Compositions and methods for restoring and maintaining dystrophin-associated protein complex (DAPC)

Abstract

To provide methods for repairing or restoring a sarcoglycan complex or DAPC, for stabilizing DAPC, restoring DAPC function, or increasing or enhancing expression of one or more components of sarcoglycan complex or DAPC in a subject suffering from a muscular dystrophy.SOLUTION: In some embodiments, a method for restoring or stabilizing a dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprises, or consists essentially of, or yet further consists of administering to the subject a polynucleotide sequence encoding (a) sarcoglycan and / or (b) the dystrophin or an abbreviated version thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 948,586, filed December 16, 2019, and U.S. Provisional Application No. 63 / 023,144, filed May 11, 2020, the contents of both of which are incorporated herein by reference in their entireties. Technical Field The present application provides methods for enhancing expression of one or more components of the sarcoglycan complex and / or dystrophin-associated protein complex (DAPC), restoring or stabilizing DAPC, restoring DAPC function, and localizing components of the sarcoglycan complex and / or DAPC complex to the cell membrane by administering a sarcoglycan, sarcospan, and / or dystrophin transgene to a subject in need thereof. [Background technology]

[0002] background Limb-girdle muscular dystrophy type 2 (LGMD2) is caused by recessive mutations in various muscle-specific genes involved in muscle cell structure and function. Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) is caused by mutations in the dystrophin (DMD) gene. A subset of LGMD type 2 (sarcoglycanopathies) involves mutations in sarcoglycan proteins (α-, β-, γ-, and δ-). These mutations ultimately lead to protein deficiency, loss of function in proteins involved in muscle homeostasis and membrane repair, and loss of stabilization of the dystrophin-associated protein complex (DAPC).

[0003] There is an urgent need for therapies that can stabilize DAPC in type 2 LGMD patients. Summary of the Invention [Means for solving the problem]

[0004] Abstract Without wishing to be bound by theory, proteins called sarcoglycans and sarcospans, along with dystrophin, are integral proteins important for stabilizing the DAPC and providing mechanical support to the sarcolemma.

[0005] Disclosed herein are methods for enhancing expression of one or more components of the sarcoglycan complex and / or dystrophin-associated protein complex (DAPC), restoring or stabilizing DAPC, restoring DAPC function, and localizing components of the sarcoglycan complex and / or DAPC complex to the cell membrane by administering a sarcoglycan, sarcospan, and / or dystrophin transgene or a truncated version thereof to a subject in need thereof.

[0006] In some embodiments, a method for restoring or stabilizing dystrophin-associated protein complexes (DAPCs) in a subject suffering from muscular dystrophy comprises, consists essentially of, or further consists of administering to the subject a polynucleotide sequence encoding (a) a sarcoglycan and / or (b) dystrophin or a truncated version thereof. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element, non-limiting examples of which are provided herein, e.g., in the Sequence Listing of the present disclosure.

[0007] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding dystrophin or a fragment of dystrophin. In some embodiments, the truncated version of dystrophin is micro-dystrophin or mini-dystrophin. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element, non-limiting examples of which are provided herein, for example, in the sequence listing of the present disclosure.

[0008] In some embodiments, the muscular dystrophy is LGMD2C. In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCG.

[0009] In some embodiments, the muscular dystrophy is LGMD2D. In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCA.

[0010] In some embodiments, the muscular dystrophy is LGMD2E. In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCB.

[0011] In some embodiments, the muscular dystrophy is LGMD2F. In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCD.

[0012] In some embodiments, a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy comprises, consists essentially of, or further comprises administering to the subject (a) a second sarcoglycan; and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof, wherein the first sarcoglycan is different from the second sarcoglycan. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element, non-limiting examples of which are provided herein. In one aspect, the polynucleotide further comprises an MHCK7 promoter polynucleotide and an alpha heavy chain enhancer.

[0013] In some embodiments, a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin on the muscle cell membrane or muscle cell membrane of a subject suffering from muscular dystrophy comprises, consists essentially of, or further comprises administering to the subject a second sarcoglycan; and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof, wherein the first sarcoglycan is different from the second sarcoglycan. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element, non-limiting examples of which are provided herein. In one aspect, the polynucleotide further comprises an MHCK7 promoter polynucleotide and an alpha heavy chain enhancer.

[0014] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD. In some embodiments, the method comprises, consists essentially of, or further consists of administering to a subject a polynucleotide encoding dystrophin or a truncated version of dystrophin. In some embodiments, the polynucleotide encoding dystrophin comprises, consists essentially of, or further consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the truncated version of dystrophin is micro-dystrophin or mini-dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or alternatively consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element; non-limiting examples of such are provided herein. In one aspect, the polynucleotide further comprises an MHCK7 promoter polynucleotide and an alpha heavy chain enhancer.

[0015] In some embodiments, the muscular dystrophy is LGMD2C. In some embodiments, the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCG. In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs:20-24 over the entire length of SEQ ID NOs:20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs:25-29 over the entire length of SEQ ID NOs:25-29. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element; non-limiting examples of such are provided herein. In one aspect, the polynucleotide further comprises an MHCK7 promoter polynucleotide and an alpha heavy chain enhancer.

[0016] In some embodiments, the muscular dystrophy is LGMD2D. In some embodiments, the method comprises, or consists essentially of, or further consists of administering to the subject a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCA. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In one aspect, the polynucleotide further comprises a promoter and / or enhancer element. In one aspect, the polynucleotide further comprises an MHCK7 promoter polynucleotide and an alpha heavy chain enhancer.

[0017] In some embodiments, the muscular dystrophy is LGMD2E. In some embodiments, the method comprises, or consists essentially of, or further consists of administering to the subject a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCB. In some embodiments, the polynucleotide encoding SGCB comprises, or consists essentially of, or further consists of, a nucleotide sequence encoding an SGCB protein that comprises, essentially consists of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In one embodiment, the polynucleotide further comprises promoter and / or enhancer elements, non-limiting examples of such are provided herein, for example, in the sequence listing of the present disclosure.

[0018] In some embodiments, the muscular dystrophy is LGMD2F. In some embodiments, the method comprises, or consists essentially of, or also consists of administering to the subject a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCD. In some embodiments, a polynucleotide encoding an SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In one aspect, the polynucleotide further comprises promoter and / or enhancer elements, non-limiting examples of which are provided herein, e.g., in the Sequence Listing of the present disclosure.

[0019] In some embodiments, the method increases or enhances the expression of the first sarcoglycan in muscle cell membranes or sarcolemma, or increases the localization of the first sarcoglycan to muscle cell membranes or sarcolemma. In some embodiments, the first sarcoglycan is SGCD. In some embodiments, the first sarcoglycan is SGCB. In some embodiments, the first sarcoglycan is SGCA. In some embodiments, the first sarcoglycan is SGCG. In some embodiments, the expression of the first sarcoglycan at or localization to the muscle cell membrane or sarcolemma is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression or localization of the first sarcoglycan before administering one or more doses of the polynucleotide. In some embodiments, the method further comprises, consists essentially of, or further consists of detecting expression of the first sarcoglycan. In some embodiments, detecting expression of the first sarcoglycan comprises, consists essentially of, or further consists of detecting protein levels of the first sarcoglycan. In some embodiments, detecting the expression of the first sarcoglycan comprises, consists essentially of, or further consists of detecting the RNA level of the first sarcoglycan. Any method known in the art can be used to detect the protein and / or RNA level of the first sarcoglycan. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. In some embodiments, detecting the expression of the first sarcoglycan comprises, consists essentially of, or further consists of performing one or more histological evaluations. Exemplary histological evaluations include, but are not limited to, hematoxylin and eosin staining.In some embodiments, histological evaluation involves hematoxylin and eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0020] In some embodiments, the method increases or enhances dystrophin expression at the muscle cell membrane or sarcolemma. In some embodiments, dystrophin expression or dystrophin localization at the muscle cell membrane or sarcolemma is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to dystrophin expression or localization before administering one or more doses of the polynucleotide. In some embodiments, the method further comprises detecting dystrophin expression. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or even further consists of detecting dystrophin protein levels. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or further consists of detecting dystrophin RNA levels. Any method known in the art can be used to detect dystrophin protein and / or RNA levels. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. In some embodiments, the dystrophin is truncated dystrophin. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or further consists of performing one or more histological evaluations. Exemplary histological evaluations include, but are not limited to, hematoxylin and eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0021] In some embodiments, the method increases or enhances the expression of sarcospan at or localizes to the muscle cell membrane. In some embodiments, the expression of sarcospan or the localization of sarcospan at or to the muscle cell membrane is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression or localization of sarcospan before administering one or more doses of the polynucleotide. In some embodiments, the method further comprises detecting sarcospan expression. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or even further consists of detecting sarcospan protein levels. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or further consists of detecting sarcospan RNA levels. Sarcospan protein and / or RNA levels can be detected using any method known in the art. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or further consists of performing one or more histological evaluations. Exemplary histological evaluations include, but are not limited to, hematoxylin and eosin staining. In some embodiments, histological evaluations include hematoxylin and eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0022] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0023] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0024] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising, consisting essentially of, or alternatively consisting of a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein, wherein the first sarcoglycan is selected from α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), and δ-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0025] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0026] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0027] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, administering to the subject a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein, wherein the first sarcoglycan is selected from alpha-sarcoglycan (SGCA), beta-sarcoglycan (SGCB), and delta-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0028] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0029] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0030] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, administering to the subject a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein, wherein the first sarcoglycan is selected from γ-sarcoglycan (SGCG), β-sarcoglycan (SGCB), and δ-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0031] In some embodiments, polynucleotide is encapsulated in nanoparticles, liposomes, or is packaged in viral vectors (i.e., viral vector particles) by encapsidation.Alternatively, or in addition, polynucleotide is contained in a vector, for example, a plasmid or viral vector.In some embodiments, viral vector is a retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, poxvirus, picornavirus, or herpes simplex virus vector.In some embodiments, viral vector is a recombinant viral vector.In some embodiments, viral vector is a recombinant AAV vector.In some embodiments, viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. In some embodiments, the viral vector is AAVrh.74. In some embodiments, the viral vector is a self-complementary vector, e.g., self-complementary AAVrh.74. In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0032] In some embodiments, the polynucleotide is administered systemically.

[0033] In some embodiments, the polynucleotide is administered locally.

[0034] In some embodiments, the polynucleotide is administered intravenously or intramuscularly.

[0035] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is selected from an MHCK7 promoter and a tMCK promoter. In some embodiments, the promoter comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:4 or 6 over the entire length of SEQ ID NO:4 or 6.

[0036] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of an intron. In some embodiments, the intron is an SV40 chimeric intron. In some embodiments, the intron comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9.

[0037] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of a polyA sequence. In some embodiments, the polyA sequence comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10.

[0038] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of an inverted terminal repeat (ITR). In some embodiments, the ITR comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or 12 over the entire length of SEQ ID NO: 11 or 12.

[0039] In some embodiments, a composition for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or even consisting of, a polynucleotide sequence encoding (a) a sarcoglycan; and / or (b) dystrophin or a truncated version thereof.

[0040] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding dystrophin or a truncated version thereof. In some embodiments, the truncated version of dystrophin is microdystrophin or minidystrophin. In some embodiments, the polynucleotide encoding dystrophin comprises, consists essentially of, or further consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or alternatively consists of, an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39.

[0041] In some embodiments, the muscular dystrophy is LGMD2C. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCG. In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29.

[0042] In some embodiments, the muscular dystrophy is LGMD2D. In some embodiments, the composition comprises, consists essentially of, or even consists of a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCA. In some embodiments, the polynucleotide encoding SGCA comprises, essentially consists of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, essentially consists of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46.

[0043] In some embodiments, the muscular dystrophy is LGMD2E. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCB. In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCB protein comprising, consisting essentially of, or further consisting of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18.

[0044] In some embodiments, the muscular dystrophy is LGMD2F. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a sarcoglycan, wherein the sarcoglycan is SGCD. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35.

[0045] In some embodiments, provided herein are compositions for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, the composition comprising, consisting essentially of, or even consisting of a polynucleotide sequence encoding (a) a second sarcoglycan; or (b) dystrophin or a truncated version thereof.

[0046] In some embodiments, a composition for enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin in a subject suffering from muscular dystrophy, the composition comprising, consisting essentially of, or even consisting of a polynucleotide sequence encoding (a) a second sarcoglycan; or (b) dystrophin or a truncated version thereof.

[0047] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD.In some embodiments, the composition comprises, or essentially consists of, or further consists of the polynucleotide encoding dystrophin or its truncated version.In some embodiments, the truncated dystrophin is micro-dystrophin or mini-dystrophin.

[0048] In some embodiments, the muscular dystrophy is LGMD2C. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCG. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCD.

[0049] In some embodiments, the muscular dystrophy is LGMD2D. In some embodiments, the composition comprises, or essentially consists of, or also consists of a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCA. In some embodiments, the first sarcoglycan is selected from SGCD, SGCB, and SGCG.

[0050] In some embodiments, the muscular dystrophy is LGMD2E. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCB. In some embodiments, the first sarcoglycan is selected from SGCA, SGCD, and SGCG.

[0051] In some embodiments, the muscular dystrophy is LGMD2F. In some embodiments, the composition comprises, consists essentially of, or further consists of a polynucleotide encoding a second sarcoglycan, wherein the second sarcoglycan is SGCD. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCG.

[0052] In some embodiments, the composition increases or enhances the expression of the first sarcoglycan in muscle cell membranes or sarcolemma, or increases the localization of the first sarcoglycan to muscle cell membranes or sarcolemma. In some embodiments, the first sarcoglycan is SGCD. In some embodiments, the first sarcoglycan is SGCB. In some embodiments, the first sarcoglycan is SGCA. In some embodiments, the first sarcoglycan is SGCG. In some embodiments, the expression or localization of the first sarcoglycan at the muscle cell membrane or sarcolemma is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression or localization of the first sarcoglycan before administering one or more doses of the polynucleotide. In some embodiments, the composition further comprises, consists essentially of, or alternatively consists of one or more reagents for detecting expression of the first sarcoglycan. In some embodiments, detecting expression of the first sarcoglycan comprises, consists essentially of, or alternatively consists of detecting protein levels of the first sarcoglycan. In some embodiments, detecting the expression of the first sarcoglycan comprises, consists essentially of, or further consists of detecting RNA levels of the first sarcoglycan. The protein and / or RNA levels of the first sarcoglycan can be detected using any method known in the art. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. Thus, the composition may further comprise an antibody or nucleic acid for detecting the first sarcoglycan. In some embodiments, detecting the expression of the first sarcoglycan comprises, consists essentially of, or further consists of performing one or more histological evaluations.Exemplary histological assessments include, but are not limited to, hematoxylin and eosin staining. In some embodiments, histological assessments include hematoxylin and eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0053] In some embodiments, the composition increases or enhances the expression of dystrophin in muscle cell membranes or sarcolemma, or increases the localization of dystrophin to muscle cell membranes or sarcolemma, after administering to a subject (a) a second sarcoglycan; and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof. In some embodiments, the expression of the first sarcoglycan or the localization of dystrophin in muscle cell membranes or sarcolemma increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression or localization of dystrophin before administering one or more doses of the polynucleotide. In some embodiments, the composition further comprises one or more reagents for detecting dystrophin expression. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or further consists of detecting dystrophin protein levels. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or further consists of detecting dystrophin RNA levels. Any method known in the art can be used to detect dystrophin protein and / or RNA levels. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. Thus, the composition may further comprise an antibody or nucleic acid for detecting dystrophin. In some embodiments, the dystrophin is truncated dystrophin. In some embodiments, detecting dystrophin expression comprises, consists essentially of, or further consists of performing one or more histological evaluations. Exemplary histological evaluations include, but are not limited to, hematoxylin and eosin staining. In some embodiments, histological evaluation involves hematoxylin and eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0054] In some embodiments, the composition increases or enhances the expression of sarcospan at the muscle cell membrane or sarcolemma, or increases the localization of sarcospan to the muscle cell membrane or sarcolemma. In some embodiments, the expression or localization of sarcospan at the muscle cell membrane or sarcolemma is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression or localization of sarcospan before administering one or more doses of the polynucleotide. In some embodiments, the composition further comprises one or more reagents for detecting sarcospan expression. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or even further consists of detecting sarcospan protein levels. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or further consists of detecting sarcospan RNA levels. Sarcospan protein and / or RNA levels can be detected using any method known in the art. Such methods include, but are not limited to, Western blot, PCR, immunofluorescence, and ELISA. Thus, the composition may further comprise an antibody or nucleic acid for detecting sarcospan. In some embodiments, detecting sarcospan expression comprises, consists essentially of, or further consists of performing one or more histological evaluations. Exemplary histological evaluations include, but are not limited to, hematoxylin-eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0055] In some embodiments, polynucleotide is encapsulated in nanoparticles, liposomes, or is packaged in viral vectors (i.e., viral vector particles) by encapsidation.Alternatively, or in addition, polynucleotide is contained in a vector, for example, a plasmid or viral vector.In some embodiments, viral vector is a retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, poxvirus, picornavirus, or herpes simplex virus vector.In some embodiments, viral vector is a recombinant viral vector.In some embodiments, viral vector is a recombinant AAV vector.In some embodiments, viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. In some embodiments, the viral vector is AAVrh.74. In some embodiments, the viral vector is a self-complementary vector, e.g., self-complementary AAVrh.74.

[0056] In some embodiments, the polynucleotide is administered systemically.

[0057] In some embodiments, the polynucleotide is administered locally.

[0058] In some embodiments, the polynucleotide is administered intravenously or intramuscularly.

[0059] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is selected from an MHCK7 promoter and a tMCK promoter. In some embodiments, the promoter comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:4 or 6 over the entire length of SEQ ID NO:4 or 6.

[0060] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of an intron. In some embodiments, the intron is an SV40 chimeric intron. In some embodiments, the intron comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9.

