PS179, PS181, and PS182; SEQ ID NO 91, 70, 110, and 117 respectively) targeting exon 46 was tested at two different concentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproducibly induced highest levels of exon 46 skipping. (M: DNA size marker)
FIG. 3. In human control myotubes, a series of AONs (PS245, PS246, PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively) targeting exon 50 was tested at 500 nM. PS248 (SEQ ID NO 127) reproducibly induced highest levels of exon 50 skipping. (M: DNA size marker; NT: non-treated cells).
FIG. 4. In human control myotubes, two novel AONs (PS232 and PS236; SEQ ID NO 246 and 299 respectively) targeting exon 52 were tested at two different concentrations (200 and 500 nM) and directly compared to a previously described AON (52-1). PS236 (SEQ ID NO 299) reproducibly induced highest levels of exon 52 skipping. (M: DNA size marker; NT: non-treated cells).
DETAILED DESCRIPTION
Method
In a first aspect, the present invention provides a method for inducing, and/or promoting skipping of at least one of exons 43, 46, 50-53 of the DMD pre-mRNA in a patient, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon. It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method.
Accordingly, a method is provided for inducing and/or promoting skipping of at least one of exons 43, 46, 50-53 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon.
As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient.
A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background. In the case of a DMD patient, an oligonucleotide used will preferably correct one mutation as present in the DMD gene of said patient and therefore will preferably create a DMD protein that will look like a BMD protein: said protein will preferably be a functional dystrophin as later defined herein. In the case of a BMD patient, an oligonucleotide as used will preferably correct one mutation as present in the BMD gene of said patient and therefore will preferably create a dystrophin which will be more functional than the dystrophin which was originally present in said BMD patient.
Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is performed by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with essential sequences such as for example the splice donor of splice acceptor sequence that required for splicing of said exon, or a molecule that is capable of interfering with an exon inclusion signal that is required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
Within the context of the invention, inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.
Preferably, a method of the invention by inducing and/or promoting skipping of at least one of the following exons 43, 46, 50-53 of the DMD pre-mRNA in one or more (muscle) cells of a patient, provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient and/or increases the production of a functional dystrophin is said patient. Providing a patient with a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in said patient is typically applied in a DMD patient. Increasing the production of a functional dystrophin is typically applied in a BMD patient.
Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient is decreased and/or wherein the production of a functional dystrophin is increased in said patient, wherein the level of said aberrant or functional dystrophin is assessed by comparison to the level of said dystrophin in said patient at the onset of the method.
Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.
Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.
Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevent synthesis of the complete protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared tot the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
A method of the invention may alleviate one or more characteristics of a myogenic or muscle cell of a patient or alleviate one or more symptoms of a DMD patient having a deletion including but not limited to exons 44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43 skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46 skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping), 13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52 (correctable by exon 51 skipping), exons 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60, (correctable by exon 52 skipping) and exons 10-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53 skipping) in the DMD gene, occurring in a total of 68% of all DMD patients with a deletion (Aartsma-Rus et al., Hum. Mut. 2009).
Alternatively, a method of the invention may improve one or more characteristics of a muscle cell of a patient or alleviate one or more symptoms of a DMD patient having small mutations in, or single exon duplications of exon 43, 46, 50-53 in the DMD gene, occurring in a total of 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut. 2009)
Furthermore, for some patients the simultaneous skipping of one of more exons in addition to exon 43, exon 46 and/or exon 50-53 is required to restore the open reading frame, including patients with specific deletions, small (point) mutations, or double or multiple exon duplications, such as (but not limited to) a deletion of exons 44-50 requiring the co-skipping of exons 43 and 51, with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, with a deletion of exons 44-52 requiring the co-skipping of exons 43 and 53, with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53, with a deletion of exons 51-54 requiring the co-skipping of exons 50 and 55, with a deletion of exons 53-54 requiring the co-skipping of exons 52 and 55, with a deletion of exons 53-56 requiring the co-skipping of exons 52 and 57, with a nonsense mutation in exon 43 or exon 44 requiring the co-skipping of exon 43 and 44, with a nonsense mutation in exon 45 or exon 46 requiring the co-skipping of exon 45 and 46, with a nonsense mutation in exon 50 or exon 51 requiring the co-skipping of exon 50 and 51, with a nonsense mutation in exon 51 or exon 52 requiring the co-skipping of exon 51 and 52, with a nonsense mutation in exon 52 or exon 53 requiring the co-skipping of exon 52 and 53, or with a double or multiple exon duplication involving exons 43, 46, 50, 51, 52, and/or 53.
In a preferred method, the skipping of exon 43 is induced, or the skipping of exon 46 is induced, or the skipping of exon 50 is induced or the skipping of exon 51 is induced or the skipping of exon 52 is induced or the skipping of exon 53 is induced. An induction of the skipping of two of these exons is also encompassed by a method of the invention. For example, preferably skipping of exons 50 and 51, or 52 and 53, or 43 and 51, or 43 and 53, or 51 and 52. Depending on the type and the identity (the specific exons involved) of mutation identified in a patient, the skilled person will know which combination of exons needs to be skipped in said patient.
In a preferred method, one or more symptom(s) of a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of one or more muscle cells from a DMD or a BMD patient is/are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.
An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD or BMD patient before treatment.
A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD or BMD patient before treatment.
A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of a muscle fiber in a same DMD or BMD patient before treatment. An alleviation of one or more symptoms may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
A treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more.
Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of a molecule or an oligonucleotide or a composition of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.
A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipient are defined in the section entitled “pharmaceutical composition”.
In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of another exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from: exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA of a patient. Molecules which can be used are depicted in any one of Table 1 to 7. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multiexon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006; 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the DMD pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other.
It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.
In case, more than one compounds or molecules are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.
Molecule
In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled “Method”. A molecule as defined herein is preferably an oligonucleotide or antisense oligonucleotide (AON).
It was found by the present investigators that any of exon 43, 46, 50-53 is specifically skipped at a high frequency using a molecule that preferably binds to a continuous stretch of at least 8 nucleotides within said exon. Although this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 8 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex. In a preferred embodiment, a molecule that binds to a continuous stretch of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides within said exon is used.
In a preferred embodiment, a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of any of exon 43, 46, 50-53 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100%. “A part of said exon” preferably means a stretch of at least 8 nucleotides. In a most preferred embodiment, an oligonucleotide of the invention consists of a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein. For example, an oligonucleotide may comprise a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.
A preferred molecule to be used in a method of the invention binds or is complementary to a continuous stretch of at least 8 nucleotides within one of the following nucleotide sequences selected from:
(SEQ ID NO: 2)
5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAU
AGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ for
skipping of exon 43;
(SEQ ID NO: 3)
5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG
AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ for
skipping of exon 46;
(SEQ ID NO: 4)
5′-GGCGGTAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUG
ACCUAGC UCCUGGACUGACCACUAUUGG-3′ for skipping of
exon 50;
(SEQ ID NO: 5)
5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU
AAGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUG
UUGGAGGUAC-3′ for skipping of exon 51;
(SEQ ID NO: 6)
5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU
UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′
for skipping of exon 52,
and
(SEQ ID NO: 7)
5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA
GCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ for skipping of
exon 53.
Of the numerous molecules that theoretically can be prepared to bind to the continuous nucleotide stretches as defined by SEQ ID NO 2-7 within one of said exons, the invention provides distinct molecules that can be used in a method for efficiently skipping of at least one of exon 43, exon 46 and/or exon 50-53. Although the skipping effect can be addressed to the relatively high density of putative SR protein binding sites within said stretches, there could be other parameters involved that favor uptake of the molecule or other, intracellular parameters such as thermodynamic, kinetic, or structural characteristics of the hybrid duplex.
It was found that a molecule that binds to a continuous stretch comprised within or consisting of any of SEQ ID NO 2-7 results in highly efficient skipping of exon 43, exon 46 and/or exon 50-53 respectively in a cell and/or in a patient provided with this molecule. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50 nucleotides within SEQ ID NO 2-7.
