Gene therapy for 2C type limb-banding muscular dystrophy

Abstract

The present disclosure relates to gene therapy vectors (e.g., AAV vectors) comprising polynucleotides encoding gamma-myoglycans (SGCG), and methods of using such gene therapy vectors to treat subjects having muscular dystrophy (e.g., LGMD2C type 2 C muscular dystrophy).

Description

[0001] This application is a divisional application of the invention patent application which entered the Chinese national phase on July 24, 2020, with application number 201980009966.2, application date January 30, 2019, and invention name “Gene therapy for limb-girdle muscular dystrophy type 2C”.

[0002] Cross-references to related patent applications

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 624,616, filed on January 31, 2018, the entirety of which is incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 25, 2019, is named 106887-7141_SL.txt, and is 18,760 bytes in size. Technical Field

[0006] The present invention relates to gene therapy. More specifically, the present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, for treating muscular dystrophy (e.g., limb-girdle muscular dystrophy type 2C (LGMD2C)). Background Art

[0007] Muscular dystrophy (MD) is a group of genetic disorders. The group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. These disorders differ in the distribution and degree of muscle weakness - some forms of MD also affect the heart muscle, age of onset, rate of progression, and inheritance pattern.

[0008] One group of MDs is the limb-girdle grouping of MDs (LGMDs). LGMDs are rare diseases that present differently in different populations in terms of age of onset, areas of muscle weakness, heart and respiratory involvement, rate of progression, and severity. LGMDs can begin in childhood, adolescence, early adulthood, or even later. Both sexes are affected equally. LGMDs cause weakness in the shoulders and pelvic girdle, and sometimes nearby thigh and arm muscles over time. Leg weakness usually precedes arm weakness. Facial muscles are usually not affected. As the disease progresses, affected individuals may have problems walking and may need to use a wheelchair for extended periods of time. Involvement of the shoulder and arm muscles may lead to difficulty raising the arms above the head and lifting objects. In some types of LGMD, heart and respiratory muscles may be involved.

[0009] LGMD2C (limb-girdle muscular dystrophy type 2C) is caused by a deficiency of gamma-sarcoglycan (SGCG). Like other sarcoglycanopathies, it presents as a progressive muscular dystrophy that begins in the limb-girdle muscles and then spreads to the lower and upper limb muscles. Presentation usually occurs in the mid- to late teens. In attempts to treat LGMD2C, no medical therapy, not even corticosteroids, has been able to alter the course of the disease.

[0010] Improvement of function in patients with LGMD2C and other muscular dystrophies requires both genetic restoration and reduction of fibrosis.There is a need in the art for compositions and methods for treating LGMD2C and other muscular dystrophies. SUMMARY OF THE INVENTION

[0012] Described herein are gene therapy vectors (e.g., recombinant adeno-associated virus (AAV) vectors) encoding gamma-sarcoglycan, and methods of delivering such vectors encoding gamma-sarcoglycan to muscle, thereby reducing or preventing fibrosis, maintaining or improving muscle function, increasing muscle strength, increasing muscle endurance, or treating gamma-sarcoglycanopathy in a mammalian subject suffering from muscular dystrophy.

[0013] Additionally, the present invention provides therapies and methods for using gene therapy vectors to deliver gamma-sarcoglycan to address the genetic defect observed in LGMD2C (limb-girdle muscular dystrophy type 2C). In one aspect, provided herein is a method for one or more of: treating gamma-sarcoglycanopathy; increasing muscle strength, muscle endurance and / or muscle mass; reducing fibrosis; reducing contraction-induced damage; reducing fatty infiltration; and / or reducing central nucleation in a subject in need thereof, and / or treating muscular dystrophy to reduce degenerated or necrotic fibers in a subject with muscular dystrophy; reducing inflammation; increasing creatine kinase levels; treating muscle fiber atrophy and hypertrophy and / or reducing dystrophic calcification, the method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector, or consisting essentially of a gene expression cassette, or consisting essentially of a gene expression cassette, the gene expression cassette comprising, or consisting essentially of, or consisting of a polynucleotide sequence encoding gamma-sarcoglycan under the transcriptional control of a promoter, the cassette being flanked by one or more AAV inverted terminal repeats.