[0061] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of a polyA sequence. In some embodiments, the polyA sequence comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10.

[0062] In some embodiments, the polynucleotide further comprises, consists essentially of, or even consists of an inverted terminal repeat (ITR). In some embodiments, the ITR comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or 12 over the entire length of SEQ ID NO: 11 or 12. In certain embodiments, for example, the following items are provided: (Item 1) A method for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising administering to the subject (a) a sarcoglycan and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof. (Item 2) Item 10. The method of claim 1, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). (Item 3) 3. The method of claim 2, comprising administering to the subject a polynucleotide encoding dystrophin or a truncated version of dystrophin. (Item 4) 4. The method of claim 2 or 3, wherein the truncated version of dystrophin is microdystrophin or minidystrophin. (Item 5) Item 2. The method of item 1, wherein the muscular dystrophy is LGMD2C. (Item 6) 6. The method of claim 5, comprising administering to the subject a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCG. (Item 7) Item 1. The method of item 1, wherein the muscular dystrophy is LGMD2D. (Item 8) 8. The method of claim 7, comprising administering to the subject a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCA. (Item 9) Item 1. The method of item 1, wherein the muscular dystrophy is LGMD2E. (Item 10) 10. The method of claim 9, comprising administering to the subject a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCB. (Item 11) Item 2. The method of item 1, wherein the muscular dystrophy is LGMD2F. (Item 12) administering to the subject a polynucleotide encoding the sarcoglycan. Item 12. The method according to Item 11, wherein the sarcoglycan is SGCD. (Item 13) A method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject (a) a second sarcoglycan; and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof, wherein the first sarcoglycan is different from the second sarcoglycan. (Item 14) A method for increasing or enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin in the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a polynucleotide sequence encoding (a) a second sarcoglycan; and / or (b) dystrophin or a truncated version thereof, wherein the first sarcoglycan is different from the second sarcoglycan. (Item 15) 15. The method of item 13 or 14, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD. (Item 16) 16. The method of claim 15, comprising administering to the subject a polynucleotide encoding dystrophin or a truncated version of dystrophin. (Item 17) 17. The method of item 3 or 16, wherein the polynucleotide encoding dystrophin comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. (Item 18) 17. The method of item 15 or 16, wherein the truncated version of dystrophin is micro-dystrophin or mini-dystrophin. (Item 19) 19. The method of item 4 or 18, wherein the polynucleotide encoding the truncated version of dystrophin comprises: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) a nucleotide sequence encoding a truncated version of dystrophin protein comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. (Item 20) 15. The method of claim 13 or 14, wherein the muscular dystrophy is LGMD2C. (Item 21) 19. The method of claim 18, comprising administering to the subject a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCG. (Item 22) 19. The method of item 6 or 18, wherein the polynucleotide encoding SGCG comprises a nucleotide sequence encoding an SGCG protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20 to 24 over the entire length of SEQ ID NOs: 20 to 24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25 to 29 over the entire length of SEQ ID NOs: 25 to 29. (Item 23) 15. The method of claim 13 or 14, wherein the muscular dystrophy is LGMD2D. (Item 24) 21. The method of claim 20, comprising administering to the subject a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCA. (Item 25) 25. The method of claim 8 or 24, wherein the polynucleotide encoding SGCA comprises a nucleotide sequence encoding an SGCA protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. (Item 26) 15. The method of claim 13 or 14, wherein the muscular dystrophy is LGMD2E. (Item 27) 23. The method of claim 22, comprising administering to the subject a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCB. (Item 28) 28. The method of item 10 or 27, wherein the polynucleotide encoding SGCB comprises a nucleotide sequence encoding an SGCB protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. (Item 29) 15. The method of claim 13 or 14, wherein the muscular dystrophy is LGMD2F. (Item 30) 25. The method of claim 24, comprising administering to the subject a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCD. (Item 31) 31. The method of claim 12 or 30, wherein the polynucleotide encoding an SGCD comprises a nucleotide sequence encoding an SGCD protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30 to 32 over the entire length of SEQ ID NOs: 30 to 32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33 to 35 over the entire length of SEQ ID NOs: 33 to 35. (Item 32) 28. The method of any one of items 13 to 27, wherein the expression of the first sarcoglycan is increased or enhanced, and the first sarcoglycan is SGCD. (Item 33) 31. The method of any one of items 13 to 24 and 29 to 30, wherein the first sarcoglycan is SGCB. (Item 34) The method of any one of items 13 to 21 and 26 to 30, wherein the first sarcoglycan is SGCA. (Item 35) 31. The method of any one of items 13 to 18 and 23 to 30, wherein the first sarcoglycan is SGCG. (Item 36) 31. The method of any one of items 13 to 14 and 20 to 30, wherein expression of dystrophin at the muscle cell membrane or sarcolemma is increased or enhanced after administering to the subject the polynucleotide sequence encoding the second sarcoglycan. (Item 37) 37. The method of any one of items 13 to 36, wherein the expression of sarcospan to the muscle cell membrane or sarcolemma is increased or enhanced. (Item 38) 38. The method of any one of items 13 to 37, wherein the expression of the first sarcoglycan, sarcospan, or dystrophin at the muscle cell membrane or sarcolemma is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% compared to the expression of the first sarcoglycan, sarcospan, or dystrophin before administering one or more doses of the polynucleotide. (Item 39) 39. The method of any one of items 1 to 38, wherein the polynucleotide is encapsulated in a nanoparticle, a liposome, or a viral vector. (Item 40) 40. The method of claim 39, wherein the viral vector is a retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, poxvirus, picornavirus, or herpes simplex virus vector. (Item 41) 41. The method of any one of items 39 to 40, wherein the viral vector is a recombinant AAV vector. (Item 42) 42. The method of any one of items 39 to 41, wherein the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. (Item 43) 43. The method of claim 42, wherein the viral vector is AAVrh.74. (Item 44) 44. The method of any one of items 39 to 43, wherein the viral vector is administered to the subject at a dosage of 1.85e13vg / kg or 7.41e13vg / kg, and the dosage is quantified by linearized PCR standard. (Item 45) 44. The method of any one of paragraphs 39 to 43, wherein the viral vector comprises a viral genome comprising a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48. (Item 46) 46. ​​The method of any one of items 1 to 45, wherein the polynucleotide is administered systemically. (Item 47) 46. ​​The method of any one of items 1 to 45, wherein the polynucleotide is administered locally. (Item 48) 48. The method of any one of items 1 to 47, wherein the polynucleotide is administered intravenously or intramuscularly. (Item 49) A composition for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising: (a) a sarcoglycan; and / or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof. (Item 50) 50. The composition of claim 49, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD. (Item 51) 51. The composition of item 50, comprising a polynucleotide encoding dystrophin or a truncated version thereof. (Item 52) 52. The composition of any one of items 49 to 51, wherein the truncated version of dystrophin is micro-dystrophin or mini-dystrophin. (Item 53) Item 49. The composition of item 49, wherein the muscular dystrophy is LGMD2C. (Item 54) 54. The composition of claim 53, comprising a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCG. (Item 55) Item 49. The composition of item 49, wherein the muscular dystrophy is LGMD2D. (Item 56) 56. The composition of claim 55, comprising a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCA. (Item 57) Item 49. The composition of item 49, wherein the muscular dystrophy is LGMD2E. (Item 58) 58. The composition according to Item 57, comprising a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCB. (Item 59) Item 49. The composition of item 49, wherein the muscular dystrophy is LGMD2F. (Item 60) 60. The composition of item 59, comprising a polynucleotide encoding the sarcoglycan, wherein the sarcoglycan is SGCD. (Item 61) A composition for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, the composition comprising: (a) a second sarcoglycan; or (b) a polynucleotide sequence encoding dystrophin or a truncated version thereof. (Item 62) A composition for enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin in a subject suffering from muscular dystrophy, the composition comprising a polynucleotide sequence encoding (a) a second sarcoglycan; or (b) dystrophin or a truncated version thereof. (Item 63) 63. The composition of item 61 or 62, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD) or BMD. (Item 64) 64. The composition of item 63, comprising a polynucleotide encoding dystrophin or a truncated version thereof. (Item 65) 65. The composition of item 51 or 64, wherein the polynucleotide encoding dystrophin comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. (Item 66) 65. The composition of any one of items 62 to 64, wherein the truncated version of dystrophin is microdystrophin or minidystrophin. (Item 67) 67. The composition of Item 52 or 66, wherein the polynucleotide encoding the truncated version of dystrophin comprises: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. (Item 68) The composition according to Item 61 or 62, wherein the muscular dystrophy is LGMD2C. (Item 69) 69. The composition of claim 68, comprising a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCG. (Item 70) 70. The method of claim 54 or 69, wherein the polynucleotide encoding SGCG comprises a nucleotide sequence encoding an SGCG protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20 to 24 over the entire length of SEQ ID NOs: 20 to 24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25 to 29 over the entire length of SEQ ID NOs: 25 to 29. (Item 71) The composition according to Item 61 or 62, wherein the muscular dystrophy is LGMD2D. (Item 72) 72. The composition of claim 71, comprising a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCA. (Item 73) 73. The composition of claim 56 or 72, wherein the polynucleotide encoding SGCA comprises a nucleotide sequence encoding an SGCA protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. (Item 74) Item 75. The composition according to Item 61 or 62, wherein the muscular dystrophy is LGMD2E. 75. The composition of claim 74, comprising a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCB. (Item 76) 76. The composition of claim 58 or 75, wherein the polynucleotide encoding SGCB comprises a nucleotide sequence encoding an SGCB protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. (Item 77) Item 78. The composition according to Item 61 or 62, wherein the muscular dystrophy is LGMD2F. 78. The composition of claim 77, comprising a polynucleotide encoding the second sarcoglycan, wherein the second sarcoglycan is SGCD. (Item 79) 79. The composition of item 60 or 78, wherein the polynucleotide encoding SGCD comprises a nucleotide sequence encoding an SGCD protein comprising: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30 to 32 over the entire length of SEQ ID NOs: 30 to 32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33 to 35 over the entire length of SEQ ID NOs: 33 to 35. (Item 80) 77. The composition of any one of items 61 to 76, wherein the first sarcoglycan is SGCD. (Item 81) 80. The composition of any one of items 61 to 73 and 77 to 79, wherein the first sarcoglycan is SGCB. (Item 82) 80. The composition of any one of items 61 to 70 and 74 to 79, wherein the first sarcoglycan is SGCA. (Item 83) 80. The composition of any one of items 61 to 67 and 71 to 79, wherein the first sarcoglycan is SGCG. (Item 84) 80. The composition of any one of items 61 to 62 and 68 to 79, which increases or enhances expression of dystrophin to the muscle cell membrane or sarcolemma. (Item 85) 85. The composition of any one of items 61 to 84, which increases or enhances the expression of sarcospan. (Item 86) the expression of the first sarcoglycan, sarcospan, or dystrophin is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1010%, 86. The composition of any one of items 61 to 85, wherein the increase in serum cholesterol levels is 20%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. (Item 87) 87. The composition of any one of items 49 to 86, wherein the polynucleotide is encapsulated in a nanoparticle, liposome, or viral vector. (Item 88) 88. The composition of item 87, wherein the viral vector is a retrovirus, adenovirus, adeno-associated virus (AAV), lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, poxvirus, picornavirus, or herpes simplex virus vector. (Item 89) Item 90. The composition of item 88, wherein the viral vector is a recombinant viral vector. 90. The composition of any one of items 87 to 89, wherein the viral vector is a recombinant AAV vector. (Item 91) 91. The composition of any one of items 87 to 90, wherein the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. (Item 92) 92. The composition of claim 91, wherein the viral vector is AAVrh.74. (Item 93) 93. The composition of any one of paragraphs 87 to 92, wherein the viral vector comprises a viral genome comprising a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48. (Item 94) 94. The composition of any one of items 49 to 93, wherein the polynucleotide is administered systemically. (Item 95) 94. The composition of any one of items 49 to 93, wherein the polynucleotide is administered topically. (Item 96) 96. The composition of any one of items 49 to 95, wherein the polynucleotide is administered intravenously or intramuscularly. (Item 97) 97. The composition of any one of items 49 to 96, wherein the polynucleotide further comprises a promoter. (Item 98) 98. The composition of claim 97, wherein the promoter is a muscle-specific promoter. (Item 99) 99. The composition of claim 98, wherein the muscle-specific promoter is selected from the group consisting of an MHCK7 promoter and a tMCK promoter. (Item 100) 99. The composition of any one of items 97 to 99, wherein the promoter comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 6 over the entire length of SEQ ID NO: 4 or 6. (Item 101) 101. The composition of any one of items 49 to 100, wherein the polynucleotide further comprises an intron. (Item 102) 102. The composition of claim 101, wherein the intron is an SV40 chimeric intron. (Item 103) 103. The composition of claim 101 or 102, wherein the intron comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. (Item 104) 104. The composition of any one of items 49 to 103, wherein the polynucleotide further comprises a polyA sequence. (Item 105) 105. The composition of claim 104, wherein the polyA sequence comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. (Item 106) 106. The composition of any one of items 49 to 105, wherein the polynucleotide further comprises an inverted terminal repeat (ITR). (Item 107) 107. The composition of claim 106, wherein the ITRs comprise a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or 12 over the entire length of SEQ ID NO: 11 or 12. (Item 108) 49. The method of any one of items 1 to 48, wherein the polynucleotide further comprises a promoter. (Item 109) 109. The method of claim 108, wherein the promoter is a muscle-specific promoter. (Item 110) 109. The method of claim 108, wherein the muscle-specific promoter is selected from the group consisting of an MHCK7 promoter and a tMCK promoter. (Item 111) 111. The method of any one of items 108 to 110, wherein the promoter comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 6 over the entire length of SEQ ID NO: 4 or 6. (Item 112) 112. The method of any one of items 1 to 48 and 108 to 111, wherein the polynucleotide further comprises an intron. (Item 113) 113. The method of claim 112, wherein the intron is an SV40 chimeric intron. (Item 114) 114. The method of claim 112 or 113, wherein the intron comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. (Item 115) 115. The method of any one of items 1 to 48 and 108 to 114, wherein the polynucleotide further comprises a polyA sequence. (Item 116) 116. The method of claim 115, wherein the polyA sequence comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. (Item 117) 117. The method of any one of items 1 to 48 and 108 to 116, wherein the polynucleotide further comprises an inverted terminal repeat (ITR). (Item 118) 118. The method of claim 117, wherein the ITRs comprise a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or 12 over the entire length of SEQ ID NO: 11 or 12. (Item 119) A method for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a beta-sarcoglycan (SGCB) protein. (Item 120) A method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, the method comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein. (Item 121) A method for increasing or enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein, wherein the first sarcoglycan is selected from α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), and δ-sarcoglycan (SGCD). (Item 122) 122. The method of any one of paragraphs 119 to 121, wherein the viral vector genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8. (Item 123) A method for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein. (Item 124) A method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, the method comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein. (Item 125) A method for increasing or enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein, wherein the first sarcoglycan is selected from alpha-sarcoglycan (SGCA), beta-sarcoglycan (SGCB) and delta-sarcoglycan (SGCD). (Item 126) 126. The method of any one of paragraphs 123 to 125, wherein the viral vector genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19. (Item 127) A method for restoring or stabilizing the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding an alpha-sarcoglycan (SGCA) protein. (Item 128) A method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding an alpha-sarcoglycan (SGCA) protein. (Item 129) A method for increasing or enhancing expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein, wherein the first sarcoglycan is selected from γ-sarcoglycan (SGCG), β-sarcoglycan (SGCB), and δ-sarcoglycan (SGCD). (Item 130) 130. The method of any one of paragraphs 127 to 129, wherein the viral vector genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 47 or 48 over the entire length of SEQ ID NO: 47 or 48. (Item 131) 131. The method of any one of paragraphs 119 to 130, wherein the viral vector genome is administered to the subject at a dosage of 1.85e13vg / kg or 7.41e13vg / kg, and the dosage is quantified by linearized PCR standard. [Brief explanation of the drawings]

[0063] [Figure 1A] Figure 1A shows histological evaluation of skeletal muscles (tibialis anterior [TA] and gastrocnemius [GAS]) from wild-type (WT), SGCB+ / - (SGCB het), and SGCB- / - (SGCB KO) mice.

[0064] [Figure 1B] Figure 1B shows quantification of central nucleation in TA and GAS of wild-type (WT), SGCB+ / − (SGCB het), and SGCB− / − (SGCB KO) mice.

[0065] [Figure 2A]Figure 2A shows SGCB mRNA levels measured by qRT-PCR in WT, SGCB het, and SGCB KO mice.

[0066] [Figure 2B] FIG. 2B shows immunofluorescence images of SGCB protein production in WT mice, SGCB het mice, and SGCB KO mice.

[0067] [Figure 2C] FIG. 2C shows SGCB protein production measured by Western blot in WT, SGCB het, and SGCB KO mice.

[0068] [Figure 3A] FIG. 3A shows the absolute force of the TA muscle of WT, SGCB het, and SGCB KO mice.

[0069] [Figure 3B] Figure 3B shows the resistance to eccentric contraction of the TA muscles of WT, SGCB het, and SGCB KO mice.

[0070] [Figure 4A] FIG. 4A shows the locomotor activity of WT, SGCB het, and SGCB KO mice.

[0071] [Figure 4B] FIG. 4B shows the vertical activity of WT, SGCB het, and SGCB KO mice.

[0072] [Figure 5A] Figure 5A shows immunofluorescence images of dystrophin and SGCB expression in the TA muscles of untreated SGCB KO mice and SGCB KO mice treated with hSGCB gene transfer.

[0073] [Figure 5B] FIG. 5B shows immunofluorescence images of SGCA and SGCB expression in the myocardium of untreated SGCB KO mice and SGCB KO mice treated with hSGCB gene transfer.