In a preferred embodiment for inducing and/or promoting the skipping of any of exon 43, exon 46 and/or exon 50-53, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences depicted in any of Tables 1 to 6. A molecule of the invention preferably comprises or consist of the antisense nucleotide sequence of SEQ ID NO 16, SEQ ID NO 65, SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO 127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ ID NO 299, SEQ ID NO:357.
A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 8 and 50 nucleotides or bases, more preferred between 10 and 50 nucleotides, more preferred between 20 and 40 nucleotides, more preferred between 20 and 30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides. A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.
Furthermore, none of the indicated sequences is derived from conserved parts of splice junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.
In one embodiment, a molecule of the invention is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the corresponding nucleotide sequence on a DMD pre-RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein incorporated by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein incorporated by reference.
A preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 2: 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAAG AAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ which is present in exon 43 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 8 to SEQ ID NO 69. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 16 and/or SEQ ID NO 65. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 65. It was found that this molecule is very efficient in modulating splicing of exon 43 of the DMD pre-mRNA in a muscle cell and/or in a patient.
Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 3: 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ which is present in exon 46 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70 to SEQ ID NO 122. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID NO 117. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 117. It was found that this molecule is very efficient in modulating splicing of exon 46 of the DMD pre-mRNA in a muscle cell or in a patient.
Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 4: 5′-GGCGGTAAACCGUUUACUUCAAGAGCU GAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3′ which is present in exon 50 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127. It was found that this molecule is very efficient in modulating splicing of exon 50 of the DMD pre-mRNA in a muscle cell and/or in a patient.
Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 5: 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′ which is present in exon 51 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 168 to SEQ ID NO 241.
Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 6: 5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ which is present in exon 52 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 299. It was found that this molecule is very efficient in modulating splicing of exon 52 of the DMD pre-mRNA in a muscle cell and/or in a patient.
Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 7: 5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ which is present in exon 53 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQ ID NO 358. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 357. It was found that this molecule is very efficient in modulating splicing of exon 53 of the DMD pre-mRNA in a muscle cell and/or in a patient.
A nucleotide sequence of a molecule of the invention may contain RNA residues, or one or more DNA residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.
It is preferred that a molecule of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
It is further preferred that that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-0,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
A preferred antisense oligonucleotide according to the invention comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
A most preferred antisense oligonucleotide according to the invention comprises of 2′-O-methyl phosphorothioate ribose.
A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing exon 43, 46, 50, 51, 52, or 53 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 43, 46, 50, 51, 52, or 53 skipping and by quantifying the amount of functional dystrophin protein. A functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analyses. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
It will be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping any of exon 43, exon 46, exon 50, exon 51, exon 52 and/or exon 53 of the human DMD pre-mRNA. It is encompassed by the present invention to use one, two, three, four, five or more oligonucleotides for skipping one of said exons (i.e. exon, 43, 46, 50, 51, 52, or 53). It is also encompassed to use at least two oligonucleotides for skipping at least two, of said exons. Preferably two of said exons are skipped. More preferably, these two exons are:
−43 and 51, or
−43 and 53, or
−50 and 51, or
−51 and 52, or
−52 and 53.
The skilled person will know which combination of exons is preferred to be skipped depending on the type, the number and the location of the mutation present in a DMD or BMD patient.
An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
A preferred antisense oligonucleotide comprises a peptide-linked PMO.
A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for inducing skipping of exon 43, 46, 50, 51, 52, or 53 of the human DMD pre-mRNA.
A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette that drives expression of a molecule as identified herein. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.
One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of exon 43, 46, 50, 51, 52 or 53 of the DMD pre-mRNA.
A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA.
Pharmaceutical Composition
If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.
Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.
A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector as defined herein which is able to induce skipping of exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA. Preferred molecules able to induce skipping of any of these exon are identified in any one of Tables 1 to 7.
Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 μM is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
More preferably, a compound preferably an oligonucleotide to be used in the invention to prevent, treat DMD or BMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
A preferred oligonucleotide as defined herein optionally comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells.
In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting a different subset of muscle cells. Therefore, in this embodiment, it is preferred to use a combination of oligonucleotides comprising different nucleotide analogs or equivalents for modulating splicing of the DMD mRNA in at least one type of muscle cells.
In a preferred embodiment, there is provided a molecule or a viral-based vector for use as a medicament, preferably for modulating splicing of the DMD pre-mRNA, more preferably for promoting or inducing skipping of any of exon 43, 46, 50-53 as identified herein.
Use
In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for modulating splicing of the DMD pre-mRNA. The splicing is preferably modulated in a human myogenic cell or muscle cell in vitro. More preferred is that splicing is modulated in a human muscle cell in vivo. Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for modulating splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD or BMD patient.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
EXAMPLES
Examples 1-4
Materials and Methods
AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
Myotube cultures derived from a healthy individual (“human control”) (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 50 nM and 150 nM (example 2), 200 nM and 500 nM (example 4) or 500 nM only (examples 1 and 3) of each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNIFectylin perm AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exons (43, 46, 50, 51, 52, or 53). PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
DMD Exon 43 Skipping.
A series of AONs targeting sequences within exon 43 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 43 herein defined as SEQ ID NO 2, was indeed capable of inducing exon 43 skipping. PS237 (SEQ ID NO: 65) reproducibly induced highest levels of exon 43 skipping (up to 66%) at 500 nM, as shown in FIG. 1. For comparison, also PS238 and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36% respectively (FIG. 1). The precise skipping of exon 43 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 43 skipping was observed in non-treated cells (NT).
DMD Exon 46 Skipping.
A series of AONs targeting sequences within exon 46 were designed and transfected in myotube cultures derived from a DMD patient carrying an exon 45 deletion in the DMD gene. For patients with such mutation antisense-induced exon 46 skipping would induce the synthesis of a novel, BMD-like dystrophin protein that may indeed alleviate one or more symptoms of the disease. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 46 herein defined as SEQ ID NO 3, was indeed capable of inducing exon 46 skipping, even at relatively low AON concentrations of 50 nM. PS182 (SEQ ID NO: 117) reproducibly induced highest levels of exon 46 skipping (up to 50% at 50 nM and 74% at 150 nM), as shown in FIG. 2. For comparison, also PS177, PS179, and PS181 are shown, inducing exon 46 skipping levels up to 55%, 58% and 42% respectively at 150 nM (FIG. 2). The precise skipping of exon 46 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 46 skipping was observed in non-treated cells (NT).
DMD Exon 50 Skipping.
A series of AONs targeting sequences within exon 50 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 50 herein defined as SEQ ID NO 4, was indeed capable of inducing exon 50 skipping. PS248 (SEQ ID NO: 127) reproducibly induced highest levels of exon 50 skipping (up to 35% at 500 nM), as shown in FIG. 3. For comparison, also PS245, PS246, and PS247 are shown, inducing exon 50 skipping levels up to 14-16% at 500 nM (FIG. 3). The precise skipping of exon 50 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 50 skipping was observed in non-treated cells (NT).
DMD Exon 51 Skipping.
A series of AONs targeting sequences within exon 51 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 51 herein defined as SEQ ID NO 5, was indeed capable of inducing exon 51 skipping. The AON with SEQ ID NO 180 reproducibly induced highest levels of exon 51 skipping (not shown).
DMD Exon 52 Skipping.
A series of AONs targeting sequences within exon 52 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 52 herein defined as SEQ ID NO 6, was indeed capable of inducing exon 52 skipping. PS236 (SEQ ID NO: 299) reproducibly induced highest levels of exon 52 skipping (up to 88% at 200 nM and 91% at 500 nM), as shown in FIG. 4. For comparison, also PS232 and AON 52-1 (previously published by Aartsma-Rus et al. Oligonucleotides 2005) are shown, inducing exon 52 skipping at levels up to 59% and 10% respectively when applied at 500 nM (FIG. 4). The precise skipping of exon 52 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 52 skipping was observed in non-treated cells (NT).
DMD Exon 53 Skipping.