[0014] In one aspect, described herein is a recombinant AAV (rAAV) vector comprising, or consisting essentially of, or consisting of a polynucleotide sequence encoding γ-sarcoglycan under the transcriptional control of a promoter. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises, or consists essentially of, or consists of a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, and encodes a protein that retains γ-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises, or consists essentially of, or consists of the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan consists of the nucleotide sequence shown in SEQ ID NO: 1, or a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, which encodes a protein that retains γ-sarcoglycan activity, which in one aspect, retains the nucleotide changes in SEQ ID NO: 1 compared to the corresponding nucleotides in the wild-type human polynucleotide encoding γ-sarcoglycan.

[0015] In another aspect, the rAAV vectors described herein comprise, consist essentially of, or consist of a polynucleotide sequence encoding a γ-sarcoglycan having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 2, and the protein retains γ-sarcoglycan activity.

[0016] γ-sarcoglycan activity is essential for muscle function. γ-sarcoglycan is one of several sarcolemmal transmembrane glycoproteins that interact with dystrophin and form a dystrophin-glycoprotein complex, which spans the sarcolemma and is composed of dystrophin, intercalated proteins, α-troponin and β-troponin, and sarcoglycans including γ-sarcoglycan. The dystrophin-glycoprotein complex provides a structural connection between the subsarcolemmal cytoskeleton and the extracellular matrix of the muscle cell. Non-limiting examples of muscle cells include heart, diaphragm, leg, pelvic girdle, shoulder and arm muscle cells. Other non-limiting examples of γ-sarcoglycan activity and the consequences of γ-sarcoglycanosis are described in Blake et al. (2002) Physiol Rev.; 82 (2): 291-329 and Tarakci et al. (2016) Front Biosci (Landmark Ed); 21: 744-56.

[0017] In another aspect, the rAAV vectors described herein can be operably linked to a promoter and / or muscle-specific control elements to restrict expression to muscle. For example, the muscle-specific control element is a human skeletal actin gene element (GenBank Accession No. NG_006672.1), a cardiac actin gene element (GenBank Accession No. NG_007553.1), a myocyte-specific enhancer binding factor MEF (GenBank Accession No. NG_016443.2), muscle creatine kinase (MCK) (GenBank Accession No. AF188002.1), 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 skeletal fast-twitch 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 steroid-inducible element, or a glucocorticoid response element (GRE).

[0018] In some embodiments, the muscle-specific promoter is MHCK7 (SEQ ID NO: 4) or its equivalent. An exemplary rAAV vector described herein is pAAV.MHCK7.hSCGC, which comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 3 or its equivalent, wherein the MHCK7 promoter spans nucleotides 136-927, the CMV intron spans nucleotides 937-1084, the γ-sarcoglycan sequence spans nucleotides 1094-1968, and the polyA spans nucleotides 1976-2028. In some cases, pAAV.MHCK7.hSCGC is packaged in an AAV rh74 capsid.

[0019] AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, or AAV rh74. In some embodiments, the rAAV vector comprises an inverted terminal repeat (ITR) sequence of AAV2.

[0020] The production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).

[0021] Also contemplated are compositions comprising or consisting essentially of any of the rAAV vectors described herein.

[0022] Also provided is a method for producing recombinant AAV vector particles, the method comprising culturing cells that have been transfected with any of the recombinant AAV vectors described herein, and recovering the recombinant AAV particles from the supernatant of the transfected cells. Also contemplated are viral particles comprising or substantially consisting of any of the recombinant AAV vectors described herein.

[0023] Also provided is a method of reducing fibrosis in a mammalian subject in need. In this regard, the method includes administering a therapeutically effective amount of an AAV vector as described herein (or a composition comprising an AAV vector as described herein or consisting essentially of the vector), or consisting essentially of the step, or consisting of the step. In some embodiments, the mammalian subject suffers from muscular dystrophy. In some embodiments, administering an AAV vector as described herein (or a composition comprising an AAV vector as described herein or consisting essentially of the vector) can reduce fibrosis in the skeletal muscle or myocardium of the subject.

[0024] In another aspect, described herein is a method of increasing muscle strength or muscle mass or muscle endurance in a mammalian subject, the method comprising, consisting essentially of, or consisting of administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising or consisting essentially of an AAV vector described herein).

[0025] In any of the methods of the present disclosure, the subject may be suffering from a muscular dystrophy, such as limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy.