[0074] [Figure 5C] FIG. 5C shows immunofluorescence images of SGCA and SGCB expression in the diaphragm of untreated SGCB KO mice and SGCB KO mice treated with hSGCB gene transfer.

[0075] [Figure 5D] Figure 5D shows immunofluorescence images of SGCB and dystrophin expression in TA muscle after scAAV.hSGCB gene transfer using the tMCK promoter.

[0076] [Figure 5E] Figure 5E shows immunofluorescence images of SGCBA and dystrophin expression in TA muscle after scAAV.hSGCB gene transfer using the tMCK promoter.

[0077] [Figure 6] FIG. 6 shows immunofluorescence staining of muscles from SGCG− / − mice.

[0078] [Figure 7] FIG. 7 shows immunofluorescence staining for sarcospan in LGMD2E mice.

[0079] [Figure 8A] FIG. 8A shows Western blotting for sarcospan in LGMD2E mice.

[0080] [Figure 8B] FIG. 8B shows normalized Western blot quantification of sarcospan.

[0081] [Figure 9A] Figure 9A shows immunofluorescence images of the expression of α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG), and δ-sarcoglycan (SGCD) in wild-type mice, SGCB− / − mice, and SGCB− / − mice treated with 1.85e13vg / kg (low dose) and 7.41e13vg / kg (high dose) of scAAV.MHCK7.hSGCB.

[0082] [Figure 9B] Figure 9B shows SGCB expression in various tissues of SGCB- / - mice treated with 1.85e13vg / kg (low dose) and 7.41e13vg / kg (high dose) of scAAV.MHCK7.hSGCB.

[0083] [Figure 9C] Figure 9C shows the expression of SGCA, SGCD, and SGCG in SGCB− / − mice treated with 1.85e13vg / kg (low dose) and 7.41e13vg / kg (high dose) of scAAV.MHCK7.hSGCB. DETAILED DESCRIPTION OF THE INVENTION

[0084] Detailed Description definition

[0085] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of this application and related art, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Unless expressly defined below, such terms should be interpreted according to their ordinary meaning.

[0086] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0087] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0088] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. By way of example, if the present specification describes a complex as comprising, consisting essentially of, or even consisting of components A, B, and C, it is expressly intended that any of A, B, or C, or combinations thereof, singly or in any combination, may be omitted and discarded.

[0089] Unless expressly indicated otherwise, all specified embodiments, features, and terms are intended to include both the described embodiment, feature, or term and its biological equivalents.

[0090] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by a variation of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%, as appropriate, and such ranges are inclusive. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It should also be understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents of such are known in the art.

[0091] Throughout this disclosure, various publications, patents and published patent specifications may be referenced. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entireties into this disclosure in order to more fully describe the state of the art to which this invention pertains.

[0092] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2002. ndedition (1989);Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987));the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0093] As used herein, the terms "increased," "decreased," "high," "low," or any grammatical variation thereof, refer to a variation of about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or even 0.1% of a reference composition, polynucleotide, polypeptide, protein, etc.

[0094] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" intend that said component, range, dosage form, etc. is suitable for the purposes of the present disclosure.

[0095] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in alternatives ("or").

[0096] Unless expressly stated and unless otherwise intended, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed that equivalents or biological equivalents of such are intended to be within the scope of the present disclosure. As used herein, the term "biological equivalent thereof" when referring to a reference protein, antibody, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof," and is intended to have minimal sequence identity while still maintaining the desired structure or functionality. Unless specifically stated herein, any polynucleotide, polypeptide, or protein referred to herein is also intended to include its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, at least about 85%, or alternatively, at least about 90%, or alternatively, at least about 95%, or alternatively, 98% homology or identity over the length of the reference sequence, and exhibit substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0097] An equivalent of a protein or polypeptide (referred to herein as a reference substance) shares 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 88%, or at least 90%, or at least 93%, or at least 95%, or at least 97%, or at least 98%, or at least 99%) identity to the reference substance and retains the function and manufacturability of the reference substance.

[0098] As used herein, the terms "function," "activity," and "enzymatic activity" are used interchangeably. Loss of sarcoglycan function can result in the protein deficiency of other sarcoglycans, dystrophin, or sarcospan, the loss of sarcoglycan complex formation, and / or the loss of stabilization of the dystrophin-associated protein complex (DAPC). For example, loss of SGCB protein also results in the loss of SGCA protein, sarcospan, and dystrophin. In another example, loss of SGCG protein results in the loss of SGCA protein, SGCB protein, and dystrophin. Examples of sarcoglycan activity include, but are not limited to, stabilizing DAPC and providing mechanical support for the sarcoglycan membrane. Functional assessment of sarcoglycan protein includes, but is not limited to, measuring the force generation and resistance to contraction-induced injury in the tibialis anterior (TA) muscle, as well as laser monitoring of open-field cage activity to evaluate overall gait (movement around the cage) and rearing activity (rearing on the hind limbs).

[0099] An equivalent of a polynucleotide (herein referred to as a reference material) shares 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 88%, or at least 90%, or at least 93%, or at least 95%, or at least 97%, or at least 98%, or at least 99%) identity to the reference material and encodes the same polypeptide as that encoded by the reference material, or in one embodiment, encodes an equivalent of the polypeptide encoded by the reference material that has the same or a similar activity or function.

[0100] To arrive at a position or continuous segment of the test sequence that is equivalent to (or corresponds to) an amino acid / nucleotide residue or continuous segment of the reference sequence, a sequence alignment is performed between the test sequence and the reference sequence. Positions or segments that are aligned with each other are determined to be equivalent.

[0101] The term "affinity tag" refers to a polypeptide that can be included within a fusion protein that can bind to the affinity tag, i.e., allows for detection and / or purification of the fusion protein from the cellular environment using a ligand with affinity for the affinity tag. The ligand may be, but is not limited to, an antibody, a resin, or a complementary polypeptide. The affinity tag may comprise a small peptide, typically approximately 4-16 amino acids in length, or it may comprise a larger polypeptide. Commonly used affinity tags include polyarginine, FLAG, V5, polyhistidine, c-Myc, Strep II, maltose-binding protein (MBP), N-utilization protein A (NusA), thioredoxin (Trx), and glutathione S-transferase (GST), among others (see, e.g., GST Gene Fusion System Handbook - Sigma-Aldrich). In embodiments, the affinity tag is a polyhistidine tag, such as a His6 tag. The inclusion of an affinity tag in a fusion protein allows the fusion protein to be purified from the cellular environment by affinity purification using an affinity medium capable of tightly and specifically binding to the affinity tag. The affinity medium may comprise, for example, a metal-charged resin or ligand covalently linked to a stationary phase (matrix) such as agarose or metal beads. For example, a polyhistidine-tagged fusion protein (also referred to as a His-tagged fusion protein) binds to Ni. 2+ or Co 2+FLAG-tagged fusion proteins can be captured by immobilized metal ion chromatography using a resin loaded with FLAG, anti-FLAG affinity gel can be used to capture FLAG-tagged fusion proteins, and glutathione cross-linked to a solid support such as agarose can be used to capture GST-tagged fusion proteins.

[0102] As used herein, the terms "purification," "purifying," or "separating" refer to the process of isolating one or more biological substances (e.g., polynucleotides, polypeptides, or viral vectors) from a complex mixture, such as a cell lysate or a mixture of polypeptides. The purification, separation, or isolation need not be complete; i.e., some components of the complex mixture may remain with one or more biological substances (e.g., polynucleotides, polypeptides, or viral vectors) after the purification process. However, the purified product should be enriched for one or more biological substances (e.g., polynucleotides, polypeptides, or viral vectors) relative to the complex mixture before purification, and a significant portion of other components originally present in the complex mixture should have been removed by the purification process.

[0103] The term "cell," as used herein, can refer to either a prokaryotic or eukaryotic cell, obtained from the subject or from a commercial source, as appropriate.

[0104] "Eukaryotic cells" include all kingdoms of life except Monera. They can be easily identified by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotes, or organisms in which cells are organized into complex structures by internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless specifically stated, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include ape, bovine, porcine, mouse, rat, avian, reptile, and human cells, such as HEK293 cells, Chinese hamster ovary (CHO) cells, 293T cells, stem cells, satellite cells, and muscle cells. Examples of muscle cells include, but are not limited to, skeletal muscle cells, cardiac muscle cells, and smooth muscle cells.

[0105] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, bacteria of the genus Bacillus, E. coli, and Salmonella.

[0106] The term "encoding," as applied to a nucleic acid sequence, refers to a polynucleotide that is said to "encode" a polypeptide if, in its natural state or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or truncated versions thereof. The antisense strand is the complement of such a nucleic acid, and a coding sequence can be deduced therefrom.

[0107] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological material, or cellular material, and are intended to have minimal homology while still maintaining the desired structure or functionality (e.g., having a similar function or activity). Even if not explicitly stated, when referring to an equivalent or biological equivalent to a reference polypeptide, protein, or polynucleotide, it should be understood that the equivalent or biological equivalent has the described structural relationship and equivalent or substantially equivalent biological activity to the reference polypeptide, protein, or polynucleotide. For example, non-limiting examples of equivalent polypeptides, proteins, or polynucleotides include polypeptides, proteins, or polynucleotides that are at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identical to the sequence of a reference polypeptide, polynucleotide, or protein over the length of the reference polypeptide, polynucleotide, or protein. Alternatively, an equivalent polypeptide is a polypeptide encoded by a polynucleotide or its complement that hybridizes to the polynucleotide encoding such a reference polypeptide sequence under high stringency conditions, and has substantially equivalent or equivalent biological activity. High stringency conditions are described herein and are incorporated by reference herein.Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or complement thereof that has at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least 97% sequence identity to a reference polynucleotide, e.g., a wild-type polynucleotide, over the length of the reference polynucleotide. Such an equivalent polypeptide has the same biological activity as the polypeptide encoded by the reference polynucleotide.

[0108] Non-limiting examples of equivalent polynucleotides include polynucleotides that have at least 60%, alternatively at least 65%, alternatively at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, or alternatively at least 97% identity to the reference polynucleotide.Equivalent also refers to polynucleotides or their complements that hybridize to the reference polynucleotide under high stringency conditions.Such equivalent polynucleotides have the same biological activity as the reference polynucleotide.

[0109] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences over the length of the reference polynucleotide. Alignment and percent homology or sequence identity can be calculated using software programs known in the art, such as Current Protocols In Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1 can be used to determine alignment. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST using default parameters. In particular, exemplary programs include BLASTN and BLASTP using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were calculated by comparing these with clustalW (web address: genome.jp / tools / clustalw / , available January 2017). This can be determined by incorporating it into the website (last accessed on the 13th).

[0110] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0111] As used herein, the term "at least 90% identical" refers to two compared sequences (polynucleotides or polypeptides) that are about 90% to about 100% identical, including at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% identical.

[0112] As used herein, the terms "retain," "similar," and "same" are used interchangeably while describing the function, activity, or functional activity of a polynucleotide, protein, and / or peptide, and refer to a functional activity of at least about 20% (including but not limited to: at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or about 100%) of the activity of the reference protein, polynucleotide, and / or peptide.

[0113] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex may contain two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0114] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6xSSC to about 10xSSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9xSSC to about 2xSSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1xSSC to about 0.1xSSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used. In one embodiment, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide or its complement. In another embodiment, an equivalent polypeptide is a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide or its complement.

[0115] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may also include splicing of the mRNA in a eukaryotic cell.

[0116] As used herein, the term "functional" may be used to modify any molecular, biological, or cellular material with the intent of achieving a particular, specified effect.

[0117] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In certain embodiments, the polynucleotide comprises and / or encodes messenger RNA (mRNA), short hairpin RNA, and / or short hairpin RNA. In one embodiment, the polynucleotide is or encodes mRNA. In certain embodiments, the polynucleotide is double-stranded (ds) DNA, such as engineered dsDNA or ds cDNA synthesized from single-stranded RNA.

[0118] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and no limit is imposed on the maximum number of amino acids that may make up the sequence of a protein or peptide. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0119] As used herein, a continuous amino acid sequence refers to a sequence having at least two amino acids.However, it should be noted that the continuous amino acid sequence of the first part and the second part is not limited to the amino acid sequence of the first part directly linked to the second part.The first part can also be linked to the second part by a third part, for example, a linkage, thereby forming a continuous amino acid sequence.

[0120] As used herein, the terms "conjugate," "conjugated," "conjugating," and "conjugation" refer to the formation of a bond between molecules, particularly between two amino acid sequences and / or two polypeptides. Conjugation may be direct (i.e., by bonding) or indirect (i.e., through an additional molecule). Conjugation may be by covalent or non-covalent bonding.

[0121] As used herein, a contiguous amino acid sequence may include two or more polypeptides conjugated to each other directly or indirectly (eg, by a linker).

[0122] As used herein, the term "recombinant expression system" refers to a genetic construct or constructs for the expression of certain genetic material formed by recombinant means.

[0123] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicle are liposome, micelle biocompatible polymer, including natural polymer and synthetic polymer; lipoprotein; lipid nanoparticle; polypeptide; polysaccharide; lipopolysaccharide; artificial virus envelope; metal particle; and bacteria, or virus such as rabies virus, flavivirus, lentivirus, baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vector and other recombinant vehicle that are typically used in the field, and are described for expression in various eukaryotic and prokaryotic hosts, and can be used for gene therapy and simple protein expression.

[0124] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, the terms "gene delivery," "gene transfer," "mRNA-based delivery," "transduction," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a transgene) into a host cell, regardless of the method used for the introduction. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein- or lipid-based gene delivery complexes, including, for example, protamine complexes, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic particles, or combinations thereof), and techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide may be unmodified or may contain one or more modifications; for example, modified mRNA may include ARCA capping; enzymatic polyadenylation to add a tail of 100 to 250 adenosine residues; and one or both of the following: substitution of cytidine with 5-methylcytidine and / or substitution of uridine with pseudouridine. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contain an origin of replication compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Several vectors are known in the art and are known to be capable of mediating gene transfer into mammalian cells, as described herein.

[0125] A "plasmid" is an extrachromosomal DNA molecule that is separate from and capable of replicating independently of chromosomal DNA. Plasmids are often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically confer a selective advantage in a given environmental condition. Plasmids can carry genes that confer resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar circumstances.

[0126] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that confers resistance to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites and facilitates the insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid carrying the gene of interest. Once the bacteria produce the protein that confers its antibiotic resistance, they can also be induced to produce large amounts of the protein from the inserted gene.

[0127] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb, up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences required for replication and storage in yeast cells. They are constructed using an initial circular plasmid, which is linearized using restriction enzymes. DNA ligase can then add sequences or genes of interest into the linear molecule using the cohesive ends. Because yeast is itself a eukaryotic cell, yeast expression vectors such as YACs, YIp (yeast integrating plasmid), and YEp (yeast episomal plasmid) are extremely useful because they can produce eukaryotic protein products with post-translational modifications. However, YACs are more unstable than BACs and have been found to produce chimeric effects.

[0128] As used herein, the term "nanoparticle" refers to a particle having a dimension less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm in size. Nanoparticles may comprise or be engineered from a variety of materials. For example, nanoparticles may comprise or be engineered from biological materials such as phospholipids, lipids, lactate, dextran, or chitosan. Alternatively, nanoparticles may comprise or be engineered from polymers, carbon, silica, and metals. Nanoparticles may be biodegradable. Exemplary nanoparticles and nanoparticle compositions are described, for example, in Jong and Borm, Int. J. Nanomedicine 3(2):133-149 and Xue et al., Nanoparticles and Nanoparticles ... al., Curr. Pharm. Des. 21(22):3140-3147.

[0129] As used herein, the term "liposome" refers to a nanoparticle composed of a phospholipid bilayer with an aqueous core. Liposomes may be prepared using biocompatible lipid / phospholipid components. Liposomes may also contain functionalized lipids, such as PEG lipids or lipids conjugated with targeting moieties for tissue-specific delivery. Exemplary liposomes are described, for example, in Xue et al., Curr. Pharm. Des. 21(22):3140-3147, the entire contents of which are incorporated by reference.

[0130] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or membrane of a viral particle. The capsid functions to encase the viral genome by encapsidation and to protect, transport, and release it into a host cell. A capsid is generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid. The capsid may be a wild-type capsid. Alternatively, the capsid may be a modified capsid. A modified capsid may differ from a wild-type capsid by one or more mutations, substitutions, or deletions in the amino acid or nucleic acid sequence of the wild-type capsid.

[0131] As used herein, the term "helper," in reference to a virus or plasmid, refers to a virus or plasmid used to provide additional components necessary for replication and packaging of viral particles or recombinant viral particles, such as the modified AAVs disclosed herein. Components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus may encode an enzyme required for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0132] In another embodiment, the recombinant AAV vectors described herein may be operably linked to a muscle-specific regulatory element, such as a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor MEF, muscle creatine kinase (MCK), tMCK (truncated MCK), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK), C5-12 (a synthetic promoter), a mouse creatine kinase enhancer element, a fast-twitch skeletal troponin C gene element, a slow-twitch cardiac troponin C gene element, a slow-twitch troponin I gene element, a hypoxia-inducible nuclear factor, a stimulatory factor (STF), ... The glucocorticoid-inducible element or glucocorticoid response element (GRE) is a steroid-inducible element.

[0133] In some embodiments, the muscle-specific promoter is MHCK7 (SEQ ID NO:4). An exemplary rAAV described herein is pAAV.MHCK7.hSCGB, which comprises, consists essentially of, or further consists of the nucleotide sequence of SEQ ID NO:3. Within the nucleotide sequence of SEQ ID NO:3, the MCHK7 promoter spans nucleotides 130-921, the SV40 chimeric intron (SEQ ID NO:9) spans nucleotides 931-1078, the β-sarcoglycan sequence (SEQ ID NO:1) spans nucleotides 1091-2047, and polyA (SEQ ID NO:10) spans nucleotides 2054-2106. In some embodiments, pAAV.MHCK7.hSCGB comprises, consists essentially of, or further consists of the nucleotide sequence of SEQ ID NO:7. Within the nucleotide sequence of SEQ ID NO:7, the MCHK7 promoter spans nucleotides 128-919, the SV40 chimeric intron spans nucleotides 929-1076, the β-sarcoglycan sequence spans nucleotides 1086-2042, and poly A spans nucleotides 2049-2101.