A series of AONs targeting sequences within exon 53 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 53 herein defined as SEQ ID NO 7, was indeed capable of inducing exon 53 skipping. The AON with SEQ ID NO 328 reproducibly induced highest levels of exon 53 skipping (not shown).
TABLE 1
oligonucleotides for skipping DMD Gene Exon 43
SEQ ID
CCACAGGCGUUGCACUUUGCAAUGC
SEQ ID NO 39
UCUUCUUGCUAUGAAUAAUGUCAAU
NO 8
SEQ ID
CACAGGCGUUGCACUUUGCAAUGCU
SEQ ID NO 40
CUUCUUGCUAUGAAUAAUGUCAAUC
NO 9
SEQ ID
ACAGGCGUUGCACUUUGCAAUGCUG
SEQ ID NO 41
UUCUUGCUAUGAAUAAUGUCAAUCC
NO 10
SEQ ID
CAGGCGUUGCACUUUGCAAUGCUGC
SEQ ID NO 42
UCUUGCUAUGAAUAAUGUCAAUCCG
NO 11
SEQ ID
AGGCGUUGCACUUUGCAAUGCUGCU
SEQ ID NO 43
CUUGCUAUGAAUAAUGUCAAUCCGA
NO 12
SEQ ID
GGCGUUGCACUUUGCAAUGCUGCUG
SEQ ID NO 44
UUGCUAUGAAUAAUGUCAAUCCGAC
NO 13
SEQ ID
GCGUUGCACUUUGCAAUGCUGCUGU
SEQ ID NO 45
UGCUAUGAAUAAUGUCAAUCCGACC
NO 14
SEQ ID
CGUUGCACUUUGCAAUGCUGCUGUC
SEQ ID NO 46
GCUAUGAAUAAUGUCAAUCCGACCU
NO 15
SEQ ID
CGUUGCACUUUGCAAUGCUGCUG
SEQ ID NO 47
CUAUGAAUAAUGUCAAUCCGACCUG
NO 16
PS240
SEQ ID
GUUGCACUUUGCAAUGCUGCUGUCU
SEQ ID NO 48
UAUGAAUAAUGUCAAUCCGACCUGA
NO 17
SEQ ID
UUGCACUUUGCAAUGCUGCUGUCUU
SEQ ID NO 49
AUGAAUAAUGUCAAUCCGACCUGAG
NO 18
SEQ ID
UGCACUUUGCAAUGCUGCUGUCUUC
SEQ ID NO 50
UGAAUAAUGUCAAUCCGACCUGAGC
NO 19
SEQ ID
GCACUUUGCAAUGCUGCUGUCUUCU
SEQ ID NO 51
GAAUAAUGUCAAUCCGACCUGAGCU
NO 20
SEQ ID
CACUUUGCAAUGCUGCUGUCUUCUU
SEQ ID NO 52
AAUAAUGUCAAUCCGACCUGAGCUU
NO 21
SEQ ID
ACUUUGCAAUGCUGCUGUCUUCUUG
SEQ ID NO 53
AUAAUGUCAAUCCGACCUGAGCUUU
NO 22
SEQ ID
CUUUGCAAUGCUGCUGUCUUCUUGC
SEQ ID NO 54
UAAUGUCAAUCCGACCUGAGCUUUG
NO 23
SEQ ID
UUUGCAAUGCUGCUGUCUUCUUGCU
SEQ ID NO 55
AAUGUCAAUCCGACCUGAGCUUUGU
NO 24
SEQ ID
UUGCAAUGCUGCUGUCUUCUUGCUA
SEQ ID NO 56
AUGUCAAUCCGACCUGAGCUUUGUU
NO 25
SEQ ID
UGCAAUGCUGCUGUCUUCUUGCUAU
SEQ ID NO 57
UGUCAAUCCGACCUGAGCUUUGUUG
NO 26
SEQ ID
GCAAUGCUGCUGUCUUCUUGCUAUG
SEQ ID NO 58
GUCAAUCCGACCUGAGCUUUGUUGU
NO 27
SEQ ID
CAAUGCUGCUGUCUUCUUGCUAUGA
SEQ ID NO 59
UCAAUCCGACCUGAGCUUUGUUGUA
NO 28
SEQ ID
AAUGCUGCUGUCUUCUUGCUAUGAA
SEQ ID NO 60
CAAUCCGACCUGAGCUUUGUUGUAG
NO 29
SEQ ID
AUGCUGCUGUCUUCUUGCUAUGAAU
SEQ ID NO 61
AAUCCGACCUGAGCUUUGUUGUAGA
NO 30
SEQ ID
UGCUGCUGUCUUCUUGCUAUGAAUA
SEQ ID NO 62
AUCCGACCUGAGCUUUGUUGUAGAC
NO 31
SEQ ID
GCUGCUGUCUUCUUGCUAUGAAUAA
SEQ ID NO 63
UCCGACCUGAGCUUUGUUGUAGACU
NO 32
SEQ ID
CUGCUGUCUUCUUGCUAUGAAUAAU
SEQ ID NO 64
CCGACCUGAGCUUUGUUGUAGACUA
NO 33
SEQ ID
UGCUGUCUUCUUGCUAUGAAUAAU
SEQ ID NO 65
CGACCUGAGCUUUGUUGUAG
NO 34
G
PS237
SEQ ID
GCUGUCUUCUUGCUAUGAAUAAUG
SEQ ID NO 66
CGACCUGAGCUUUGUUGUAGACUAU
NO 35
U
PS238
SEQ ID
CUGUCUUCUUGCUAUGAAUAAUGUC
SEQ ID NO 67
GACCUGAGCUUUGUUGUAGACUAUC
NO 36
SEQ ID
UGUCUUCUUGCUAUGAAUAAUGUC
SEQ ID NO 68
ACCUGAGCUUUGUUGUAGACUAUCA
NO 37
A
SEQ ID
GUCUUCUUGCUAUGAAUAAUGUCA
SEQ ID NO 69
CCUGA GCUUU GUUGU AGACU AUC
NO 38
A
TABLE 2
oligonucleotides for skipping DMD Gene Exon 46
SEQ ID
GCUUUUCUUUUAGUUGCUGCUCUUU
SEQ ID NO 97
CCAGGUUCAAGUGGGAUACUAGCAA
NO 70
PS179
SEQ ID
CUUUUCUUUUAGUUGCUGCUCUUUU
SEQ ID NO 98
CAGGUUCAAGUGGGAUACUAGCAAU
NO 71
SEQ ID
UUUUCUUUUAGUUGCUGCUCUUUUC
SEQ ID NO 99
AGGUUCAAGUGGGAUACUAGCAAUG
NO 72
SEQ ID
UUUCUUUUAGUUGCUGCUCUUUUCC
SEQ ID NO
GGUUCAAGUGGGAUACUAGCAAUGU
NO 73
100
SEQ ID
UUCUUUUAGUUGCUGCUCUUUUCCA
SEQ ID NO
GUUCAAGUGGGAUACUAGCAAUGUU
NO 74
101
SEQ ID
UCUUUUAGUUGCUGCUCUUUUCCAG
SEQ ID NO
UUCAAGUGGGAUACUAGCAAUGUUA
NO 75
102
SEQ ID
CUUUUAGUUGCUGCUCUUUUCCAGG
SEQ ID NO
UCAAGUGGGAUACUAGCAAUGUUAU
NO 76
103
SEQ ID
UUUUAGUUGCUGCUCUUUUCCAGGU
SEQ ID NO
CAAGUGGGAUACUAGCAAUGUUAUC
NO 77
104
SEQ ID
UUUAGUUGCUGCUCUUUUCCAGGUU
SEQ ID NO
AAGUGGGAUACUAGCAAUGUUAUCU
NO 78
105
SEQ ID
UUAGUUGCUGCUCUUUUCCAGGUUC
SEQ ID NO
AGUGGGAUACUAGCAAUGUUAUCUG
NO 79
106
SEQ ID
UAGUUGCUGCUCUUUUCCAGGUUCA
SEQ ID NO
GUGGGAUACUAGCAAUGUUAUCUGC
NO 80
107
SEQ ID
AGUUGCUGCUCUUUUCCAGGUUCAA