[0026] Also provided is a method of treating muscular dystrophy in a mammalian subject, the method comprising administering to the mammalian subject a therapeutically effective amount of an AAV vector as described herein (or a composition comprising or consisting essentially of an AAV vector as described herein). In some embodiments, the muscular dystrophy is limb-girdle muscular dystrophy.

[0027] In any method of the present disclosure, rAAV is administered by any suitable mode of administration, such as intramuscular injection or intravenous injection. In addition, in any method of the present disclosure, rAAV is administered systemically, such as parenterally by injection, infusion or implantation.

[0028] The compositions of the present disclosure are formulated for intramuscular or intravenous injection. Additionally, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation.

[0029] In addition, any composition for administration to a subject suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other muscular dystrophy associated with dystrophin) is formulated. Combination therapy is also described herein, comprising one or more compositions disclosed herein and corticosteroids, or consisting essentially of them. A host cell comprising an rAAV vector disclosed herein is provided herein. A kit is further provided herein, comprising any one of one or more embodiments disclosed herein and instructions for use. The kit may comprise one or more compositions disclosed herein and corticosteroids or one or more combination therapies provided herein, or consisting essentially of them. In any application of the present disclosure, the drug is formulated for administration, such as intramuscular injection or intravenous injection. In addition, in any application of the present disclosure, the drug is formulated for systemic administration, such as parenteral administration by injection, infusion or implantation. In addition, any drug can be prepared for administration to a subject suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other muscular dystrophy associated with dystrophin).

[0030] The preceding paragraphs are not intended to define every aspect of the invention, and other aspects are described in other sections such as the "Detailed Description." The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, paragraph, or section of this document. As an additional aspect, the present invention includes all embodiments of the invention whose scope is narrower in any respect than the variants defined in the specific paragraphs above. For example, in the case of certain aspects of the invention described as a genus, it should be understood that each member of the genus is respectively an aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Depicted is an AAV vector (scAAVrh74.MHCK7.hSGCG) comprising a codon-optimized full-length human γ-sarcoglycan (hSCGB) cDNA (SEQ ID NO: 1). The construct is flanked by two ˜100 bp AAV inverted terminal repeats (ITRs), including a codon-optimized human γ-sarcoglycan cDNA (hSGCG), a chimeric intron (intron), a synthetic polyadenylation signal (pA), and driven by the skeletal and cardiac muscle-specific MHCK7 promoter.

[0032] Figure 2 Depicted are hematoxylin and eosin (H&E) staining of the tibialis anterior (TA) muscles of 8-week-old BL6 WT and γ-sarcoglycan knockout (γ-SGKO) mice, showing the dystrophic phenotype of the diseased mice.

[0033] Figures 3A-3C In vivo vector potency is depicted. scAAVrh74.MHCK7.hSGCG was injected into the tibialis anterior (TA) muscle of γ-SG KO mice at a total dose of 3e10 vg. Figure 3A Immunofluorescence staining of TA muscle in γ-SG KO mice is shown. Nearly 100% γ-sarcoglycan protein expression at the sarcolemma is due to vector delivery. Figure 3B Western blot of γ-sarcoglycan expression in injected TA muscles from treated mice #794, 795 is shown. Figure 3C Shown are immunofluorescence staining of TA muscles in control wild-type mice ("BL6 WT TA") or uninjected control γ-SG KO mice ("GSG KO TA"), as well as unstained samples ("GSG KO No Primary Antibody").

[0034] Figure 4A-4B In vivo vector titer and toxicity in BL6 wild-type (WT) mice are depicted. Figure 4AImmunofluorescence staining is shown, demonstrating overexpression of γ-sarcoglycan by membrane and intracellular staining. Figure 4B Western blots are shown, demonstrating overexpression of γ-sarcoglycan in injected LTA muscles.

[0035] Figure 5 In vivo vector titer and toxicity in BL6 wild-type (WT) mice are depicted. H&E staining of uninjected and injected BL6 WT TA muscles showed no toxicity, with a complete absence of any central cores, necrotic fibers, inflammatory infiltrates, or fibrotic tissue.

[0036] Figure 6 Immunofluorescence staining of γ-sarcoglycan on the TA, gastrocnemius (GAS), quadriceps (QUAD), gluteal (GLUT), PSOAS, TRICEP, diaphragm, and myocardium is depicted, confirming widespread expression of γ-sarcoglycan.