[0134] In some embodiments, pAAV.MHCK7.hSCGB comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, and 8 over the entire length of SEQ ID NOs: 3, 7, and 8. In some embodiments, pAAV.MHCK7.hSCGB comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 and 17 over the entire length of SEQ ID NOs: 1 and 17. In some embodiments, pAAV.MHCK7.hSCGB comprises, consists essentially of, or even consists of a nucleotide sequence encoding a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2 and 18 over the entire length of SEQ ID NOs: 2 and 18. In one embodiment, the polynucleotide sequence encodes a protein that retains sarcoglycan activity, including beta and / or alpha-sarcoglycan activity. In another embodiment, the polynucleotide sequence encodes a protein that retains beta-sarcoglycan activity.

[0135] In some embodiments, the muscle-specific promoter is tMCK (SEQ ID NO: 6). An exemplary rAAV described herein is pAAV.tMCK.hSCGB, which comprises, consists essentially of, or alternatively consists of the nucleotide sequence of SEQ ID NO: 5. Within the nucleotide sequence of SEQ ID NO: 5, the tMCK promoter spans nucleotides 141-854, the SV40 chimeric intron spans nucleotides 886-1018, the β-sarcoglycan sequence spans nucleotides 1058-2014, and polyA spans nucleotides 2021-2073. In some embodiments, the polynucleotide sequence encoding pAAV.tMCK.hSCGB comprises, consists essentially of, or even consists of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to the nucleotide sequence set forth in SEQ ID NO:5, for example, where the polynucleotide sequence encodes a protein that retains sarcoglycan activity, including, but not limited to, beta and / or alpha-sarcoglycan activity. In some embodiments, pAAV.tMCK.hSCGB comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 and 17 over the entire length of SEQ ID NOs: 1 and 17.In some embodiments, pAAV.tMCK.hSCGB comprises, consists essentially of, or even consists of a nucleotide sequence encoding a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2 and 18 over the entire length of SEQ ID NOs: 2 and 18.

[0136] As used herein, a biological sample, or sample, can be obtained from a subject, a cell line, or cultured cells or tissues. Exemplary samples include, but are not limited to, cell samples, tissue samples, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculate, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / vaginal washings, synovial fluid, mucosal secretions, stool water, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyl cavity fluid, or umbilical cord blood. Cell samples The sample may include cells from a tissue or organ. Exemplary organs include, but are not limited to, heart, lung, liver, eye, stomach, spleen, kidney, pancreas, and gallbladder. Exemplary tissues include, but are not limited to, connective tissue, epithelial tissue, muscle tissue, and nerve tissue. The cell sample may include muscle cells or components of muscle cells. The components of muscle cells may include muscle cell membranes or sarcolemma. In some embodiments, the cell membrane is a skeletal muscle cell membrane or sarcolemma. The tissue sample may include muscle tissue.

[0137] As used herein, the term "muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle, e.g., derived from the gastrointestinal tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0138] As used herein, the term "detectable marker" refers to at least one marker capable of directly or indirectly producing a detectable signal. This non-exhaustive list of markers includes enzymes that produce detectable signals, for example, by colorimetry, fluorescence, or luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase, etc.; chromophores, such as fluorescent or luminescent dyes; electron-dense groups that are detected by electron microscopy or their electrical properties, such as conductivity, amperometry, voltammetry, or impedance; for example, detectable groups whose molecules are large enough to induce detectable modifications of their physical and / or chemical properties, and such detection can be achieved by optical methods, such as diffraction, surface plasmon resonance, surface fluctuations, changes in contact angle, or atomic force spectroscopy, tunneling effect, or the like. 32 P, 35 S, 89 Zr or 125 This can be achieved by physical methods such as radioactive molecules such as I.

[0139] As used herein, the term "purification marker" refers to at least one marker useful for purification or identification. A non-exhaustive list of markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluor, FITC, TRITC, or any other fluorescent dye or hapten.

[0140] As used herein, an epitope tag is a biological structure or sequence, such as a protein or carbohydrate, that acts as an antigen recognized by an antibody. In certain embodiments, epitope tags are used interchangeably with purification markers and / or affinity tags.

[0141] A "composition" is intended to mean a combination of two or more compounds, e.g., a combination of an active polypeptide, polynucleotide, viral vector, or antibody with another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0142] A "pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide, vector or antibody with an inert or active carrier, such as a solid support or a liquid carrier, to make a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0143] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0144] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, mice, rats, rabbits, apes, cattle, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans. In addition to being useful for treating humans, the present invention is also useful for the veterinary treatment of domesticated animals, including companion animals, exotic animals, and mammals, rodents, susceptible to muscular dystrophy. In one embodiment, mammals include horses, dogs, and cats. In other embodiments of the present invention, the human is an adult human over the age of 18, an adolescent human between the ages of 13 and 18, or a child human under the age of 13, or under the age of 10, or under the age of 8, or under the age of 6, or under the age of 4, or under the age of 2. In some embodiments, the subject or human is male. In some embodiments, the subject or human is female.

[0145] "Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e., preventing the development of clinical symptoms of the disease in a patient who is predisposed to the disease but who has not yet experienced or displayed symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) palliating the disease, i.e., causing the disease or its clinical symptoms to regress. In one aspect, the term "treating" excludes prevention or prophylaxis.

[0146] The term "suffering," in relation to the term "treatment," refers to a subject who has been diagnosed with or is predisposed to a disease. In one embodiment, the subject is diagnosed with muscular dystrophy. In one embodiment, the subject is predisposed to muscular dystrophy. A subject predisposed to muscular dystrophy is a subject, individual, or patient who has one or more mutations or alterations in the genes responsible for healthy muscle structure and function. A subject suffering from muscular dystrophy may exhibit one or more symptoms or signs of muscular dystrophy. Exemplary symptoms or signs of muscular dystrophy include, but are not limited to, enlarged gastrocnemius muscles, difficulty walking or running, gait abnormalities (such as a wobbling gait), difficulty swallowing, cardiac problems such as arrhythmia and heart failure (cardiomyopathy), learning disabilities, stiff or wobbly joints, muscle pain, a curved spine (scoliosis), and breathing problems. Alternatively, a subject suffering from muscular dystrophy may have one or more mutations or alterations in the genes responsible for healthy muscle structure and function.

[0147] The term "muscular dystrophy" as used herein refers to a disorder that gradually reduces strength and muscle mass. Non-limiting examples of muscular dystrophy include Becker muscular dystrophy (BMD), tibial muscular dystrophy, Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanopathy, congenital muscular dystrophy such as partial LAMA2 deficiency congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, type 1D congenital muscular dystrophy (type ID congenital muscular dystrophy), and Fukuyama congenital muscular dystrophy. , can include limb-girdle muscular dystrophy type 1A, limb-girdle muscular dystrophy type 2A, limb-girdle muscular dystrophy type 2B, limb-girdle muscular dystrophy type 2C, limb-girdle muscular dystrophy type 2D, limb-girdle muscular dystrophy type 2E, limb-girdle muscular dystrophy type 2F, limb-girdle muscular dystrophy type 2G, limb-girdle muscular dystrophy type 2H, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2J, limb-girdle muscular dystrophy type 2K, limb-girdle IC muscular dystrophy, ankylosing spinal muscular dystrophy with epidermolysis bullosa simplex, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy and Ullrich scleroatonic muscular dystrophy.In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject has limb-girdle muscular dystrophy type 2E (LGMD2E).

[0148] An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount may be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the duration for which individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, the specific dosage level of a therapeutic agent of the present invention for any particular subject will vary depending on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity and administration form of the particular disorder being treated. Treatment dosages can generally be titrated to optimize safety and efficacy. Typically, dose-effect relationships from in vitro and / or in vivo studies can provide useful guidance initially regarding appropriate dosages for patient administration. In general, it is desirable to administer an amount of compound effective to achieve serum levels commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to treat muscular dystrophies, such as LGMD and DMD, ex vivo, in vitro or in vivo.

[0149] The term administration includes, but is not limited to, oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implantation), inhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), which may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The present invention is not limited by the route of administration, formulation, or dosing schedule.

[0150] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, Handbook of Parvoviruses 1:169-228, 1989, and Berns, Virology 1743-1764, 1999. However, various serotypes have different gene sequences. It is fully expected that these same principles will be applicable to additional AAV serotypes, since they are known to be very closely related, both structurally and functionally, even at the level of their respective serotypes (see, e.g., Blacklowe, Parvoviruses and Human Disease 165-174, 1988, J.R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61, 1974). (See references below.) For example, all AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication functions in each serotype are under similar regulatory control.

[0151] As described herein, the term "short version of dystrophin" refers to a protein that is shorter than the full-length dystrophin protein but maintains at least part of the function of the dystrophin protein.In one embodiment, the short version is a mini-dystrophin or a micro-dystrophin.In one embodiment, the micro-dystrophin protein is about 1 / 3 the size of the full-length dystrophin protein.For example, one embodiment of a micro-dystrophin protein can be found in WO2017181015, which is incorporated by reference.In another embodiment, the sequence of a mini-dystrophin protein can be found in U.S. Patent No. 6,869,777, which is incorporated by reference.

[0152] Without wishing to be bound by theory, in skeletal and cardiac muscles, dystrophin is part of a group of proteins (DAPC) that work together to strengthen muscle fibers and protect them from injury when the muscles contract and relax. In some embodiments, the dystrophin protein transmits the force of muscle contraction from the inside of the muscle cell to the outside of the cell membrane. Absence or reduced expression of dystrophin or many of the DAPC components causes muscular dystrophies, a group of genetic diseases in which repeated muscle damage leads to atrophy and fibrosis, ultimately resulting in muscle degradation.

[0153] "AAV expression cassette," as used herein, refers to a nucleotide sequence comprising, consisting essentially of, or even consisting of, one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such an AAV expression cassette can be replicated and packaged into infectious viral particles (e.g., AAV vectors) when a vector encoding and expressing the rep and cap gene products is present in a transfected host cell.

[0154] An "AAV virion" or "AAV vector" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and a polynucleotide AAV expression cassette encapsidated by encapsidation. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, the production of an AAV vector particle necessarily includes the production of an AAV expression cassette, since the cassette is contained within the AAV vector particle. An AAV vector may be a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector. In an ssAAV vector, the coding sequence and complementary sequence of the transgene expression cassette are present on separate strands and are packaged into separate viral capsids. In an scAAV vector, both the coding sequence and complementary sequence of the transgene expression cassette are present on each of the plus and minus strands of the genome.

[0155] Adeno-associated virus (AAV) is a replication-deficient parvovirus, whose single-stranded DNA genome is approximately 4.7 kb long and contains 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is shown in Srivastava et al., J Virol, 45: 555-564 (1983), as amended by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the AAV-5 genome is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least portions of AAV-7 and AAV-8 are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8); the AAV-9 genome is described in Gao et al., J. Virol., 78: 6381-6388. (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and AAV-11 is provided in Virology, 330(2): 375-383 (2004). The cloning of the AVrh.74 serotype is described in Rodino-Klapac., et al., Journal of Translational Medicine 5, 45 (2007). Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging, and integration into host cell chromosomes are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 in reference to their relative map locations) drive expression of two AAV internal open reading frames encoding the rep and cap genes. With differential splicing of a single AAV intron (e.g., AAV2 nucleotides 2107 and 2227), the two rep promoters (p5 and p19) drive the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately drive viral genome replication. The cap gene is expressed from the p40 promoter, which encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at position 95 of the AAV genome map. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0156] The recombinant AAV (rAAV) genome of the present disclosure comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome may be derived from any AAV serotype that can be derived from a recombinant virus, including, but not limited to, AAV serotypes AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In some embodiments, AAV1, AAV6, AAV8, or AAVrh.74 are used to promote skeletal muscle-specific expression. In some embodiments, the rAAV genome comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0157] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells (including mixtures thereof).

[0158] As used herein, the terms "comprising" or "comprises" are intended to mean that compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, means excluding other elements of any essential significance to the combination for the described purpose. Thus, a composition consisting essentially of elements as defined herein does not exclude trace amounts of contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" means excluding more than trace amounts of other ingredients, and substantial method steps for administering a composition of the invention or process steps for producing the composition or achieving an intended result. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0159] The term "isolated," as used herein with respect to nucleic acids, such as DNA or RNA, refers to a molecule separated from other DNAs or RNAs, respectively, present in the natural source of the macromolecule. The term "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur naturally as fragments. The term "isolated" is also used herein to refer to polypeptides, proteins, and / or host cells that have been isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" refers to a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, separated from the cellular and other components with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a dissimilar phenotype or genotype. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0160] The term "recombinant," as used herein with respect to a polypeptide or a polynucleotide, such as DNA or RNA, refers to a molecule formed by laboratory methods of recombination, such as molecular cloning. Molecular cloning techniques are known in the art and may include, but are not limited to, PCR amplification of a polynucleotide, enzymatic digestion of a polynucleotide, ligation of a polynucleotide into an expression cassette (e.g., a mammalian expression cassette), transformation, transfection or transduction of a polynucleotide into a cell, and expression of the polynucleotide to produce a polypeptide. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 2012. The term "recombinant polynucleotide" is intended to include a fragment of a polynucleotide that encodes a protein. For example, a recombinant polynucleotide may include a fragment of a polynucleotide encoding a human sarcoglycan protein. A recombinant polynucleotide may be generated by PCR amplification of a fragment of a polynucleotide that encodes a protein. A recombinant polypeptide may be generated by expression of one or more recombinant polynucleotides.