SEQ ID NO
UGGGAUACUAGCAAUGUUAUCUGCU
NO 81
108
SEQ ID
GUUGCUGCUCUUUUCCAGGUUCAAG
SEQ ID NO
GGGAUACUAGCAAUGUUAUCUGCUU
NO 82
109
SEQ ID
UUGCUGCUCUUUUCCAGGUUCAAGU
SEQ ID NO
GGAUACUAGCAAUGUUAUCUGCUUC
NO 83
110
PS181
SEQ ID
UGCUGCUCUUUUCCAGGUUCAAGUG
SEQ ID NO
GAUACUAGCAAUGUUAUCUGCUUCC
NO 84
111
SEQ ID
GCUGCUCUUUUCCAGGUUCAAGUGG
SEQ ID NO
AUACUAGCAAUGUUAUCUGCUUCCU
NO 85
112
SEQ ID
CUGCUCUUUUCCAGGUUCAAGUGGG
SEQ ID NO
UACUAGCAAUGUUAUCUGCUUCCUC
NO 86
113
SEQ ID
UGCUCUUUUCCAGGUUCAAGUGGGA
SEQ ID NO
ACUAGCAAUGUUAUCUGCUUCCUCC
NO 87
114
SEQ ID
GCUCUUUUCCAGGUUCAAGUGGGAC
SEQ ID NO
CUAGCAAUGUUAUCUGCUUCCUCCA
NO 88
115
SEQ ID
CUCUUUUCCAGGUUCAAGUGGGAUA
SEQ ID NO
UAGCAAUGUUAUCUGCUUCCUCCAA
NO 89
116
SEQ ID
UCUUUUCCAGGUUCAAGUGGGAUAC
SEQ ID NO
AGCAAUGUUAUCUGCUUCCUCCAAC
NO 90
117
PS182
SEQ ID
UCUUUUCCAGGUUCAAGUGG
SEQ ID NO
GCAAUGUUAUCUGCUUCCUCCAACC
NO 91
118
PS177
SEQ ID
CUUUUCCAGGUUCAAGUGGGAUACU
SEQ ID NO
CAAUGUUAUCUGCUUCCUCCAACCA
NO 92
119
SEQ ID
UUUUCCAGGUUCAAGUGGGAUACU
SEQ ID NO
AAUGUUAUCUGCUUCCUCCAACCAU
NO 93
A
120
SEQ ID
UUUCCAGGUUCAAGUGGGAUACUA
SEQ ID NO
AUGUUAUCUGCUUCCUCCAACCAUA
NO 94
G
121
SEQ ID
UUCCAGGUUCAAGUGGGAUACUAGC
SEQ ID NO
UGUUAUCUGCUUCCUCCAACCAUAA
NO 95
122
SEQ ID
UCCAGGUUCAAGUGGGAUACUAGCA
NO 96
TABLE 3
oligonucleotides for skipping DMD Gene Exon 50
SEQ ID
CCAAUAGUGGUCAGUCCAGGAGCUA
SEQ ID NO
CUAGGUCAGGCUGCUUUGCCCUCAG
NO 123
146
SEQ ID
CAAUAGUGGUCAGUCCAGGAGCUAG
SEQ ID NO
UAGGUCAGGCUGCUUUGCCCUCAGC
NO 124
147
SEQ ID
AAUAGUGGUCAGUCCAGGAGCUAGG
SEQ ID NO
AGGUCAGGCUGCUUUGCCCUCAGCU
NO 125
148
SEQ ID
AUAGUGGUCAGUCCAGGAGCUAGGU
SEQ ID NO
GGUCAGGCUGCUUUGCCCUCAGCUC
NO 126
149
SEQ ID
AUAGUGGUCAGUCCAGGAGCU
SEQ ID NO
GUCAGGCUGCUUUGCCCUCAGCUCU
NO 127
150
PS248
SEQ ID
UAGUGGUCAGUCCAGGAGCUAGGUC
SEQ ID NO
UCAGGCUGCUUUGCCCUCAGCUCUU
NO 128
151
SEQ ID
AGUGGUCAGUCCAGGAGCUAGGUCA
SEQ ID NO
CAGGCUGCUUUGCCCUCAGCUCUUG
NO 129
152
SEQ ID
GUGGUCAGUCCAGGAGCUAGGUCAG
SEQ ID NO
AGGCUGCUUUGCCCUCAGCUCUUGA
NO 130
153
SEQ ID
UGGUCAGUCCAGGAGCUAGGUCAGG
SEQ ID NO
GGCUGCUUUGCCCUCAGCUCUUGAA
NO 131
154
SEQ ID
GGUCAGUCCAGGAGCUAGGUCAGGC
SEQ ID NO
GCUGCUUUGCCCUCAGCUCUUGAAG
NO 132
155
SEQ ID
GUCAGUCCAGGAGCUAGGUCAGGCU
SEQ ID NO
CUGCUUUGCCCUCAGCUCUUGAAGU
NO 133
156
SEQ ID
UCAGUCCAGGAGCUAGGUCAGGCUG
SEQ ID NO
UGCUUUGCCCUCAGCUCUUGAAGUA
NO 134
157
SEQ ID
CAGUCCAGGAGCUAGGUCAGGCUGC
SEQ ID NO
GCUUUGCCCUCAGCUCUUGAAGUAA
NO 135
158
SEQ ID
AGUCCAGGAGCUAGGUCAGGCUGCU
SEQ ID NO
CUUUGCCCUCAGCUCUUGAAGUAAA
NO 136
159
SEQ ID
GUCCAGGAGCUAGGUCAGGCUGCUU
SEQ ID NO
UUUGCCCUCAGCUCUUGAAGUAAAC
NO 137
160
SEQ ID
UCCAGGAGCUAGGUCAGGCUGCUUU
SEQ ID NO
UUGCCCUCAGCUCUUGAAGUAAACG
NO 138
161
SEQ ID
CCAGGAGCUAGGUCAGGCUGCUUUG
SEQ ID NO
UGCCCUCAGCUCUUGAAGUAAACGG
NO 139
162
SEQ ID
CAGGAGCUAGGUCAGGCUGCUUUGC
SEQ ID NO
GCCCUCAGCUCUUGAAGUAAACGGU
NO 140
163
SEQ ID
AGGAGCUAGGUCAGGCUGCUUUGCC
SEQ ID NO
CCCUCAGCUCUUGAAGUAAACGGUU
NO 141
164
SEQ ID
GGAGCUAGGUCAGGCUGCUUUGCCC
SEQ ID NO
CCUCAGCUCUUGAAGUAAAC
NO 142
165
PS246
SEQ ID
GAGCUAGGUCAGGCUGCUUUGCCCU
SEQ ID NO
CCUCAGCUCUUGAAGUAAACG
NO 143
166
PS247
SEQ ID
AGCUAGGUCAGGCUGCUUUGCCCUC
SEQ ID NO
CUCAGCUCUUGAAGUAAACG
NO 144
167
PS245
SEQ ID
GCUAGGUCAGGCUGCUUUGCCCUCA
SEQ ID NO
CCUCAGCUCUUGAAGUAAACGGUUU
NO 145
529
SEQ ID
CUCAGCUCUUGAAGUAAACGGUUUA
SEQ ID NO
UCAGCUCUUGAAGUAAACGGUUUAC
NO 530
531
SEQ ID
CAGCUCUUGAAGUAAACGGUUUACC
SEQ ID NO
AGCUCUUGAAGUAAACGGUUUACCG
NO 532
533
SEQ ID
GCUCUUGAAGUAAACGGUUUACCGC
SEQ ID NO
CUCUUGAAGUAAACGGUUUACCGCC
NO 534
535
TABLE 4
oligonucleotides for skipping DMD Gene Exon 51
SEQ ID
GUACCUCCAACAUCAAGGAAGAUGG
SEQ ID NO
GAGAUGGCAGUUUCCUUAGUAACCA
NO 168
205
SEQ ID
UACCUCCAACAUCAAGGAAGAUGGC
SEQ ID NO
AGAUGGCAGUUUCCUUAGUAACCAC
NO 169
206
SEQ ID
ACCUCCAACAUCAAGGAAGAUGGCA
SEQ ID NO
GAUGGCAGUUUCCUUAGUAACCACA
NO 170
207
SEQ ID
CCUCCAACAUCAAGGAAGAUGGCAU
SEQ ID NO
AUGGCAGUUUCCUUAGUAACCACAG