[0161] Modes for carrying out the present disclosure

[0162] Disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in patients suffering from muscular dystrophy. In some embodiments, the method comprises, consists essentially of, or even consists of administering to a subject a polynucleotide sequence encoding (a) sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin. As described herein, the term "truncated version of dystrophin" refers to a protein that is shorter than the full-length dystrophin protein but maintains at least part of the function of dystrophin protein. In one embodiment, the truncated version is mini-dystrophin or micro-dystrophin. In one embodiment, micro-dystrophin protein is about 1 / 3 the size of full-length dystrophin protein. For example, one embodiment of micro-dystrophin protein can be found in WO2017181015, which is incorporated by reference. In another embodiment, the sequence of mini-dystrophin protein can be found in U.S. Patent No. 6,869,777, which is incorporated by reference. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide sequence encoding dystrophin. In some embodiments, the dystrophin-encoding polynucleotide comprises, consists essentially of, or further consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide sequence encoding a truncated version of dystrophin.In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG.In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB. In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB.In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises administering a viral vector comprising, consisting essentially of, or consisting of a viral genome comprising, consisting essentially of, or consisting of a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin. In some embodiments, the viral vector comprises, consisting essentially of, or consisting of a viral genome comprising, consisting essentially of, or consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0163] Disclosed herein is a method for localizing a first sarcoglycan, sarcospan, or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy.In some embodiments, the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide sequence encoding (a) a second sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin, wherein the first sarcoglycan is different from the second sarcoglycan.In some embodiments, the first sarcoglycan is SGCA, and the second sarcoglycan is selected from SGCB, SGCD, and SGCG.In some embodiments, the first sarcoglycan is SGCB, and the second sarcoglycan is selected from SGCA, SGCD, and SGCG.In some embodiments, the first sarcoglycan is SGCD, and the second sarcoglycan is selected from SGCB, SGCA, and SGCG. In some embodiments, the first sarcoglycan is SGCG, and the second sarcoglycan is selected from SGCB, SGCD, and SGCA. In some embodiments, the first sarcoglycan is selected from SGCA, SGCD, and SGCG, and the second sarcoglycan is SGCB. In some embodiments, the first sarcoglycan is selected from SGCB, SGCD, and SGCG, and the second sarcoglycan is SGCA. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCG, and the second sarcoglycan is SGCD. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCD, and the second sarcoglycan is SGCG. In some embodiments, the method comprises, consists essentially of, or even further consists of administering a polynucleotide sequence encoding dystrophin.In some embodiments, the polynucleotide encoding dystrophin comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the method comprises, consists essentially of, or even consists of administering a polynucleotide sequence encoding a truncated version of dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding SGCG.In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB.In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises, or consists essentially of, or consists of, administering a viral vector comprising, or consisting essentially of, or consisting of a viral genome comprising, or consisting essentially of, or consisting of, a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin.In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0164] Disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, or dystrophin on muscle cell membranes or muscle cell membranes in a subject suffering from muscular dystrophy.In some embodiments, the method comprises, or essentially consists of, or also consists of administering to the subject a polynucleotide sequence encoding (a) a second sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin, wherein the first sarcoglycan is different from the second sarcoglycan.In some embodiments, the first sarcoglycan is SGCA, and the second sarcoglycan is selected from SGCB, SGCD, and SGCG.In some embodiments, the first sarcoglycan is SGCB, and the second sarcoglycan is selected from SGCA, SGCD, and SGCG.In some embodiments, the first sarcoglycan is SGCD, and the second sarcoglycan is selected from SGCB, SGCA, and SGCG. In some embodiments, the first sarcoglycan is SGCG, and the second sarcoglycan is selected from SGCB, SGCD, and SGCA. In some embodiments, the first sarcoglycan is selected from SGCA, SGCD, and SGCG, and the second sarcoglycan is SGCB. In some embodiments, the first sarcoglycan is selected from SGCB, SGCD, and SGCG, and the second sarcoglycan is SGCA. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCG, and the second sarcoglycan is SGCD. In some embodiments, the first sarcoglycan is selected from SGCA, SGCB, and SGCD, and the second sarcoglycan is SGCG. In some embodiments, the method comprises, consists essentially of, or even further consists of administering a polynucleotide sequence encoding dystrophin.In some embodiments, the polynucleotide encoding dystrophin comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the method comprises, consists essentially of, or even consists of administering a polynucleotide sequence encoding a truncated version of dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding SGCG.In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB.In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises, or consists essentially of, or consists of, administering a viral vector comprising, or consisting essentially of, or consisting of a viral genome comprising, or consisting essentially of, or consisting of, a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin.In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0165] Disclosed herein is a composition for restoring or stabilizing dystrophin-associated protein complex (DAPC) in patients suffering from muscular dystrophy. In some embodiments, the composition comprises, essentially consists of, or even consists of a polynucleotide sequence encoding (a) sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin. In one embodiment, the truncated version is mini-dystrophin or micro-dystrophin. In some embodiments, the method comprises, essentially consists of, or even consists of administering a polynucleotide sequence encoding dystrophin. In some embodiments, the polynucleotide encoding dystrophin comprises, essentially consists of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the method comprises administering, or consists essentially of, or further consists of, a polynucleotide sequence encoding a truncated version of dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or further consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the methods comprise, consist essentially of, or even consist of administering a polynucleotide encoding SGCG.In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB.In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises, or consists essentially of, or consists of, administering a viral vector comprising, or consisting essentially of, or consisting of a viral genome comprising, or consisting essentially of, or consisting of, a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin.In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0166] Disclosed herein is a composition that localizes a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or muscle cell membrane of a subject suffering from muscular dystrophy.In some embodiments, the composition comprises, essentially consists of, or even consists of a polynucleotide sequence encoding (a) a second sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin.In one embodiment, the truncated version is mini-dystrophin or micro-dystrophin.In some embodiments, the method comprises, essentially consists of, or even consists of administering a polynucleotide sequence encoding dystrophin.In some embodiments, the polynucleotide encoding dystrophin comprises, essentially consists of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:36 or 37 over the entire length of SEQ ID NO:36 or 37. In some embodiments, the method comprises administering, or consists essentially of, or further consists of, a polynucleotide sequence encoding a truncated version of dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or further consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39.In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein comprising, consisting essentially of, or further consisting of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCA. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB.In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises, or consists essentially of, or consists of, administering a viral vector comprising, or consisting essentially of, or consisting of a viral genome comprising, or consisting essentially of, or consisting of, a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin.In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0167] Disclosed herein is a composition for enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin in a subject suffering from muscular dystrophy. In some embodiments, the composition comprises, essentially consists of, or further consists of a polynucleotide sequence encoding (a) a second sarcoglycan; (b) dystrophin; or (c) a truncated version of dystrophin. In one embodiment, the truncated version is mini-dystrophin or micro-dystrophin. In some embodiments, the method comprises, essentially consists of, or further consists of administering a polynucleotide sequence encoding dystrophin. In some embodiments, the polynucleotide encoding dystrophin comprises, essentially consists of, or further consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 36 or 37 over the entire length of SEQ ID NO: 36 or 37. In some embodiments, the method comprises administering, or consists essentially of, or further consists of, a polynucleotide sequence encoding a truncated version of dystrophin. In some embodiments, the polynucleotide encoding the truncated version of dystrophin comprises, consists essentially of, or further consists of a nucleotide sequence encoding a truncated version of dystrophin protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39.In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCG. In some embodiments, the polynucleotide encoding SGCG comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCG protein comprising, consisting essentially of, or further consisting of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCA. In some embodiments, the polynucleotide encoding SGCA comprises, consists essentially of, or further consists of a nucleotide sequence encoding an SGCA protein that comprises, consists essentially of, or further consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the method comprises, consists essentially of, or further consists of administering a polynucleotide encoding SGCB.In some embodiments, the polynucleotide encoding SGCB comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCB protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18. In some embodiments, the method comprises, consists essentially of, or alternatively consists of administering a polynucleotide encoding an SGCB. In some embodiments, the polynucleotide encoding SGCD comprises, consists essentially of, or alternatively consists of a nucleotide sequence encoding an SGCD protein that comprises, consists essentially of, or alternatively consists of: (a) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32; or (b) an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the method comprises, or consists essentially of, or consists of, administering a viral vector comprising, or consisting essentially of, or consisting of a viral genome comprising, or consisting essentially of, or consisting of, a polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin.In some embodiments, the viral vector comprises, consists essentially of, or even consists of a viral genome comprising, consisting essentially of, or even consisting of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0168] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0169] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0170] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising, consisting essentially of, or alternatively consisting of a polynucleotide sequence encoding a β-sarcoglycan (SGCB) protein, wherein the first sarcoglycan is selected from α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), and δ-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, and 8 over the entire length of SEQ ID NOs: 3, 5, 7, and 8.

[0171] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0172] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0173] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising, consisting essentially of, or further consisting of a polynucleotide sequence encoding a gamma-sarcoglycan (SGCG) protein, wherein the first sarcoglycan is selected from alpha-sarcoglycan (SGCA), beta-sarcoglycan (SGCB), and delta-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or further consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:19 over the entire length of SEQ ID NO:19.

[0174] Further disclosed herein is a method for restoring or stabilizing dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0175] Further disclosed herein is a method for localizing a first sarcoglycan, sarcospan, and / or dystrophin to the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising, consisting essentially of, or consisting of, a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein to the subject. In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0176] Further disclosed herein is a method for increasing or enhancing the expression of a first sarcoglycan, sarcospan, and / or dystrophin, on the muscle cell membrane or sarcolemma of a subject suffering from muscular dystrophy, comprising administering to the subject a viral vector genome comprising, consisting essentially of, or consisting of a polynucleotide sequence encoding an α-sarcoglycan (SGCA) protein, wherein the first sarcoglycan is selected from γ-sarcoglycan (SGCG), β-sarcoglycan (SGCB), and δ-sarcoglycan (SGCD). In some embodiments, the viral vector genome comprises, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:47 or 48 over the entire length of SEQ ID NO:47 or 48.

[0177] Sarcoglycan complex and DAPC

[0178] Proteins called sarcoglycan and sarcospan, together with dystrophin, are essential proteins that stabilize DAPC and provide mechanical support to muscle cell membranes.Autosomal recessive mutations in sarcoglycan lead to protein deficiency, loss of sarcoglycan complex formation, and loss of stabilization of dystrophin-associated protein complex (DAPC).Disclosed herein are methods for using any of the polynucleotides, expression cassettes, viral vectors, and compositions disclosed herein to repair sarcoglycan complexes, restore or increase sarcoglycan expression, restore or increase dystrophin expression, restore or increase sarcospan expression, stabilize DAPC or muscle cell membrane, or restore or increase the function of DAPC or muscle cell membrane.Methods for determining such things are known in the art.

[0179] Sarcoglycan

[0180] Sarcoglycans are transmembrane proteins found in plasma membrane-associated complexes known as sarcoglycan complexes, which are subcomplexes of DAPC. The exact function of the sarcoglycan complex is unknown, but it may have both mechanical and non-mechanical roles in stabilizing the plasma membrane of cardiac and skeletal muscle. Variants of the sarcoglycan complex are found in vascular smooth muscle and some non-muscle cells and tissue types. Thus, the sarcoglycan complex plays an important role in both muscle and non-muscle tissues.

[0181] Examples of sarcoglycans include, but are not limited to, α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG), δ-sarcoglycan (SGCD), ε-sarcoglycan (SGCE), and ζ-sarcoglycan (SGCZ). Autosomal recessive mutations in several sarcoglycan genes, α, β, γ, and δ, cause disruption of the sarcoglycan complex, resulting in sarcoglycanopathy such as limb-girdle muscular dystrophy type 2 (LGMD2). For example, mutations in SGCG can result in LGMD2C, mutations in SGCA can result in LGMD2D, mutations in SGCB can result in LGMD2E, and mutations in SGCD can result in LGMD2F.

[0182] In some embodiments, the methods disclosed herein comprise, consist essentially of, or further consist of administering a polynucleotide encoding SGCA to a subject suffering from muscular dystrophy. In some embodiments, the methods disclosed herein comprise, consist essentially of, or further consist of administering a polynucleotide encoding an SGCA protein to a subject suffering from LGMD2D. In some embodiments, the sarcoglycan is SGCA. Polynucleotides encoding SGCA proteins, for example, polynucleotides encoding the amino acid sequences described below, are known in the art. For example, the polynucleotide encoding the SGCA protein comprises, consists essentially of, or further consists of the SGCA nucleotide sequence disclosed in International Application No. PCT / US2020 / 47339 (corresponding to SEQ ID NO: 45), GenBank Accession No. NM_000023.4 (corresponding to SEQ ID NO: 13), or GenBank Accession No. NM_001135697.3 (corresponding to SEQ ID NO: 14), each of which is incorporated by reference in its entirety. In some embodiments, the polynucleotide encoding the SGCA protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence encoding SGCA disclosed in International Application No. PCT / US2020 / 47339 (corresponding to SEQ ID NO: 45), GenBank Accession No. NM_000023.4 (corresponding to SEQ ID NO: 13), or GenBank Accession No. NM_001135697.3 (corresponding to SEQ ID NO: 14) over the entire length of the nucleotide sequence encoding SGCA. In some embodiments, the polynucleotide encoding the SGCA protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45.In some embodiments, a polynucleotide encoding an SGCA protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45. In some embodiments, a polynucleotide encoding an SGCA protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45. In some embodiments, a polynucleotide encoding an SGCA protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45. In some embodiments, the polynucleotide encoding the SGCA protein comprises, consists essentially of, or further consists of a nucleotide sequence that is 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 13, 14, and 45 over the entire length of SEQ ID NOs: 13, 14, and 45. SGCA protein sequences are known in the art. For example, the SGCA protein comprises, consists essentially of, or further consists of the SGCA amino acid sequence disclosed in International Application No. PCT / US2020 / 47339 (corresponding to SEQ ID NO: 46), GenBank Accession No. NP_000014.1 (corresponding to SEQ ID NO: 15), or NCBI Reference Sequence NP_0011292169.1 (corresponding to SEQ ID NO: 16), each of which is incorporated by reference in its entirety.In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the SGCA amino acid sequence disclosed in International Application No. PCT / US2020 / 47339 (corresponding to SEQ ID NO: 46), GenBank Accession No. NP_000014.1 (corresponding to SEQ ID NO: 15), or NCBI Reference Sequence NP_0011292169.1 (corresponding to SEQ ID NO: 16) over the entire length of the SGCA amino acid sequence. In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 80% identical to the SGCA amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 85% identical to the SGCA amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 90% identical to the SGCA amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 95% identical to the SGCA amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the SGCA protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 100% identical to the SGCA amino acid sequence of any one of SEQ ID NOs: 15, 16, and 46 over the entire length of SEQ ID NOs: 15, 16, and 46. In some embodiments, the polynucleotide encoding the SGCA protein is codon-optimized.

[0183] Disclosed herein is a method for increasing or restoring the expression of SGCA in a subject in need thereof, for example, a subject suffering from muscular dystrophy. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein.

[0184] In some embodiments, the expression level of SGCA is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCA before administration of the first dose of a polynucleotide encoding a truncated version of SGCG, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCA is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCA before administration of one or more subsequent doses of a polynucleotide encoding a truncated version of an SGCG, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCA is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCA in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene (e.g., SGCD, SGCB, or SGCG). In some embodiments, the expression level of SGCA is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the expression level of SGCA in reference sample, wherein the reference sample is derived from one or more healthy subjects (for example, the subjects who are not affected by the muscular dystrophy caused by the mutation in dystrophin or sarcoglycan gene).The expression level of SGCA can be detected by any known method for detecting or quantifying the level of protein or mRNA.For example, such method may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR). In some embodiments, PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, SGCA expression is detected in a sample from a subject. In some embodiments, the sample comprises, or essentially consists of, or also consists of skeletal muscle cells or cardiac muscle cells.

[0185] Disclosed herein is a method for localizing SGCA to the cell membrane of a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCG protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCD protein.In some embodiments, the muscular dystrophy is DMD or Becker muscular dystrophy (BMD), and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.In some embodiments, the cell membrane is a muscle cell membrane or a muscle cell membrane. In some embodiments, the cell membrane is a skeletal muscle cell membrane or a sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or a sarcolemma.

[0186] In some embodiments, the methods disclosed herein comprise, or consist essentially of, or further consist of administering a polynucleotide encoding SGCB to a subject suffering from muscular dystrophy. In some embodiments, the methods disclosed herein comprise, or consist essentially of, or further consist of administering a polynucleotide encoding an SGCB protein to a subject suffering from LGMD2E. In some embodiments, the sarcoglycan is SGCB. Polynucleotides encoding SGCB proteins are known in the art. For example, the polynucleotide encoding the SGCB protein comprises, consists essentially of, or further consists of the SGCB nucleotide sequence disclosed in International Application No. PCT / US2020 / 19892 (corresponding to SEQ ID NO: 1) or GenBank Accession No. NM_000232.5 (corresponding to SEQ ID NO: 17), each of which is incorporated by reference in its entirety. In some embodiments, the polynucleotide encoding the SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence encoding SGCB disclosed in International Application No. PCT / US2020 / 19892 (corresponding to SEQ ID NO: 1) or GenBank Accession No. NM_000232.5 (corresponding to SEQ ID NO: 17) over the entire length of the nucleotide sequence encoding SGCB. In some embodiments, the polynucleotide encoding the SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17. In some embodiments, the polynucleotide encoding the SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17.In some embodiments, a polynucleotide encoding an SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17. In some embodiments, a polynucleotide encoding an SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17. In some embodiments, a polynucleotide encoding an SGCB protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 100% identical to the nucleotide sequence of SEQ ID NO: 1 or 17 over the entire length of SEQ ID NO: 1 or 17. SGCB protein sequences are known in the art. For example, the SGCB protein may comprise, consist essentially of, or even consist of the SGCB amino acid sequence disclosed in International Application No. PCT / US2020 / 19892 (corresponding to SEQ ID NO:2) or GenBank Accession No. NP_000223.1 (corresponding to SEQ ID NO:18), each of which is incorporated by reference in its entirety. In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of the SGCB amino acid sequence to the SGCB amino acid sequence disclosed in International Application No. PCT / US2020 / 19892 (corresponding to SEQ ID NO:2) or GenBank Accession No. NP_000223.1 (corresponding to SEQ ID NO:18). In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 2 or 18 over the entire length of SEQ ID NO: 2 or 18.In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:2 or 18 over the entire length of SEQ ID NO:2 or 18. In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:2 or 18 over the entire length of SEQ ID NO:2 or 18. In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:2 or 18 over the entire length of SEQ ID NO:2 or 18. In some embodiments, the SGCB protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 100% identical to the amino acid sequence of SEQ ID NO:2 or 18 over the entire length of SEQ ID NO:2 or 18. In some embodiments, the polynucleotide encoding the SGCB protein is codon-optimized.

[0187] Disclosed herein is a method for increasing or restoring the expression of SGCB in a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCG protein.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCA protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCD protein.In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein.

[0188] In some embodiments, the expression level of SGCB is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCB before administration of the first dose of a polynucleotide encoding a truncated version of SGCA, SGCG, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCB is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCB before administration of one or more subsequent doses of a polynucleotide encoding a truncated version of an SGCA, SGCG, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCB is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCB in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene (e.g., SGCA, SGCG, or SGCD). In some embodiments, the expression level of SGCB is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the expression level of SGCB in reference sample, wherein the reference sample is derived from one or more healthy subjects (for example, subjects who do not suffer from muscular dystrophy caused by mutations in dystrophin or sarcoglycan genes).The expression level of SGCB can be detected by any known method for detecting or quantifying protein or mRNA levels.For example, such methods may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR). In some embodiments, PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, SGCB expression is detected in a sample from a subject. In some embodiments, the sample comprises, essentially consists of, or even consists of skeletal muscle cells or cardiac muscle cells.

[0189] Disclosed herein is a method for localizing SGCB to the cell membrane of a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCG protein.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCD protein.In some embodiments, the muscular dystrophy is DMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.In some embodiments, the cell membrane is a muscle cell membrane or muscle cell membrane. In some embodiments, the cell membrane is a skeletal muscle cell membrane or a sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or a sarcolemma.