NO 171
208
SEQ ID
CUCCAACAUCAAGGAAGAUGGCAUU
SEQ ID NO
UGGCAGUUUCCUUAGUAACCACAGG
NO 172
209
SEQ ID
UCCAACAUCAAGGAAGAUGGCAUUU
SEQ ID NO
GGCAGUUUCCUUAGUAACCACAGGU
NO 173
210
SEQ ID
CCAACAUCAAGGAAGAUGGCAUUUC
SEQ ID NO
GCAGUUUCCUUAGUAACCACAGGUU
NO 174
211
SEQ ID
CAACAUCAAGGAAGAUGGCAUUUCU
SEQ ID NO
CAGUUUCCUUAGUAACCACAGGUUG
NO 175
212
SEQ ID
AACAUCAAGGAAGAUGGCAUUUCUA
SEQ ID NO
AGUUUCCUUAGUAACCACAGGUUGU
NO 176
213
SEQ ID
ACAUCAAGGAAGAUGGCAUUUCUAG
SEQ ID NO
GUUUCCUUAGUAACCACAGGUUGUG
NO 177
214
SEQ ID
CAUCAAGGAAGAUGGCAUUUCUAGU
SEQ ID NO
UUUCCUUAGUAACCACAGGUUGUGU
NO 178
215
SEQ ID
AUCAAGGAAGAUGGCAUUUCUAGUU
SEQ ID NO
UUCCUUAGUAACCACAGGUUGUGUC
NO 179
216
SEQ ID
UCAAGGAAGAUGGCAUUUCUAGUUU
SEQ ID NO
UCCUUAGUAACCACAGGUUGUGUCA
NO 180
217
SEQ ID
CAAGGAAGAUGGCAUUUCUAGUUUG
SEQ ID NO
CCUUAGUAACCACAGGUUGUGUCAC
NO 181
218
SEQ ID
AAGGAAGAUGGCAUUUCUAGUUUGG
SEQ ID NO
CUUAGUAACCACAGGUUGUGUCACC
NO 182
219
SEQ ID
AGGAAGAUGGCAUUUCUAGUUUGGA
SEQ ID NO
UUAGUAACCACAGGUUGUGUCACCA
NO 183
220
SEQ ID
GGAAGAUGGCAUUUCUAGUUUGGAG
SEQ ID NO
UAGUAACCACAGGUUGUGUCACCAG
NO 184
221
SEQ ID
GAAGAUGGCAUUUCUAGUUUGGAGA
SEQ ID NO
AGUAACCACAGGUUGUGUCACCAGA
NO 185
222
SEQ ID
AAGAUGGCAUUUCUAGUUUGGAGAU
SEQ ID NO
GUAACCACAGGUUGUGUCACCAGAG
NO 186
223
SEQ ID
AGAUGGCAUUUCUAGUUUGGAGAUG
SEQ ID NO
UAACCACAGGUUGUGUCACCAGAGU
NO 187
224
SEQ ID
GAUGGCAUUUCUAGUUUGGAGAUGG
SEQ ID NO
AACCACAGGUUGUGUCACCAGAGUA
NO 188
225
SEQ ID
AUGGCAUUUCUAGUUUGGAGAUGGC
SEQ ID NO
ACCACAGGUUGUGUCACCAGAGUAA
NO 189
226
SEQ ID
UGGCAUUUCUAGUUUGGAGAUGGCA
SEQ ID NO
CCACAGGUUGUGUCACCAGAGUAAC
NO 190
227
SEQ ID
GGCAUUUCUAGUUUGGAGAUGGCAG
SEQ ID NO
CACAGGUUGUGUCACCAGAGUAACA
NO 191
228
SEQ ID
GCAUUUCUAGUUUGGAGAUGGCAGU
SEQ ID NO
ACAGGUUGUGUCACCAGAGUAACAG
NO 192
229
SEQ ID
CAUUUCUAGUUUGGAGAUGGCAGUU
SEQ ID NO
CAGGUUGUGUCACCAGAGUAACAGU
NO 193
230
SEQ ID
AUUUCUAGUUUGGAGAUGGCAGUUU
SEQ ID NO
AGGUUGUGUCACCAGAGUAACAGUC
NO 194
231
SEQ ID
UUUCUAGUUUGGAGAUGGCAGUUUC
SEQ ID NO
GGUUGUGUCACCAGAGUAACAGUCU
NO 195
232
SEQ ID
UUCUAGUUUGGAGAUGGCAGUUUCC
SEQ ID NO
GUUGUGUCACCAGAGUAACAGUCUG
NO 196
233
SEQ ID
UCUAGUUUGGAGAUGGCAGUUUCCU
SEQ ID NO
UUGUGUCACCAGAGUAACAGUCUGA
NO 197
234
SEQ ID
CUAGUUUGGAGAUGGCAGUUUCCUU
SEQ ID NO
UGUGUCACCAGAGUAACAGUCUGAG
NO 198
235
SEQ ID
UAGUUUGGAGAUGGCAGUUUCCUUA
SEQ ID NO
GUGUCACCAGAGUAACAGUCUGAGU
NO 199
236
SEQ ID
AGUUUGGAGAUGGCAGUUUCCUUAG
SEQ ID NO
UGUCACCAGAGUAACAGUCUGAGUA
NO 200
237
SEQ ID
GUUUGGAGAUGGCAGUUUCCUUAGU
SEQ ID NO
GUCACCAGAGUAACAGUCUGAGUAG
NO 201
238
SEQ ID
UUUGGAGAUGGCAGUUUCCUUAGUA
SEQ ID NO
UCACCAGAGUAACAGUCUGAGUAGG
NO 202
239
SEQ ID
UUGGAGAUGGCAGUUUCCUUAGUAA
SEQ ID NO
CACCAGAGUAACAGUCUGAGUAGGA
NO 203
240
SEQ ID
UGGAGAUGGCAGUUUCCUUAGUAAC
SEQ ID NO
ACCAGAGUAACAGUCUGAGUAGGAG
NO 204
241
TABLE 5
oligonucleotides for skipping DMD Gene Exon 52
SEQ ID
AGCCUCUUGAUUGCUGGUCUUGUUU
SEQ ID NO
UUGGGCAGCGGUAAUGAGUUCUUCC
NO 242
277
SEQ ID
GCCUCUUGAUUGCUGGUCUUGUUUU
SEQ ID NO
UGGGCAGCGGUAAUGAGUUCUUCCA
NO 243
278
SEQ ID
CCUCUUGAUUGCUGGUCUUGUUUUU
SEQ ID NO
GGGCAGCGGUAAUGAGUUCUUCCAA
NO 244
279
SEQ ID
CCUCUUGAUUGCUGGUCUUG
SEQ ID NO
GGCAGCGGUAAUGAGUUCUUCCAAC
NO 245
280
SEQ ID
CUCUUGAUUGCUGGUCUUGUUUUUC
SEQ ID NO
GCAGCGGUAAUGAGUUCUUCCAACU
NO 246
281
PS232
SEQ ID
UCUUGAUUGCUGGUCUUGUUUUUCA
SEQ ID NO
CAGCGGUAAUGAGUUCUUCCAACUG
NO 247
282
SEQ ID
CUUGAUUGCUGGUCUUGUUUUUCAA
SEQ ID NO
AGCGGUAAUGAGUUCUUCCAACUGG
NO 248
283
SEQ ID
UUGAUUGCUGGUCUUGUUUUUCAAA
SEQ ID NO
GCGGUAAUGAGUUCUUCCAACUGGG
NO 249
284
SEQ ID
UGAUUGCUGGUCUUGUUUUUCAAAU
SEQ ID NO
CGGUAAUGAGUUCUUCCAACUGGGG
NO 250
285
SEQ ID
GAUUGCUGGUCUUGUUUUUCAAAUU
SEQ ID NO
GGUAAUGAGUUCUUCCAACUGGGGA
NO 251
286
SEQ ID
GAUUGCUGGUCUUGUUUUUC
SEQ ID NO
GGUAAUGAGUUCUUCCAACUGG
NO 252
287
SEQ ID
AUUGCUGGUCUUGUUUUUCAAAUUU
SEQ ID NO
GUAAUGAGUUCUUCCAACUGGGGAC
NO 253
288
SEQ ID
UUGCUGGUCUUGUUUUUCAAAUUUU
SEQ ID NO
UAAUGAGUUCUUCCAACUGGGGACG