[0190] In some embodiments, the methods disclosed herein comprise, or consist essentially of, or even consist of administering a polynucleotide encoding SGCG to a subject suffering from muscular dystrophy. In some embodiments, the methods disclosed herein comprise, or consist essentially of, or even consist of administering a polynucleotide encoding an SGCG protein to a subject suffering from LGMD2C. In some embodiments, the sarcoglycan is SGCG. Polynucleotides encoding SGCG proteins are known in the art. For example, a polynucleotide encoding an SGCG protein may comprise, consist essentially of, or even consist of the SGCG nucleotide sequence disclosed in International Application No. PCT / US2019 / 015779 (corresponding to SEQ ID NO:20), GenBank Accession No. NM_000231.3 (corresponding to SEQ ID NO:21), GenBank Accession No. NM_001378244.1 (corresponding to SEQ ID NO:22), GenBank Accession No. NM_001378245.1 (corresponding to SEQ ID NO:23), or GenBank Accession No. NM_001378246.1 (corresponding to SEQ ID NO:24), each of which is incorporated by reference in its entirety. In some embodiments, the polynucleotide encoding the SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence encoding the SGCG disclosed in International Application No. PCT / US2019 / 015779 (corresponding to SEQ ID NO:20), GenBank Accession No. NM_000231.3 (corresponding to SEQ ID NO:21), GenBank Accession No. NM_001378244.1 (corresponding to SEQ ID NO:22), GenBank Accession No. NM_001378245.1 (corresponding to SEQ ID NO:23), or GenBank Accession No. NM_001378246.1 (corresponding to SEQ ID NO:24) over the entire length of the nucleotide sequence encoding the SGCG.In some embodiments, a polynucleotide encoding an SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any one of SEQ ID NOs:20-24 over the entire length of SEQ ID NOs:20-24. In some embodiments, a polynucleotide encoding an SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any one of SEQ ID NOs:20-24 over the entire length of SEQ ID NOs:20-24. In some embodiments, a polynucleotide encoding an SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of any one of SEQ ID NOs:20-24 over the entire length of SEQ ID NOs:20-24. In some embodiments, a polynucleotide encoding an SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of any one of SEQ ID NOs:20-24 over the entire length of SEQ ID NOs:20-24. In some embodiments, a polynucleotide encoding an SGCG protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 20-24 over the entire length of SEQ ID NOs: 20-24. SGCG protein sequences are known in the art.For example, the SGCG protein may comprise, consist essentially of, or even further consist of the SGCG amino acid sequence disclosed in International Application No. PCT / US2019 / 015779 (corresponding to SEQ ID NO:25), GenBank Accession No. NP_000222.2 (corresponding to SEQ ID NO:26), GenBank Accession No. NP_001365173.1 (corresponding to SEQ ID NO:27), GenBank Accession No. NP_001365174.1 (corresponding to SEQ ID NO:28), or GenBank Accession No. NP_001365175.1 (corresponding to SEQ ID NO:29), each of which is incorporated by reference in its entirety. In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of the SGCG amino acid sequence to the SGCG amino acid sequence disclosed in International Application No. PCT / US2019 / 015779 (corresponding to SEQ ID NO:25), GenBank Accession No. NP_000222.2 (corresponding to SEQ ID NO:26), GenBank Accession No. NP_001365173.1 (corresponding to SEQ ID NO:27), GenBank Accession No. NP_001365174.1 (corresponding to SEQ ID NO:28), or GenBank Accession No. NP_001365175.1 (corresponding to SEQ ID NO:29). In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29.In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the SGCG protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 100% identical to the amino acid sequence of any one of SEQ ID NOs: 25-29 over the entire length of SEQ ID NOs: 25-29. In some embodiments, the polynucleotide encoding the SGCG protein is codon-optimized.

[0191] Disclosed herein is a method for increasing or restoring the expression of SGCG in a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCD protein.In some embodiments, the muscular dystrophy is DMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.

[0192] In some embodiments, the expression level of SGCG is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCG before administration of the first dose of a polynucleotide encoding a truncated version of SGCA, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCG is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCG before administration of one or more subsequent doses of a polynucleotide encoding a truncated version of an SGCA, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of SGCG is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCG in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene (e.g., SGCA, SGCB, or SGCD). In some embodiments, the expression level of SGCG is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the expression level of SGCG in reference sample, wherein the reference sample is derived from one or more healthy subjects (for example, subjects who do not suffer from muscular dystrophy caused by mutation in dystrophin or sarcoglycan gene).The expression level of SGCG can be detected by any known method for detecting or quantifying the level of protein or mRNA.For example, such methods may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR). In some embodiments, PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, SGCG expression is detected in a sample from a subject. In some embodiments, the sample comprises, essentially consists of, or even consists of skeletal muscle cells or cardiac muscle cells.

[0193] Disclosed herein is a method for localizing SGCG to the cell membrane of a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCD protein.In some embodiments, the muscular dystrophy is DMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.In some embodiments, the cell membrane is a muscle cell membrane or muscle cell membrane. In some embodiments, the cell membrane is a skeletal muscle cell membrane or a sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or a sarcolemma.

[0194] In some embodiments, the methods disclosed herein comprise, or consist essentially of, or further consist of administering a polynucleotide encoding SGCD to a subject suffering from muscular dystrophy. In some embodiments, the methods disclosed herein comprise, or consist essentially of, or further consist of administering a polynucleotide encoding an SGCD protein to a subject suffering from LGMD2F. In some embodiments, the sarcoglycan is SGCD. Polynucleotides encoding SGCD proteins are known in the art. For example, the polynucleotide encoding the SGCD protein may comprise, essentially consist of, or further consist of GenBank Accession No. NM_000337.5 (corresponding to SEQ ID NO: 30), GenBank Accession No. NM_001128209.2 (corresponding to SEQ ID NO: 31), or GenBank Accession No. NM_172244.3 (corresponding to SEQ ID NO: 32), each of which is incorporated by reference in its entirety. In some embodiments, a polynucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence encoding an SGCD disclosed in GenBank Accession No. NM_000337.5 (corresponding to SEQ ID NO: 30), GenBank Accession No. NM_001128209.2 (corresponding to SEQ ID NO: 31), or GenBank Accession No. NM_172244.3 (corresponding to SEQ ID NO: 32) over the entire length of the nucleotide sequence encoding the SGCD. In some embodiments, a polynucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32.In some embodiments, a polynucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32. In some embodiments, a polynucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32. In some embodiments, a polynucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32. In some embodiments, a nucleotide encoding an SGCD protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-32 over the entire length of SEQ ID NOs: 30-32. SGCD protein sequences are known in the art. For example, the SGCD protein may comprise, consist essentially of, or even consist of the SGCD amino acid sequence disclosed in GenBank Accession No. NP_000328.2 (corresponding to SEQ ID NO: 33), GenBank Accession No. NP_001121681.1 (corresponding to SEQ ID NO: 34), or GenBank Accession No. NP_758447.1 (corresponding to SEQ ID NO: 35), each of which is incorporated by reference in its entirety.In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the SGCD amino acid sequence disclosed in GenBank Accession No. NP_000328.2 (corresponding to SEQ ID NO: 33), GenBank Accession No. NP_001121681.1 (corresponding to SEQ ID NO: 34), or GenBank Accession No. NP_758447.1 (corresponding to SEQ ID NO: 35) over the entire length of the SGCD amino acid sequence. In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the SGCD protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33-35 over the entire length of SEQ ID NOs: 33-35. In some embodiments, the polynucleotide encoding the SGCD protein is codon-optimized.

[0195] Disclosed herein is a method for increasing or restoring the expression of SGCD in a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCG protein.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.

[0196] In some embodiments, the expression level of SGCD is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCD before administration of the first dose of a polynucleotide encoding a truncated version of SGCA, SGCG, SGCB, or dystrophin protein. In some embodiments, the expression level of SGCD is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCD before administration of one or more subsequent doses of a polynucleotide encoding a truncated version of SGCA, SGCG, SGCB, or dystrophin protein. In some embodiments, the expression level of SGCD is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of SGCD in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene (e.g., SGCA, SGCB, or SGCG). In some embodiments, the expression level of SGCD is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the expression level of SGCD in reference sample, wherein the reference sample is derived from one or more healthy subjects (for example, subjects who do not suffer from muscular dystrophy caused by mutation in dystrophin or sarcoglycan gene).The expression level of SGCD can be detected by any known method for detecting or quantifying the level of protein or mRNA.For example, such methods may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR). In some embodiments, PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, SGCD expression is detected in a sample from a subject. In some embodiments, the sample comprises, essentially consists of, or even consists of skeletal muscle cells or cardiac muscle cells. Disclosed herein is a method for localizing SGCD to the cell membrane of a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCG protein.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding dystrophin protein or a truncated version of dystrophin protein.In some embodiments, the cell membrane is a muscle cell membrane or muscle cell membrane. In some embodiments, the cell membrane is a skeletal muscle cell membrane or a sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or a sarcolemma.

[0197] Sarcospan

[0198] Sarcospan is a component of DAPC. Sarcospan expression can be reduced or lost in subjects suffering from muscular dystrophy. Disclosed herein are methods for increasing or restoring sarcospan expression in a subject in need thereof, for example, a subject suffering from muscular dystrophy. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, consists essentially of, or further consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, consists essentially of, or even further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein.

[0199] In some embodiments, the expression level of sarcospan is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcospan before administration of the first dose of a polynucleotide encoding a truncated version of SGCA, SGCG, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of sarcospan is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcospan before administration of one or more subsequent doses of a polynucleotide encoding a truncated version of SGCA, SGCG, SGCB, SGCD, or dystrophin protein. In some embodiments, the expression level of sarcospan is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcospan in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene. In some embodiments, the expression level of sarcospan is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the expression level of sarcospan in a reference sample, wherein the reference sample is derived from one or more healthy subjects (e.g., subjects not suffering from muscular dystrophy caused by mutations in the dystrophin or sarcoglycan gene). The expression level of sarcospan can be detected by any known method for detecting or quantifying protein or mRNA levels.For example, such methods may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR). In some embodiments, the PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, sarcospan expression is detected in a sample from a subject. In some embodiments, the sample comprises, consists essentially of, or even consists of skeletal muscle cells or cardiac muscle cells.

[0200] Disclosed herein is a method for localizing sarcospan to the cell membrane of a subject in need thereof, for example, a subject suffering from muscular dystrophy. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or consists essentially of, or further consists of, or consists essentially of, or further consists of, a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein to the subject. In some embodiments, the cell membrane is a muscle cell membrane or sarcolemma. In some embodiments, the cell membrane is a skeletal muscle cell membrane or sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or sarcolemma.

[0201] dystrophin

[0202] In some embodiments, the method disclosed herein comprises, or essentially consists of, or further consists of administering a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein to a subject in need thereof, for example, a subject suffering from muscular dystrophy. In some embodiments, the method disclosed herein comprises, or essentially consists of, or further consists of administering a polynucleotide encoding a dystrophin protein or a truncated version of a dystrophin protein to a subject suffering from DMD or BMD. Polynucleotides encoding dystrophin proteins are known in the art. For example, the polynucleotide encoding dystrophin comprises, or essentially consists of, or further consists of the nucleotide sequence disclosed in GenBank Accession No. AH003182.2, the entirety of which is incorporated by reference. In some embodiments, a polynucleotide encoding a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of GenBank Accession No. AH003182.2 (corresponding to SEQ ID NO: 36) over the entire length of SEQ ID NO: 36. In some embodiments, a polynucleotide encoding a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 36 over the entire length of SEQ ID NO: 36. In some embodiments, a polynucleotide encoding a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 36 over the entire length of SEQ ID NO: 36.In some embodiments, the polynucleotide encoding the dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 36 over the entire length of SEQ ID NO: 36. In some embodiments, the polynucleotide encoding the dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 36 over the entire length of SEQ ID NO: 36. In some embodiments, the polynucleotide encoding the dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 100% identical to the nucleotide sequence of SEQ ID NO: 36 over the entire length of SEQ ID NO: 36.

[0203] The sequence of dystrophin proteins is known in the art. For example, dystrophin proteins may comprise, essentially consist of, or even further consist of the amino acid sequence of GenBank Accession No. AAA74506.1 (corresponding to SEQ ID NO:37), the entirety of which is incorporated by reference. In some embodiments, dystrophin proteins comprise, essentially consist of, or even further consist of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of GenBank Accession No. AAA74506.1 (corresponding to SEQ ID NO:37) over the entire length of SEQ ID NO:37. In some embodiments, dystrophin proteins comprise, essentially consist of, or even further consist of an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:37 over the entire length of SEQ ID NO:37. In some embodiments, the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 37 over the entire length of SEQ ID NO: 37. In some embodiments, the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 37 over the entire length of SEQ ID NO: 37. In some embodiments, the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 37 over the entire length of SEQ ID NO: 37. In some embodiments, the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 100% identical to the amino acid sequence of SEQ ID NO: 37 over the entire length of SEQ ID NO: 37. In some embodiments, the polynucleotide encoding the dystrophin protein is codon-optimized. Polynucleotides encoding truncated versions of dystrophin proteins are known in the art.For example, a polynucleotide encoding a truncated version of a dystrophin protein may comprise, consist essentially of, or alternatively consist of the micro- or mini-dystrophin gene disclosed in Fabb et al., Hum Mol Genet, 11(7):733-741, 2002; Gregorevic et al., Mol Ther, 16(4):657-64, 2008; Zhang and Duan, Hum Gene Ther, 23(1):98-103, 2012; or Decrouy et al., Gene Ther, 5(1):59-64, 1998, which are incorporated by reference in their entireties. In some embodiments, the polynucleotide encoding the truncated version of the dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a micro- or mini-dystrophin gene disclosed in Fabb et al., Hum Mol Genet, 11(7):733-741, 2002; Gregorevic et al., Mol Ther, 16(4):657-64, 2008; Zhang and Duan, Hum Gene Ther, 23(1):98-103, 2012; or Decrouy et al., Gene Ther, 5(1):59-64, 1998. In some embodiments, the polynucleotide encoding the truncated version of the dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44.In some embodiments, a polynucleotide encoding a truncated version of a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44. In some embodiments, a polynucleotide encoding a truncated version of a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44. In some embodiments, a polynucleotide encoding a truncated version of a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44. In some embodiments, a polynucleotide encoding a truncated version of a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44. In some embodiments, a polynucleotide encoding a truncated version of a dystrophin protein comprises, consists essentially of, or even consists of a nucleotide sequence that is at least about 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 38 and 40-44 over the entire length of SEQ ID NOs: 38 and 40-44. Sequences of micro-dystrophin and mini-dystrophin proteins are known in the art. For example, sequences of micro-dystrophin or mini-dystrophin proteins are described in Fabb et al. al., Hum Mol Genet, 11(7):733-741, 2002, Gregorevich et al., Mol Ther, 16(4):657-64, 2008, Zhang and Duan, Hum Gene Ther, 23(1):98-103, 2012 or the amino acid sequences disclosed in Decrouy et al., Gene Ther, 5(1):59-64, 1998. In some embodiments, the truncated version of a dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a micro- or mini-dystrophin disclosed in Fabb et al., Hum Mol Genet, 11(7):733-741, 2002; Gregorevic et al., Mol Ther, 16(4):657-64, 2008; Zhang and Duan, Hum Gene Ther, 23(1):98-103, 2012; or Decrouy et al., Gene Ther, 5(1):59-64, 1998. In some embodiments, truncated versions of dystrophin proteins comprise, consist essentially of, or even consist of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, truncated versions of dystrophin proteins comprise, consist essentially of, or even consist of an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, truncated versions of dystrophin proteins comprise, consist essentially of, or even consist of an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, truncated versions of dystrophin proteins comprise, consist essentially of, or even consist of an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39.In some embodiments, the truncated version of the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the truncated version of the dystrophin protein comprises, consists essentially of, or even consists of an amino acid sequence that is at least about 100% identical to the amino acid sequence of SEQ ID NO: 39 over the entire length of SEQ ID NO: 39. In some embodiments, the polynucleotide encoding the truncated version of the dystrophin protein is codon-optimized.

[0204] Disclosed herein is a method for increasing or restoring dystrophin expression in a subject in need thereof, for example, a subject suffering from muscular dystrophy.In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCG protein.In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCA protein.In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCB protein.In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding SGCD protein.

[0205] In some embodiments, the level of dystrophin expression is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the level of dystrophin expression before administration of the first dose of a polynucleotide encoding an SGCA, SGCG, SGCB, or SGCD. In some embodiments, the level of dystrophin expression is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the level of dystrophin expression before administration of one or more subsequent doses of a polynucleotide encoding an SGCA, SGCG, SGCB, or SGCD. In some embodiments, the expression level of dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of dystrophin in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a sarcoglycan gene (e.g., SGCA, SGCB, SGCG, or SGCD). In some embodiments, the expression level of dystrophin is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the expression level of dystrophin in reference sample, wherein the reference sample is derived from one or more healthy subjects (for example, subjects who do not suffer from muscular dystrophy caused by mutation in sarcoglycan gene).The expression level of dystrophin can be detected by any known method for detecting or quantifying the level of protein or mRNA.For example, such method may include immunofluorescence staining, Western blot, or polymerase chain reaction (PCR).In some embodiments, PCR is quantitative reverse transcription PCR (qRT-PCR).In some embodiments, dystrophin expression is detected in a sample from a subject.In some embodiments, the sample comprises, or essentially consists of, or also consists of skeletal muscle cells or cardiac muscle cells. Disclosed herein is a method for localizing dystrophin to the cell membrane of a subject suffering from muscular dystrophy. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the cell membrane is a muscle cell membrane or sarcolemma. In some embodiments, the cell membrane is a skeletal muscle cell membrane or sarcolemma. In some embodiments, the cell membrane is a cardiac muscle cell membrane or a sarcolemma.

[0206] Sarcoglycan complex

[0207] In some embodiments, the method for repairing sarcoglycan complexes in a subject suffering from muscular dystrophy comprises, consists of, or even essentially consists of administering to the subject a polynucleotide encoding a truncated version of a sarcoglycan protein or a dystrophin protein. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of the dystrophin protein. In some embodiments, the method further comprises, or essentially consists of, or further consists of detecting the expression level of at least one protein selected from sarcoglycan, dystrophin, and sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG).In some embodiments, the detecting comprises, consists essentially of, or further consists of at least one of immunofluorescence staining, Western blotting, or polymerase chain reaction (PCR). In some embodiments, the PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, the expression level of sarcoglycan, dystrophin, or sarcospan is determined from a sample derived from the subject. In some embodiments, the sample comprises, consists essentially of, or further consists of skeletal muscle cells or cardiac muscle cells. In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in the dystrophin or sarcoglycan gene.In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin before administration of a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene. In some embodiments, the sarcoglycan complex is restored when the expression level of sarcoglycan, sarcospan, or dystrophin is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more healthy subjects (e.g., subjects not suffering from muscular dystrophy caused by a mutation in the dystrophin or sarcoglycan gene).

[0208] In some embodiments, a method for restoring or stabilizing dystrophin-associated protein complexes (DAPCs) in a subject suffering from muscular dystrophy comprises, consists of, or even essentially consists of administering to the subject a polynucleotide encoding a truncated version of a sarcoglycan protein or a dystrophin protein. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of the dystrophin protein. In some embodiments, the method further comprises, or essentially consists of, or further consists of detecting the expression level of at least one protein selected from sarcoglycan, dystrophin, and sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG).In some embodiments, the detecting comprises, consists essentially of, or further consists of at least one of immunofluorescence staining, Western blotting, or polymerase chain reaction (PCR). In some embodiments, the PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, the expression level of sarcoglycan, dystrophin, or sarcospan is determined from a sample derived from the subject. In some embodiments, the sample comprises, consists essentially of, or further consists of skeletal muscle cells or cardiac muscle cells. In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in the dystrophin or sarcoglycan gene.In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin before administration of a polynucleotide encoding a truncated version of a sarcoglycan protein or dystrophin protein. In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene. In some embodiments, DAPCs are restored or stabilized when the expression level of sarcoglycan, sarcospan, or dystrophin is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more healthy subjects (e.g., subjects not suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene).

[0209] In some embodiments, a method for restoring or increasing the function of the dystrophin-associated protein complex (DAPC) in a subject suffering from muscular dystrophy comprises, consists of, or even essentially consists of administering to the subject a polynucleotide encoding a truncated version of a sarcoglycan protein or a dystrophin protein. In some embodiments, the muscular dystrophy is LGMD2C, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCG protein. In some embodiments, the muscular dystrophy is LGMD2D, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCA protein. In some embodiments, the muscular dystrophy is LGMD2E, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCB protein. In some embodiments, the muscular dystrophy is LGMD2F, and the method comprises, consists essentially of, or even consists of administering to the subject a polynucleotide encoding an SGCD protein. In some embodiments, the muscular dystrophy is DMD or BMD, and the method comprises, or essentially consists of, or further consists of administering to the subject a polynucleotide encoding a dystrophin protein or a truncated version of the dystrophin protein. In some embodiments, the method further comprises, or essentially consists of, or further consists of detecting the expression level of at least one protein selected from sarcoglycan, dystrophin, and sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG).In some embodiments, the detecting comprises, consists essentially of, or further consists of at least one of immunofluorescence staining, Western blotting, or polymerase chain reaction (PCR). In some embodiments, the PCR is quantitative reverse transcription PCR (qRT-PCR). In some embodiments, the expression level of sarcoglycan, dystrophin, or sarcospan is determined from a sample derived from the subject. In some embodiments, the sample comprises, consists essentially of, or further consists of skeletal muscle cells or cardiac muscle cells. In some embodiments, the function of DAPCs is increased or restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, the function of DAPCs is increased or restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan before administering a polynucleotide encoding a truncated version of the sarcoglycan protein or dystrophin protein. In some embodiments, the function of DAPCs is increased or restored when the expression level of sarcoglycan, dystrophin, or sarcospan protein is increased compared to the expression level of sarcoglycan, dystrophin, or sarcospan in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in the dystrophin or sarcoglycan gene.In some embodiments, the function of the DAPCs is increased or restored when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin before administration of a polynucleotide encoding a truncated version of a sarcoglycan protein or dystrophin protein. In some embodiments, DAPC function is increased or restored when the expression level of sarcoglycan, sarcospan, or dystrophin is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more compared to the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more subjects suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene. In some embodiments, the function of DAPCs is increased or restored when the expression level of sarcoglycan, sarcospan, or dystrophin is at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the expression level of sarcoglycan, sarcospan, or dystrophin in a reference sample, wherein the reference sample is derived from one or more healthy subjects (e.g., subjects not suffering from muscular dystrophy caused by a mutation in a dystrophin or sarcoglycan gene).

[0210] Mode of Administration

[0211] In some embodiments, polynucleotides encoding truncated versions of sarcoglycan or dystrophin proteins are administered orally, parenterally (e.g., intramuscularly, intraperitoneally, intravenously, ICV, intracisternal injection or infusion, subcutaneous injection, or implantation), by inhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.). In some embodiments, the polynucleotides are administered intramuscularly or intravenously.

[0212] In some embodiments, polynucleotide is administered in nanoparticles, liposomes, or is packaged in viral vectors (i.e., viral vector particles) by encapsidation.Alternatively, or in addition, polynucleotide is contained in vectors, for example, plasmids or viral vectors.In some embodiments, viral vectors are retroviruses, adenoviruses, adeno-associated viruses (AAV), lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, poxviruses, picornaviruses, or herpes simplex virus vectors.In some embodiments, viral vectors are recombinant viral vectors.In some embodiments, viral vectors are recombinant AAV vectors.

[0213] In some embodiments, the polynucleotide is administered in an adeno-associated virus (AAV) expression cassette, an AAV genome, or an AAV vector. In some embodiments, the AAV is AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAVrh.10, AAV-11, AAV-12, or AAV-13. In some embodiments, the AAV is AAVrh.74. In some embodiments, the AAV genome is a single-stranded (ss) AAV genome. In some embodiments, the AAV genome is a self-complementary (sc) AAV genome. In some embodiments, the AAV is self-complementary AAVrh.74. In some embodiments, the AAV is a scAAVrh.74 vector containing an MHCK7 promoter. In some embodiments, the AAV genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48 over the entire length of SEQ ID NOs: 3, 5, 7, 8, 19, 47, and 48.

[0214] In another embodiment, the polynucleotide or recombinant AAV vector described herein may be operably linked to a muscle-specific regulatory element and / or enhancer element. For example, the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF) element, a muscle creatine kinase (MCK) promoter, a tMCK (truncated MCK) element, a myosin heavy chain (MHC) element, an MHCK7 (a hybrid version of MHC and MCK) promoter, a C5-12 (synthetic promoter), a mouse creatine kinase enhancer element, a fast-twitch skeletal troponin C gene element, a slow-twitch cardiac troponin C gene element, a slow-twitch troponin I gene element, a hypoxia-inducible nuclear factor element, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0215] In some embodiments, the muscle-specific promoter is the MHCK7 promoter. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 95% to the nucleotide sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. In some embodiments, the MHCK7 promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 100% to the nucleotide sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4.

[0216] In some embodiments, the muscle-specific promoter is a tMCK promoter. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 95% to the nucleotide sequence of SEQ ID NO: 6 over the entire length of SEQ ID NO: 6. In some embodiments, the tMCK promoter comprises, consists essentially of, or alternatively consists of a nucleotide sequence that is at least about 100% to the nucleotide sequence of SEQ ID NO: 6 over the entire length of SEQ ID NO: 6.

[0217] An exemplary rAAV described herein is pAAV.MHCK7.hSGCB, which comprises the nucleotide sequence of SEQ ID NO: 3. Within the nucleotide sequence of SEQ ID NO: 3, the MCHK7 promoter spans nucleotides 130-921, the SV40 chimeric intron (SEQ ID NO: 9) spans nucleotides 931-1078, the SGCB sequence (SEQ ID NO: 1) spans nucleotides 1091-2047, and polyA (SEQ ID NO: 10) spans nucleotides 2054-2106. In some embodiments, the rAAV pAAV.MHCK7.hSGCB comprises the nucleotide sequence of SEQ ID NO: 7. Within the nucleotide sequence of SEQ ID NO: 7, the MCHK7 promoter spans nucleotides 128-919, the SV40 chimeric intron spans nucleotides 929-1076, the SGCB sequence spans nucleotides 1086-2042, and polyA spans nucleotides 2049-2101.

[0218] In some embodiments, the rAAV is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:7. or a nucleotide sequence encoding a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:2. 1. An exemplary rAAV vector described herein is pAAV.MHCK7.hSGCG, which contains the nucleotide sequence of SEQ ID NO: 19; the MCHK7 promoter spans nucleotides 136-927, the intron spans nucleotides 937-1084, the SGCG sequence spans nucleotides 1094-1969, and polyA spans nucleotides 1976-2028. In some cases, the only viral sequences contained in the rAAV vector are inverted terminal repeats, which are required for viral DNA replication and packaging. In some embodiments, pAAV.MHCK7.hSGCG is packaged into AAV rh.74 capsids. In one embodiment, the AAV vector is administered to a subject at a dosage of 1.85e13vg / kg or 7.41e13vg / kg, where the dosage is quantified by linearized PCR standards.

[0219] In some embodiments, the rAAV comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence set forth in SEQ ID NO:19.

[0220] An exemplary rAAV is scAAVrh74.tMCK.hSGCA (SEQ ID NO:47). In some embodiments, the rAAV comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence set forth in SEQ ID NO:47.

[0221] In one embodiment, the AAV is the pAAV.tMCK.hSGCA.KAN plasmid (SEQ ID NO: 48). In another embodiment, the rAAV comprises a nucleotide sequence that is at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO: 48.

[0222] The titer of the AAV vector administered in the methods of the present invention will vary depending, for example, on the particular AAV, the mode of administration, the purpose of treatment, the individual, and the cell type being targeted, and can be determined by standard methods in the art. The titer of the AAV should be at least about 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 Dosages may range from 1 x 10 to 1 x 10 or more DNase-resistant particles (DRPs). Dosages may also be expressed in units of viral genomes (vg). For example, the dosage of AAV may range from at least about 1 x 10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 ~Approx. 1×10 14In some embodiments, the dosage may be expressed in units of viral genomes per kilogram of subject mass (vg / kg). For example, the dosage of AAV may range from about 1 x 10 6 ~1×10 16 vg / kg, approx. 1×10 8 ~1×10 15 vg / kg, approx. 1×10 10 ~1×10 14 vg / kg, approx. 1×10 12 ~1×10 14 vg / kg, approx. 1×10 13 ~1×10 14 vg / kg, or approximately 1.80 × 10 13 ~7.5×10 13 In another embodiment, the dosage is about at least 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 2×10 12 , about 4×10 12 , about 6×10 12 , about 8×10 12 , about 1×10 13 , about 2×10 13 , about 2.4×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 1×10 15 , or at least about 1 × 10 16 In one embodiment, the dosage is at least 2×10 12 , 4×10 12 , 6×10 12 , 8×10 12 , 1×10 13 , 2 × 10 13 , 2.4 × 10 13 , 3×1013 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , or 8 × 10 13 In some embodiments, the dosage is at least about 1.85×10 vg / kg. 13 In some embodiments, the dosage is at least about 7.41×10 vg / kg. 13 vg / kg. In some embodiments, the dosage is quantified by linearized PCR standards. Alternatively, in some embodiments, the dosage is quantified by supercoiled PCR standards.

[0223] A therapeutically effective amount of a rAAV vector is from about 1e13vg / kg to about 5e14vg / kg, or from about 1e13vg / kg to about 2e13vg / kg, or from about 1e13vg / kg to about 3e13vg / kg, or from about 1e13vg / kg to about 4e13vg / kg, or from about 1e13vg / kg to about 5e13vg / kg, or from about 1e13vg / kg to about 6e13vg / kg, or from about 1e13vg / kg to about 7e13vg / kg, or from about 1e13vg / kg to about 8e13vg / kg, or from about 1e13vg / kg to about 9e13vg / kg, or about 1e13vg / kg to about 1e13vg / kg. e13vg / kg to about 1e14vg / kg, or about 1e13vg / kg to about 2e14vg / kg, or 1e13vg / kg to about 3e14vg / kg, or about 1e13 to about 4e14vg / kg, or about 3e13vg / kg to about 4e13vg / kg, or about 3e13vg / kg to about 5e13vg / kg, or about 3e13vg / kg to about 6e13vg / kg, or about 3e13vg / kg to about 7e13vg / kg, or about 3e13vg / kg to about 8e13vg / kg, or about 3e13vg / kg to about 9e13vg / kg, or about 3e13vg / kg to about 1e14vg / kg, or about 3e13vg / kg to about 2e14vg / kg, or 3e13vg / kg to about 3e14vg / kg, or about 3e13 to about 4e14vg / kg, or about 3e13vg / kg to about 5e14vg / kg, or about 5e13vg / kg to about 6e13vg / kg, or about 5e13vg / kg to about 7e13vg / kg, or about 5e13vg / kg to about 8e13vg / kg, or about 5e13vg / kg to about 9e13vg / kg, or about 5e13vg / kg to about 1e14vg / kg, or The dose of rAAV ranges from about 5e13vg / kg to about 2e14vg / kg, or 5e13vg / kg to about 3e14vg / kg, or about 5e13 to about 4e14vg / kg, or about 5e13vg / kg to about 5e14vg / kg, or about 1e14vg / kg to about 2e14vg / kg, or 1e14vg / kg to about 3e14vg / kg, or about 1e14 to about 4e14vg / kg, or about 1e14vg / kg to about 5e14vg / kg, 6e14vg / kg, 7e14vg / kg, 8e14vg / kg, or 9e14vg / kg.The present invention also includes compositions comprising these ranges of rAAV vectors.

[0224] For example, the therapeutically effective amount of rAAV vector is 1e13vg / kg, about 2e13vg / kg, about 3e13vg / kg, about 4e13vg / kg, about 5e13vg / kg, about 6e13vg / kg, about 7e13vg / kg, about 7.4e13vg / kg, about 8e13vg / kg, about 9e13vg / kg, about 1e14vg / kg, about 2e14vg / kg, about 3e14vg / kg, about 4e14vg / kg and 5e14vg / kg. The titer or dosage of AAV vector can vary based on the physical form of plasmid DNA as a quantitative standard. For example, the titer or dosage value can vary based on supercoiled standard qPCR titration method or linear standard qPCR titration method. In one embodiment, the therapeutically effective amount of rAAV is a 5e13vg / kg dose based on a supercoiled plasmid as a quantitative standard or a 1.85e13vg / kg dose based on a linearized plasmid as a quantitative standard, hi another embodiment, the therapeutically effective amount of rAAV is a 2e14vg / kg dose based on a supercoiled plasmid as a quantitative standard or a 7.41e13vg / kg dose based on a linearized plasmid as a quantitative standard. In another embodiment, a therapeutically effective amount of scAAVrh74.MHCK7.hSGCB is from about 1e13 vg / kg to about 5e14 vg / kg, or from about 1e13 vg / kg to about 2e13 vg / kg, or from about 1e13 vg / kg to about 3e13 vg / kg, or from about 1e13 vg / kg to about 4e13 vg / kg, or from about 1e13 vg / kg to about 5e13 vg / kg, or from about 1e13 vg / kg to about 6e13 vg / kg, or from about 1e13 vg / kg to about 7e13 vg / kg, or 1e13vg / kg to about 8e13vg / kg, or about 1e13vg / kg to about 9e13vg / kg, or about 1e13vg / kg to about 1e14vg / kg, or about 1e13vg / kg to about 2e14vg / kg, or 1e13vg / kg to about 3e14vg / kg, or about 1e13 to about 4e14vg / kg, or about 3e13vg / kg to about 4e13vg / kg, or about 3e13vg / kg to about 5e13vg / kg, or about 3e13vg / kg to about 6e13vg / kg, or about 3e13vg / kg to about 7e13vg / kg,or about 3e13vg / kg to about 8e13vg / kg, or about 3e13vg / kg to about 9e13vg / kg, or about 3e13vg / kg to about 1e14vg / kg, or about 3e13vg / kg to about 2e14vg / kg, or 3e13vg / kg to about 3e14vg / kg, or about 3e13 to about 4e14vg / kg, or about 3e13vg / kg to about 5e14vg / kg, or about 5e13vg / kg to about 6e13vg / kg, or about 5e13vg / kg to about 7e13vg / kg, or about 5e13vg / kg to about 8e13vg / kg, or about 5e13vg / kg to about 9e13vg / kg g, or about 5e13 vg / kg to about 1e14 vg / kg, or about 5e13 vg / kg to about 2e14 vg / kg, or 5e13 vg / kg to about 3e14 vg / kg, or about 5e13 to about 4e14 vg / kg, or about 5e13 vg / kg to about 5e14 vg / kg, or about 1e14 vg / kg to about 2e14 vg / kg, or 1e14 vg / kg to about 3e14 vg / kg, or about 1e14 to about 4e14 vg / kg, or about 1e14 vg / kg to about 5e14 vg / kg, 6e14 vg / kg, 7e14 vg / kg, 8e14 vg / kg, or 9e14 vg / kg. The present invention also includes compositions comprising these doses of rAAV vector.

[0225] In some embodiments, the methods disclosed herein provide for the delivery of at least about 1 x 10 mAb in a total volume of 1.5 ml per injection. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13In some embodiments, the methods disclosed herein comprise, or consist essentially of, or even consist of administering at least about 1 x 10 vg. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 , about 2×10 13 , about 5×10 13 , about 7×10 13 , about 1×10 14 The method may comprise, consist essentially of, or even consist of administering a total daily dose of 0.05 mg of encapsidated vector genomes. One exemplary method for determining the titer of encapsidated vector genomes uses quantitative PCR, such as the method described in Pozsgai et al., Mol. Ther. 25(4): 855-869, 2017, which is incorporated by reference in its entirety.

[0226] In some embodiments, any of the methods disclosed herein comprises administering to a subject about 5.0 x 10 IgG based on a supercoiled plasmid as a quantitative standard. 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantitative standard. 13 vg / kg or 7.41 x 10 13 The method may comprise, consist essentially of, or alternatively further consist of administering an rAAV intravenous infusion at a dose of 100 mg / kg over approximately 1-2 hours.

[0227] In some embodiments, the dose and dosage of rAAV administered using the intravenous route is about 1.0 x 10 based on supercoiled plasmid as a quantitative standard. 13 vg / kg ~ approx. 5×10 14 or approximately 1.0 x 10 based on linearized plasmid as a quantitative standard 13 vg / kg ~ approx. 1.0×10 14 vg / kg.

[0228] Furthermore, the dose of rAAV administered is approximately 1.5 × 10 13 vg ~ approx. 2×10 16 vg, or 1.5 × 10 13 vg~1×10 16 vg, or approximately 1.5 × 10 13 vg ~ approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 15 vg. Furthermore, in any of the methods, compositions and uses, the dose of rAAV is administered at a concentration of about 10 mL / kg.