NO 254
289
SEQ ID
UGCUGGUCUUGUUUUUCAAAUUUUG
SEQ ID NO
AAUGAGUUCUUCCAACUGGGGACGC
NO 255
290
SEQ ID
GCUGGUCUUGUUUUUCAAAUUUUGG
SEQ ID NO
AUGAGUUCUUCCAACUGGGGACGCC
NO 256
291
SEQ ID
CUGGUCUUGUUUUUCAAAUUUUGGG
SEQ ID NO
UGAGUUCUUCCAACUGGGGACGCCU
NO 257
292
SEQ ID
UGGUCUUGUUUUUCAAAUUUUGGGC
SEQ ID NO
GAGUUCUUCCAACUGGGGACGCCUC
NO 258
293
SEQ ID
GGUCUUGUUUUUCAAAUUUUGGGCA
SEQ ID NO
AGUUCUUCCAACUGGGGACGCCUCU
NO 259
294
SEQ ID
GUCUUGUUUUUCAAAUUUUGGGCAG
SEQ ID NO
GUUCUUCCAACUGGGGACGCCUCUG
NO 260
295
SEQ ID
UCUUGUUUUUCAAAUUUUGGGCAGC
SEQ ID NO
UUCUUCCAACUGGGGACGCCUCUGU
NO 261
296
SEQ ID
CUUGUUUUUCAAAUUUUGGGCAGCG
SEQ ID NO
UCUUCCAACUGGGGACGCCUCUGUU
NO 262
297
SEQ ID
UUGUUUUUCAAAUUUUGGGCAGCGG
SEQ ID NO
CUUCCAACUGGGGACGCCUCUGUUC
NO 263
298
SEQ ID
UGUUUUUCAAAUUUUGGGCAGCGGU
SEQ ID NO
UUCCAACUGGGGACGCCUCUGUUCC
NO 264
299
PS236
SEQ ID
GUUUUUCAAAUUUUGGGCAGCGGUA
SEQ ID NO
UCCAACUGGGGACGCCUCUGUUCCA
NO 265
300
SEQ ID
UUUUUCAAAUUUUGGGCAGCGGUAA
SEQ ID NO
CCAACUGGGGACGCCUCUGUUCCAA
NO 266
301
SEQ ID
UUUUCAAAUUUUGGGCAGCGGUAAU
SEQ ID NO
CAACUGGGGACGCCUCUGUUCCAAA
NO 267
302
SEQ ID
UUUCAAAUUUUGGGCAGCGGUAAUG
SEQ ID NO
AACUGGGGACGCCUCUGUUCCAAAU
NO 268
303
SEQ ID
UUCAAAUUUUGGGCAGCGGUAAUGA
SEQ ID NO
ACUGGGGACGCCUCUGUUCCAAAUC
NO 269
304
SEQ ID
UCAAAUUUUGGGCAGCGGUAAUGAG
SEQ ID NO
CUGGGGACGCCUCUGUUCCAAAUCC
NO 270
305
SEQ ID
CAAAUUUUGGGCAGCGGUAAUGAGU
SEQ ID NO
UGGGGACGCCUCUGUUCCAAAUCCU
NO 271
306
SEQ ID
AAAUUUUGGGCAGCGGUAAUGAGUU
SEQ ID NO
GGGGACGCCUCUGUUCCAAAUCCUG
NO 272
307
SEQ ID
AAUUUUGGGCAGCGGUAAUGAGUUC
SEQ ID NO
GGGACGCCUCUGUUCCAAAUCCUGC
NO 273
308
SEQ ID
AUUUUGGGCAGCGGUAAUGAGUUCU
SEQ ID NO
GGACGCCUCUGUUCCAAAUCCUGCA
NO 274
309
SEQ ID
UUUUGGGCAGCGGUAAUGAGUUCUU
SEQ ID NO
GACGCCUCUGUUCCAAAUCCUGCAU
NO 275
310
SEQ ID
UUUGGGCAGCGGUAAUGAGUUCUUC
NO 276
TABLE 6
oligonucleotides for skipping DMD Gene Exon 53
SEQ ID
CUCUGGCCUGUCCUAAGACCUGCUC
SEQ ID NO
CAGCUUCUUCCUUAGCUUCCAGCCA
NO 311
335
SEQ ID
UCUGGCCUGUCCUAAGACCUGCUCA
SEQ ID NO
AGCUUCUUCCUUAGCUUCCAGCCAU
NO 312
336
SEQ ID
CUGGCCUGUCCUAAGACCUGCUCAG
SEQ ID NO
GCUUCUUCCUUAGCUUCCAGCCAUU
NO 313
337
SEQ ID
UGGCCUGUCCUAAGACCUGCUCAGC
SEQ ID NO
CUUCUUCCUUAGCUUCCAGCCAUUG
NO 314
338
SEQ ID
GGCCUGUCCUAAGACCUGCUCAGCU
SEQ ID NO
UUCUUCCUUAGCUUCCAGCCAUUGU
NO 315
339
SEQ ID
GCCUGUCCUAAGACCUGCUCAGCUU
SEQ ID NO
UCUUCCUUAGCUUCCAGCCAUUGUG
NO 316
340
SEQ ID
CCUGUCCUAAGACCUGCUCAGCUUC
SEQ ID NO
CUUCCUUAGCUUCCAGCCAUUGUGU
NO 317
341
SEQ ID
CUGUCCUAAGACCUGCUCAGCUUCU
SEQ ID NO
UUCCUUAGCUUCCAGCCAUUGUGUU
NO 318
342
SEQ ID
UGUCCUAAGACCUGCUCAGCUUCUU
SEQ ID NO
UCCUUAGCUUCCAGCCAUUGUGUUG
NO 319
343
SEQ ID
GUCCUAAGACCUGCUCAGCUUCUUC
SEQ ID NO
CCUUAGCUUCCAGCCAUUGUGUUGA
NO 320
344
SEQ ID
UCCUAAGACCUGCUCAGCUUCUUCC
SEQ ID NO
CUUAGCUUCCAGCCAUUGUGUUGAA
NO 321
345
SEQ ID
CCUAAGACCUGCUCAGCUUCUUCCU
SEQ ID NO
UUAGCUUCCAGCCAUUGUGUUGAAU
NO 322
346
SEQ ID
CUAAGACCUGCUCAGCUUCUUCCUU
SEQ ID NO
UAGCUUCCAGCCAUUGUGUUGAAUC
NO 323
347
SEQ ID
UAAGACCUGCUCAGCUUCUUCCUUA
SEQ ID NO
AGCUUCCAGCCAUUGUGUUGAAUCC
NO 324
348
SEQ ID
AAGACCUGCUCAGCUUCUUCCUUAG
SEQ ID NO
GCUUCCAGCCAUUGUGUUGAAUCCU
NO 325
349
SEQ ID
AGACCUGCUCAGCUUCUUCCUUAGC
SEQ ID NO
CUUCCAGCCAUUGUGUUGAAUCCUU
NO 326
350
SEQ ID
GACCUGCUCAGCUUCUUCCUUAGCU
SEQ ID NO
UUCCAGCCAUUGUGUUGAAUCCUUU
NO 327
351
SEQ ID
ACCUGCUCAGCUUCUUCCUUAGCUU
SEQ ID NO
UCCAGCCAUUGUGUUGAAUCCUUUA
NO 328
352
SEQ ID
CCUGCUCAGCUUCUUCCUUAGCUUC
SEQ ID NO
CCAGCCAUUGUGUUGAAUCCUUUAA
NO 329
353
SEQ ID
CUGCUCAGCUUCUUCCUUAGCUUCC
SEQ ID NO
CAGCCAUUGUGUUGAAUCCUUUAAC
NO 330
354
SEQ ID
UGCUCAGCUUCUUCCUUAGCUUCCA
SEQ ID NO
AGCCAUUGUGUUGAAUCCUUUAACA
NO 331
355
SEQ ID
GCUCAGCUUCUUCCUUAGCUUCCAG
SEQ ID NO
GCCAUUGUGUUGAAUCCUUUAACAU
NO 332
356
SEQ ID
CUCAGCUUCUUCCUUAGCUUCCAGC
SEQ ID NO
CCAUUGUGUUGAAUCCUUUAACAUU
NO 333
357
SEQ ID
UCAGCUUCUUCCUUAGCUUCCAGCC
SEQ ID NO
CAUUGUGUUGAAUCCUUUAACAUUU
NO 334
358
TABLE 7