[0229] In some embodiments, any of the polynucleotides encoding truncated versions of sarcoglycan or dystrophin proteins is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day. In some embodiments, any of the polynucleotides or compositions disclosed herein is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times per week. In some embodiments, any of the polynucleotides or compositions disclosed herein is administered to a subject at least 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, or 31 times per month. In some embodiments, any of the polynucleotides or compositions disclosed herein are administered to a subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, any of the polynucleotides or compositions disclosed herein are administered to a subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks. In some embodiments, any of the polynucleotides or compositions disclosed herein are administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, any of the polynucleotides or compositions disclosed herein are administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks. In some embodiments, any of the polynucleotides or compositions disclosed herein is administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months.

[0230] In some embodiments, the methods disclosed herein comprise, consist essentially of, or even consist of systemic administration of any of the polynucleotides or compositions disclosed herein. For example, systemic administration is administered to the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration by injection, infusion, or implantation.

[0231] In some embodiments, the method disclosed herein comprises, or essentially consists of, or further consists of locally administering any of the polynucleotides or compositions disclosed herein.In some embodiments, the method disclosed herein comprises, or essentially consists of, or further consists of administering any of the polynucleotides or compositions disclosed herein to one or more tissues.In some embodiments, the tissue is selected from muscle tissue, epithelial tissue, connective tissue, and nervous tissue.In some embodiments, the tissue is muscle tissue.

[0232] Combination therapy is also contemplated by the present invention. Combination, as used herein, includes both simultaneous and sequential treatment. Combination of the methods of the present invention with standard medical treatments (e.g., corticosteroids) is specifically contemplated, as is combination with novel therapies.

[0233] In some embodiments, the methods disclosed herein further comprise, consist essentially of, or even consist of detecting the presence or absence of a mutation in a sarcoglycan gene or a dystrophin gene in a subject before or after administering any of the polynucleotides or compositions disclosed herein to the subject. In some embodiments, any of the polynucleotides or compositions disclosed herein is administered to a subject upon detecting the presence of a mutation in a sarcoglycan gene or a dystrophin gene.

[0234] In some embodiments, the methods disclosed herein further comprise, or consist essentially of, or even consist of detecting the level of sarcoglycan, sarcospan, or dystrophin protein in a subject before or after administering any of the polynucleotides or compositions disclosed herein to the subject. In some embodiments, the methods disclosed herein further comprise, or consist essentially of, or even consist of detecting the level of sarcoglycan, sarcospan, or dystrophin protein in a subject after administering any of the polynucleotides or compositions disclosed herein to the subject. In some embodiments, detecting the level of sarcoglycan, sarcospan, or dystrophin comprises, consists essentially of, or even consists of detecting the expression of a sarcoglycan, sarcospan, or dystrophin gene. Detecting the expression of a sarcoglycan, sarcospan, or dystrophin gene may comprise, consist essentially of, or even consist of quantifying the level of sarcoglycan, sarcospan, or dystrophin DNA or RNA. Alternatively, or in addition, detecting the level of sarcoglycan, sarcospan, or dystrophin protein comprises, consists essentially of, or even consists of quantifying the level of sarcoglycan, sarcospan, or dystrophin protein. In some embodiments, the level of sarcoglycan, sarcospan, or dystrophin protein is detected in a sample derived from a subject. In some embodiments, the sample is a body fluid sample. Examples of body fluid samples include, but are not limited to, blood, urine, sweat, saliva, stool, and synovial fluid. In some embodiments, the blood sample is a plasma or serum sample.

[0235] In some embodiments, the method disclosed herein further comprises, or consists essentially of, or further consists of modifying the dose or administration frequency of any of the polynucleotides or compositions administered to the subject.In some embodiments, modifying the dose or administration frequency is based on detecting the level of sarcoglycan, sarcospan, and / or sarcoglycan, sarcospan, or dystrophin protein.In some embodiments, the dose or administration frequency is reduced when the level of sarcoglycan, sarcospan, and / or sarcoglycan, sarcospan, or dystrophin protein in the subject increases compared to the level of sarcoglycan, sarcospan, and / or sarcoglycan, sarcospan, or dystrophin protein in the subject from an earlier time point (for example, before administering the polynucleotide or composition, or after administering the first dose of the polynucleotide or composition but before administering the next dose of the polynucleotide or composition).

[0236] Promoter, intron, polyA sequence, and inverted terminal repeats

[0237] In some embodiments, any of the polynucleotides, viral genomes, or expression cassettes disclosed herein comprise, consist essentially of, or even consist of (a) a first polynucleotide encoding a sarcoglycan, dystrophin, or a truncated version of dystrophin; and (b) one or more of a promoter, an intron, a polyA sequence, and an inverted terminal repeat sequence.

[0238] In some embodiments, the polynucleotide, viral genome, or expression cassette comprises, consists essentially of, or even consists of a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is selected from the group consisting of an MHCK7 promoter and a tMCK promoter. In some embodiments, the promoter comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:4 or 6 over the entire length of SEQ ID NO:4 or 6.

[0239] In some embodiments, the polynucleotide, viral genome, or expression cassette comprises, consists essentially of, or even consists of an intron. In some embodiments, the intron is an SV40 chimeric intron. In some embodiments, the intron comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9.

[0240] In some embodiments, the polynucleotide, viral genome, or expression cassette comprises, consists essentially of, or even consists of a polyA sequence. In some embodiments, the polyA sequence comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10.

[0241] In some embodiments, the polynucleotide, viral genome, or expression cassette comprises, consists essentially of, or even consists of an inverted terminal repeat (ITR). In some embodiments, the ITR comprises, consists essentially of, or even consists of a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11 or 12 over the entire length of SEQ ID NO: 11 or 12.

[0242] composition

[0243] In yet a further aspect, compositions are provided that comprise, alternatively consist essentially of, or even consist of one or more of the polynucleotides disclosed herein. In some embodiments, the compositions comprise, consist of, or even consist essentially of any of the polynucleotides disclosed herein. In some embodiments, the compositions comprise, consist of, or even consist essentially of one, two, three, four, or five or more polynucleotides encoding one, two, three, four, or five or more proteins selected from sarcoglycan, dystrophin, or sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG). In some embodiments, the compositions comprise, consist of, or even consist essentially of a polynucleotide encoding SGCB. In some embodiments, the composition comprises, consists of, or even further essentially consists of one, two, three, four, or five or more nanoparticles, liposomes, or viral vectors that comprise, consist essentially of, or even further consist of one, two, three, four, or five or more polynucleotides encoding one, two, three, four, or five or more proteins selected from sarcoglycan, dystrophin, or sarcospan.

[0244] In some embodiments, the composition provides one or more dosage units vg / kg as described above and incorporated herein by reference.

[0245] kit

[0246] In still further aspects, kits are provided that comprise, alternatively consist essentially of, or alternatively consist of, either one or more of the polynucleotides or compositions and instructions for use, hi one aspect, either one or more of the polynucleotides or compositions are detectably labeled, or further comprise, consist essentially of, or alternatively consist of, a purification or detectable marker.

[0247] In some embodiments, the kit comprises, consists of, or even consists essentially of any of the polynucleotides or compositions disclosed herein and instructions for in vitro or in vivo use. In some embodiments, the kit comprises, consists of, or even consists of one, two, three, four, or five or more polynucleotides encoding one, two, three, four, or five or more proteins selected from sarcoglycan, dystrophin, or sarcospan. In some embodiments, the kit comprises, consists of, or even consists of one, two, three, four, or five or more compositions comprising, consisting essentially of, or even consisting of one or more polynucleotides encoding one, two, three, four, or five or more proteins selected from sarcoglycan, dystrophin, or sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG). In some embodiments, the kit comprises, consists of, or even further essentially consists of a polynucleotide encoding SGCB or a composition comprising, consisting essentially of, or alternatively consisting of a polynucleotide encoding SGCB. In some embodiments, the kit comprises, consists of, or even further essentially consists of one, two, three, four, or five or more nanoparticles, liposomes, or viral vectors comprising, consisting essentially of, or alternatively consisting of one, two, three, four, or five or more proteins selected from sarcoglycan, dystrophin, or sarcospan. In some aspects, the composition provides one or more dosage units vg / kg as described above and incorporated herein by reference.

[0248] In some embodiments, the kit further comprises, consists essentially of, or even consists of instructions for detecting the expression level of at least one protein selected from sarcoglycan, dystrophin, and sarcospan. In some embodiments, the sarcoglycan is selected from α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), δ-sarcoglycan (SGCD), or γ-sarcoglycan (SGCG). [Example]

[0249] Example 1 SGCB gene transfer restores DAPCs

[0250] This example investigates whether DAPC function is restored in SGCB- / - mice after SGCB gene transfer, and whether the expression of other sarcoglycans and sarcospan can serve as surrogate markers for functional recovery of DAPCs. By characterizing SGCB+ / - mice, this example also demonstrates the existence of expression-function correlations for defining dose-response / expression level thresholds for clinical (functional) benefit. Specifically, the objectives were: (a) to evaluate the ability of the SGCB transgene to restore sarcoglycan and sarcospan expression in SGCB- / - mice; and (b) to test their usefulness as surrogate markers for DAPC recovery.

[0251] Limb-girdle muscular dystrophy type 2E (LGMD2E) is an autosomal recessive disease caused by mutations in β-sarcoglycan (SGCB) that result in protein deficiency, loss of sarcoglycan complex formation, and loss of stabilization of the dystrophin-associated protein complex (DAPC).

[0252] Individuals with a single pathogenic variant are asymptomatic (carriers) and thus can supplement with a copy of the defective gene.

[0253] Sarcoglycanopathies present as progressive muscular dystrophies that begin in the limb-girdle muscles before spreading to the lower and upper limb muscles, and can also involve the diaphragm and heart, leading to respiratory and cardiac failure in certain patient subtypes.

[0254] Sarcoglycans and sarcospans are integral proteins important for stabilizing the DAPC and providing mechanical support to the sarcolemma.

[0255] Adeno-associated virus (AAV)-mediated gene replacement therapy has shown early signs of potential for treating sarcoglycanopathies. Important considerations include planned and staged evaluation of safety, transduction, expression, localization, cellular effects, and clinical function.

[0256] Based on these findings, we designed a self-complementary (sc) AAV.MHCK7.hSGCB construct to restore functional β-sarcoglycan to muscle. The scAAV.MHCK7.hSGCB construct contains (a) the AAVrh74 vector, which exhibits strong muscle (skeletal and cardiac) tissue tropism and possesses relatively low levels of pre-existing immunity; (b) the MHCK7 promoter, which selectively regulates and drives transgene expression in skeletal and cardiac muscle and contains the alpha myosin heavy chain enhancer, driving particularly strong expression in cardiac muscle; and (c) an hSGCB transgene carrying the full-length β-sarcoglycan cDNA.

[0257] SGCB- / - mice were shown to concomitantly exhibit loss of additional sarcoglycans (α, γ, and δ), and evidence suggests that sarcospan may also be lost in the absence of SGCB.

[0258] method

[0259] The transcriptional and translational regulation of SGCB, along with functional outputs, was assessed in normal wild-type (WT) mice, heterozygous SGCB+ / - mice, and homozygous knockout (KO) SGCB- / - mice.

[0260] The transcript levels of SGCB in mouse skeletal muscle were measured using quantitative reverse transcription PCR (qRT-PCR).

[0261] Sarcoglycan and sarcospan protein expression in skeletal and cardiac muscles from naive and vector-treated SGCB− / − mice was assessed by immunofluorescence staining and Western blot.

[0262] Histological evaluation included hematoxylin-eosin staining of skeletal muscle (tibialis anterior [TA] and gastrocnemius [GAS]) and quantification of central nucleation.

[0263] Functional assessment included measurements of force production and resistance to contraction-induced injury in TA muscles, as well as laser monitoring of open-field cage activity to assess overall ambulation (movement around the cage) and rearing activity (hindlimb rearing).

[0264] Animal model: C57BL6 wild-type (WT), SGCB+ / - (SGCB het), and SGCB- / - (SGCB KO) mice were maintained under standardized conditions with food and water ad libitum and a 12:12-h light:dark cycle.

[0265] result

[0266] Expression-function correlation

[0267] SGCB+ / - mice were found to have a significant dystrophin phenotype compared to wild-type mice, as indicated by histology (Figure 1A) and quantification of central nucleation in the TA and GAS (Figure 1B). As shown in Figure 1B, the level of central nucleation for SGCB het mice is similar to WT and dramatically different from SGCB KO mice.

[0268] Sarcoglycan expression was determined at the transcriptional and protein levels in SGCB+ / -, SGCB- / -, and WT mice. Transcript mRNA levels were measured by qRT-PCR (Figure 2A), and protein production was measured by Western blot (Figure 2C) (immunofluorescence images are also shown in Figure 2B). SGCB expression is significantly reduced in SGCB- / - mice, as expected. SGCB mRNA levels are reduced in SGCB+ / - mice compared to WT mice, but no detectable difference in protein production is observed.

[0269] Analysis of functional output in SGCB het mice

[0270] Absolute force and resistance to eccentric contraction were similar in TA muscles of SGCB+ / - compared to WT mice and significantly different compared to SGCB- / - mice (Figures 3A-3B). Analysis of activity in an open field cage indicates that both gait and rearing activity were unaffected in SGCB+ / - mice compared to WT mice (Figures 4A-4B).

[0271] Restoration of DAPC

[0272] Dystrophin and SGCA expression was restored in both the TA and myocardium after aav.hSGCB gene transfer in SGCB- / - mice (Figures 5A-5C). As shown in Figure 5A, loss of SGCB resulted in reduced dystrophin in TA muscle. Restoration of SGCB protein levels in TA resulted in restoration of dystrophin in TA muscle. As shown in Figure 5B, loss of SGCB resulted in reduced SGCA in myocardium. Restoration of SGCB protein levels in myocardium resulted in restoration of SGCA in myocardium. As shown in Figure 5C, loss of SGCB resulted in reduced SGCA in the diaphragm. Restoration of SGCB protein levels in the diaphragm also resulted in restoration of SGCA in the diaphragm. As shown in Figures 5D-5E, restoration of SGCB protein levels and subsequent restoration of dystrophin and SGCA is not limited to a specific promoter, as similar results were observed using a scAAV genome containing the tMCK promoter and human SGCB polynucleotide sequence. As shown in Figure 5D, loss of SGCB results in a reduction of dystrophin. Restoration of SGCB protein levels at the sarcolemma results in the restoration of dystrophin at the sarcolemma. As shown in Figure 5E, loss of SGCB results in the loss of SGCA and dystrophin, which is restored after AAV.hSGCB gene transfer.

[0273] The expression of alpha-sarcoglycan, beta-sarcoglycan, and dystrophin was restored in the TA muscles of SGCG- / - mice after aav.hSGCG gene transfer (Figure 6). As shown in Figure 6, loss of SGCG resulted in a reduction of SGCA and dystrophin (DYS) in TA muscles (middle row). Restoration of SGCG protein levels in TA muscles resulted in the restoration of SGCA and DYS in TA muscles (bottom row).

[0274] Sarcospan as a surrogate biomarker for DAPC recovery

[0275] Sarcospan was reduced or absent in TA muscles of SGCB− / − mice and was restored in SGCB− / − mice after aav.hSGCB gene transfer, as measured by immunofluorescence (Figure 7) and Western blot (Figures 8A-8B).

[0276] conclusion

[0277] Overall, SGCB+ / - mice present with a normal muscle phenotype similar to WT mice and do not develop any dystrophin histopathology.

[0278] The RNA transcript levels of SGCB in het mice were found to be approximately half of those in WT mice, as expected, whereas the protein levels were normal and similar to those in WT mice.

[0279] Colocalization studies confirmed the recovery of DAPCs after gene therapy with SGCB or SGCG. This data demonstrates that additional sarcoglycans and sarcospan may serve as surrogate markers for functional recovery of DAPCs.

[0280] Example 2 SGCB gene transfer restores DAPCs

[0281] This example demonstrates that DAPC function was restored after SGCB gene transfer in SGCB- / - mice, and that expression of other sarcoglycans and sarcospan can serve as surrogate markers for functional recovery of DAPCs.

[0282] Figures 9A-9C show restoration of DAPC protein in SGCB- / - mice following administration of scAAV.MHCK7.hSGCB at 1.85e13 vg / kg and 7.41e13 vg / kg based on linearized PCR standards.

[0283] Muscle cells from SGCB- / - mice exhibit absent or reduced sarcolemmal expression of α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG), and δ-sarcoglycan (SGCD), components of the dystrophin-associated protein complex (DAPC) (Figure 9A). Systemic IV administration of scAAV.MHCK7.hSGCB at 1.85e13vg / kg and 7.41e13vg / kg (quantified by linearized PCR standards) to SGCB- / - mice not only increased SGCB expression but also increased sarcolemmal expression of SGCA, SGCG, and SGCD subunits in SGCB- / - mice, demonstrating dose-dependent restoration of DAPC protein by scAAV.MHCK7.hSGCB (Figures 9B-9C). In Figure 9B, TA refers to the tibialis anterior; GAS refers to the gastrocnemius; QD refers to the quadriceps; TRI refers to the triceps; GLUT refers to the gluteal muscles; PSO refers to the psoas major; and DIA refers to the diaphragm.

[0284] equivalent

[0285] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0286] The technology illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., are to be interpreted expansively and without limitation. Furthermore, the terms and expressions used herein are used as descriptive terms and without limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, but recognizes that various modifications are possible within the scope of the claimed technology of the present invention.

[0287] Thus, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present technology.

[0288] The inventive technology has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the inventive technology. This includes generic descriptions of the inventive technology with a provisio or negative limitation that removes any subject matter from the genus, whether or not the removed subject matter is specifically described herein.

[0289] Furthermore, where features or aspects of the present technology are described in terms of a Markush group, one of skill in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0290] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0291] Other aspects are within the scope of the following claims. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Claims

[Claim 1] The invention described in this specification.

Images