oligonucleotides for skipping other exons of the DMD gene as identified
DMD Gene Exon 6
SEQ ID
CAUUUUUGACCUACAUGUGG
SEQ ID NO
AUUUUUGACCUACAUGGGAAAG
NO 359
364
SEQ ID
UUUGACCUACAUGUGGAAAG
SEQ ID NO
UACGAGUUGAUUGUCGGACCCAG
NO 360
365
SEQ ID
UACAUUUUUGACCUACAUGUGGAA
SEQ ID NO
GUGGUCUCCUUACCUAUGACUGUGG
NO 361
A G
366
SEQ ID
GGUCUCCUUACCUAUGA
SEQ ID NO
UGUCUCAGUAAUCUUCUUACCUAU
NO 362
367
SEQ ID
UCUUACCUAUGACUAUGGAUGAGA
NO 363
DMD Gene Exon 7
SEQ ID
UGCAUGUUCCAGUCGUUGUGUGG
SEQ ID NO 370
AUUUACCAACCUUCAGGAUCGAGU
NO 368
A
SEQ ID
CACUAUUCCAGUCAAAUAGGUCUGG
SEQ ID NO 371
GGCCUAAAACACAUACACAUA
NO 369
DMD Gene Exon 11
SEQ ID
CCCUGAGGCAUUCCCAUCUUGAAU
SEQ ID
CUUGAAUUUAGGAGAUUCAUCU
NO 372
NO 374
G
SEQ ID
AGGACUUACUUGCUUUGUUU
SEQ ID
CAUCUUCUGAUAAUUUUCCUGUU
NO 373
NO 375
DMD Gene Exon 17
SEQ ID
CCAUUACAGUUGUCUGUGUU
SEQ ID
UAAUCUGCCUCUUCUUUUGG
NO 376
NO 378
SEQ ID
UGACAGCCUGUGAAAUCUGUGAG
NO 377
DMD Gene Exon 19
SEQ ID
CAGCAGUAGUUGUCAUCUGC
SEQ ID
GCCUGAGCUGAUCUGCUGGCAUC
NO 379
NO 381
UUGCAGUU
SEQ ID
GCCUGAGCUGAUCUGCUGGCAUCUUGC
SEQ ID
UCUGCUGGCAUCUUGC
NO 380
NO 382
DMD Gene Exon 21
SEQ ID
GCCGGUUGACUUCAUCCUGUGC
SEQ ID
CUGCAUCCAGGAACAUGGGUCC
NO 383
NO 386
SEQ ID
GUCUGCAUCCAGGAACAUGGGUC
SEQ ID
GUUGAAGAUCUGAUAGCCGGUUGA
NO 384
NO 387
SEQ ID
UACUUACUGUCUGUAGCUCUUUCU
NO 385
DMD Gene Exon 44
SEQ ID
UCAGCUUCUGUUAGCCACUG
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 388
NO 413
SEQ ID
UUCAGCUUCUGUUAGCCACU
SEQ ID
CAGCUUCUGUUAGCCACUGAUUAAA
NO 389
NO 414
SEQ ID
UUCAGCUUCUGUUAGCCACUG
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 390
NO 415
SEQ ID
UCAGCUUCUGUUAGCCACUGA
SEQ ID
AGCUUCUGUUAGCCACUGAU
NO 391
NO 416
SEQ ID
UUCAGCUUCUGUUAGCCACUGA
SEQ ID
GCUUCUGUUAGCCACUGAUU
NO 392
NO 417
SEQ ID
UCAGCUUCUGUUAGCCACUGA
SEQ ID
AGCUUCUGUUAGCCACUGAUU
NO 393
NO 418
SEQ ID
UUCAGCUUCUGUUAGCCACUGA
SEQ ID
GCUUCUGUUAGCCACUGAUUA
NO 394
NO 419
SEQ ID
UCAGCUUCUGUUAGCCACUGAU
SEQ ID
AGCUUCUGUUAGCCACUGAUUA
NO 395
NO 420
SEQ ID
UUCAGCUUCUGUUAGCCACUGAU
SEQ ID
GCUUCUGUUAGCCACUGAUUAA
NO 396
NO 421
SEQ ID
UCAGCUUCUGUUAGCCACUGAUU
SEQ ID
AGCUUCUGUUAGCCACUGAUUAA
NO 397
NO 422
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUU
SEQ ID
GCUUCUGUUAGCCACUGAUUAAA
NO 398
NO 423
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUA
SEQ ID
AGCUUCUGUUAGCCACUGAUUAAA
NO 399
NO 424
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUA
SEQ ID
GCUUCUGUUAGCCACUGAUUAAA
NO 400
NO 425
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID
CCAUUUGUAUUUAGCAUGUUCCC
NO 401
NO 426
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUUAA
SEQ ID
AGAUACCAUUUGUAUUUAGC
NO 402
NO 427
SEQ ID
UCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID
GCCAUUUCUCAACAGAUCU
NO 403
NO 428
SEQ ID
UUCAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID
GCCAUUUCUCAACAGAUCUGUCA
NO 404
NO 429
SEQ ID
CAGCUUCUGUUAGCCACUG
SEQ ID
AUUCUCAGGAAUUUGUGUCUUUC
NO 405
NO 430
SEQ ID
CAGCUUCUGUUAGCCACUGAU
SEQ ID
UCUCAGGAAUUUGUGUCUUUC
NO 406
NO 431
SEQ ID
AGCUUCUGUUAGCCACUGAUU
SEQ ID
GUUCAGCUUCUGUUAGCC
NO 407
NO 432
SEQ ID
CAGCUUCUGUUAGCCACUGAUU
SEQ ID
CUGAUUAAAUAUCUUUAUAUC
NO 408
NO 433
SEQ ID
AGCUUCUGUUAGCCACUGAUUA
SEQ ID
GCCGCCAUUUCUCAACAG
NO 409
NO 434
SEQ ID
CAGCUUCUGUUAGCCACUGAUUA
SEQ ID
GUAUUUAGCAUGUUCCCA
NO 410
NO 435
SEQ ID
AGCUUCUGUUAGCCACUGAUUAA
SEQ ID
CAGGAAUUUGUGUCUUUC
NO 411
NO 436
SEQ ID
CAGCUUCUGUUAGCCACUGAUUAA
NO 412
DMD Gene Exon 45
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID
GUUGCAUUCAAUGUUCUGACAACAG
NO 437
NO 470
SEQ ID
AUUCAAUGUUCUGACAACAGUUUGC
SEQ ID
UUGCAUUCAAUGUUCUGACAACAGU
NO 438
NO 471
SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID
UGCAUUCAAUGUUCUGACAACAGUU
NO 439
NO 472
SEQ ID
CAGUUGCAUUCAAUGUUCUGAC
SEQ ID
GCAUUCAAUGUUCUGACAACAGUUU
NO 440
NO 473
SEQ ID
AGUUGCAUUCAAUGUUCUGA
SEQ ID
CAUUCAAUGUUCUGACAACAGUUUG
NO 441
NO 474
SEQ ID
GAUUGCUGAAUUAUUUCUUCC
SEQ ID
AUUCAAUGUUCUGACAACAGUUUGC
NO 442
NO 475
SEQ ID
GAUUGCUGAAUUAUUUCUUCCCCAG
SEQ ID
UCAAUGUUCUGACAACAGUUUGCCG
NO 443
NO 476
SEQ ID
AUUGCUGAAUUAUUUCUUCCCCAGU
SEQ ID
CAAUGUUCUGACAACAGUUUGCCGC
NO 444
NO 477
SEQ ID
UUGCUGAAUUAUUUCUUCCCCAGUU
SEQ ID
AAUGUUCUGACAACAGUUUGCCGCU
NO 445
NO 478
SEQ ID
UGCUGAAUUAUUUCUUCCCCAGUUG
SEQ ID
AUGUUCUGACAACAGUUUGCCGCUG
NO 446
NO 479
SEQ ID
GCUGAAUUAUUUCUUCCCCAGUUGC
SEQ ID
UGUUCUGACAACAGUUUGCCGCUGC
NO 447
NO 480
SEQ ID
CUGAAUUAUUUCUUCCCCAGUUGCA
SEQ ID
GUUCUGACAACAGUUUGCCGCUGCC
NO 448
NO 481
SEQ ID
UGAAUUAUUUCUUCCCCAGUUGCAU
SEQ ID
UUCUGACAACAGUUUGCCGCUGCCC
NO 449
NO 482
SEQ ID
GAAUUAUUUCUUCCCCAGUUGCAUU
SEQ ID
UCUGACAACAGUUUGCCGCUGCCCA
NO 450
NO 483
SEQ ID
AAUUAUUUCUUCCCCAGUUGCAUUC
SEQ ID
CUGACAACAGUUUGCCGCUGCCCAA
N0451
NO 484
SEQ ID
AUUAUUUCUUCCCCAGUUGCAUUCA
SEQ ID
UGACAACAGUUUGCCGCUGCCCAAU
NO 452
NO 485
SEQ ID
UUAUUUCUUCCCCAGUUGCAUUCAA
SEQ ID
GACAACAGUUUGCCGCUGCCCAAUG
NO 453
NO 486
SEQ ID
UAUUUCUUCCCCAGUUGCAUUCAAU
SEQ ID
ACAACAGUUUGCCGCUGCCCAAUGC
NO 454
NO 487
SEQ ID
AUUUCUUCCCCAGUUGCAUUCAAUG
SEQ ID
CAACAGUUUGCCGCUGCCCAAUGCC
NO 455
NO 488
SEQ ID
UUUCUUCCCCAGUUGCAUUCAAUGU
SEQ ID
AACAGUUUGCCGCUGCCCAAUGCCA
NO 456
NO 489
SEQ ID
UUCUUCCCCAGUUGCAUUCAAUGUU
SEQ ID
ACAGUUUGCCGCUGCCCAAUGCCAU
NO 457
NO 490
SEQ ID
UCUUCCCCAGUUGCAUUCAAUGUUC
SEQ ID
CAGUUUGCCGCUGCCCAAUGCCAUC
NO 458
NO 491
SEQ ID
CUUCCCCAGUUGCAUUCAAUGUUCU
SEQ ID
AGUUUGCCGCUGCCCAAUGCCAUCC
NO 459
NO 492
SEQ ID
UUCCCCAGUUGCAUUCAAUGUUCUG
SEQ ID
GUUUGCCGCUGCCCAAUGCCAUCCU
NO 460
NO 493
SEQ ID
UCCCCAGUUGCAUUCAAUGUUCUGA
SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG
NO 461
NO 494
SEQ ID
CCCCAGUUGCAUUCAAUGUUCUGAC
SEQ ID
UUGCCGCUGCCCAAUGCCAUCCUGG
NO 462
NO 495
SEQ ID
CCCAGUUGCAUUCAAUGUUCUGACA
SEQ ID
UGCCGCUGCCCAAUGCCAUCCUGGA
NO 463
NO 496
SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA
SEQ ID
GCCGCUGCCCAAUGCCAUCCUGGAG
NO 464
NO 497
SEQ ID
CAGUUGCAUUCAAUGUUCUGACAAC
SEQ ID
CCGCUGCCCAAUGCCAUCCUGGAGU
NO 465
NO 498
SEQ ID
AGUUGCAUUCAAUGUUCUGACAACA
SEQ ID
CGCUGCCCAAUGCCAUCCUGGAGUU
NO 466
NO 499
SEQ ID
UCC UGU AGA AUA CUG GCA UC
SEQ ID
UGUUUUUGAGGAUUGCUGAA
NO 467
NO 500
SEQ ID
UGCAGACCUCCUGCCACCGCAGAUUCA
SEQ ID
UGUUCUGACAACAGUUUGCCGCU
NO 468
NO 501
GCCCAAUGCCAUCCUGG
SEQ ID
UUGCAGACCUCCUGCCACCGCAGAUUC
NO 469
AGGCUUC
DMD Gene Exon 55
SEQ ID
CUGUUGCAGUAAUCUAUGAG
SEQ ID
UGCCAUUGUUUCAUCAGCUCUUU
NO 502
NO 505
SEQ ID
UGCAGUAAUCUAUGAGUUUC
SEQ ID
UCCUGUAGGACAUUGGCAGU
NO 503
NO 506
SEQ ID
GAGUCUUCUAGGAGCCUU
SEQ ID
CUUGGAGUCUUCUAGGAGCC
NO 504
NO 507
DMD Gene Exon 57
SEQ ID
UAGGUGCCUGCCGGCUU
SEQ ID
CUGAACUGCUGGAAAGUCGCC
NO 508
NO 510
SEQ ID
UUCAGCUGUAGCCACACC
SEQ ID
CUGGCUUCCAAAUGGGACCUGAA
NO 509
NO 511
AAAGAAC
DMD Gene Exon 59
SEQ ID
CAAUUUUUCCCACUCAGUAUU
SEQ ID
UCCUCAGGAGGCAGCUCUAAAU
NO 512
NO 514
SEQ ID
UUGAAGUUCCUGGAGUCUU
NO 513
DMD Gene Exon 62
SEQ ID
UGGCUCUCUCCCAGGG
SEQ ID
GGGCACUUUGUUUGGCG
NO 515
NO 517
SEQ ID
GAGAUGGCUCUCUCCCAGGGACCCUGG
NO 516
DMD Gene Exon 63
SEQ ID
GGUCCCAGCAAGUUGUUUG
SEQ ID
GUAGAGCUCUGUCAUUUUGGG
NO 518
NO 520
SEQ ID
UGGGAUGGUCCCAGCAAGUUGUUUG
NO 519
DMD Gene Exon 65
SEQ ID
GCUCAAGAGAUCCACUGCAAAAAAC
SEQ ID
UCUGCAGGAUAUCCAUGGGCUGGUC
NO 521
NO 523
SEQ ID
GCCAUACGUACGUAUCAUAAACAUUC
NO 522
DMD Gene Exon 66
SEQ ID
GAUCCUCCCUGUUCGUCCCCUAUUAUG
NO 524
DMD Gene Exon 69
SEQ ID
UGCUUUAGACUCCUGUACCUGAUA
NO 525
DMD Gene Exon 75
SEQ ID
GGCGGCCUUUGUGUUGAC
SEQ ID
CCUUUAUGUUCGUGCUGCU
NO 526
NO 528
SEQ ID
GGACAGGCCUUUAUGUUCGUGCUGC
NO 527
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16024558
|
biomarin technologies b.v.
|
USA
|
B2
|
Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
|
Open
|
|
|
Apr 1st, 2022 05:10PM
|
Apr 1st, 2022 05:10PM
|
BioMarin Pharmaceutical
|
Health Care
|
Pharmaceuticals & Biotechnology
|