[0037] Figure 7 Immunofluorescence staining of IV titer tissues is depicted. IF staining of γ-sarcoglycan in various skeletal muscles, diaphragm, and heart demonstrated strong expression with few negative fibers 6 weeks after systemic delivery of scAAVrh.74.MHCK7.hSGCG.

[0038] Figures 8A-8B The expression of SGCG in IV treated animals is shown. Representative 20X images show immunofluorescence imaging of skeletal muscle, diaphragm and heart of SGCG- / - mice injected intravenously with a total dose of 1e13 vg scAAVrh.74.MHCK7.hSGCG ( Fig. 8A Western blotting showed that hSGCG was expressed in all skeletal muscles and heart in mice injected intravenously with scAAVrh.74.MHCK7.hSGCG ( Figure 8B ).

[0039] Figures 9A-9B Histological evaluation of tissues after systemic treatment is shown. Hematoxylin and eosin staining of TRI and DIA skeletal muscle in BL6 WT, untreated SGCG- / -, and AAV.MHCK7.hSGCG treated SGCG- / - mice showed reversal of dystrophic pathology after treatment ( Fig.9A ). Quantification of the percentage of fibers with central nucleation showed a decrease in treated muscles. BL6 WT (n=5), untreated SGCG- / - (n=6), AAV.MHCK7.hSGCG treated (n=5) ( Fig. 9B ). ***=p<0.001, ****=p<0.0001.

[0040] Figures 10A-10FQuantification of fiber diameter is shown. GAS ( Fig. 10A )、PSOAS( Fig. 10B ) and TRI( Fig. 10C ) were used to quantify fiber diameters and the fiber diameter distribution was normalized after treatment. Fig. 10D )、PSOAS( Fig.10E ) and TRI( Fig.10F ) muscles, the average fiber diameter decreased, whereas after AAV.MHCK7.hSGCG treatment in SGCG- / - mice, the average fiber diameter per muscle increased to WT levels. ****=p<0.0001.

[0041] Figures 11A-11C TA and diaphragm physiology are shown. TA and DIA muscles of BL6 WT (n=5), untreated SGCG- / - (n=6), and AAV.MHCK7.hSGCG-treated (n=5) mice were subjected to measurements of normalized specific force generation. TA muscles were subjected to an eccentric contraction injury protocol ( Fig.11A Improved TA specific force output and resistance to contraction-induced injury were observed in treated SGCG- / - mice ( Fig. 11B In treated SGCG- / - mice, DIA specific force output was restored to WT levels ( Fig. 11C ). *=p<0.05, ****=p<0.0001.

[0042] Fig.12 Laser monitoring of open field cage activity is shown. Overall ambulation in the x and y planes was reduced in SGCG- / - mice and improved in mice treated with AAV.MCHK7.hSGCG. BL6 WT (n=6), untreated SGCG- / - (n=6), and treated with AAV.MHCK7.hSGCG (n=5).

[0043] Fig.13 Biodistribution of vector genomes is shown. The vector genome distribution as average vg copy number per microgram genomic DNA (gDNA) was measured in various tissues of two SGCG- / - mice 3 months after IV delivery of a total dose of 1e13 vg of scAAVrh.74.MHCK7.hSGCG.

[0044] Figures 14A-14BComparison of serum ALT and AST is shown. The levels of biochemical components of serum from BL6 WT mice (n=6), untreated SGCG- / - mice (n=6), and SGCG- / - mice treated with AAV.MHCK7.hSGCG IV (n=5) (1e13 vg total dose) were analyzed. Liver enzymes alkaline aminotransferase (ALT, Fig.14A ) and aspartate aminotransferase (AST, Fig. 14B ) was elevated in diseased SGCG- / - mice and recovered to near WT levels after treatment. * = p < 0.05. Dashed lines indicate the lower and upper limits of the normal range. Detailed description

[0046] The present disclosure relates to the administration of a recombinant adeno-associated virus (rAAV) vector comprising a polynucleotide expressing gamma-sarcoglycan to reduce or completely reverse muscle fibrosis in individuals with limb-girdle muscular dystrophy. As demonstrated in the "Examples" section, administration of the rAAV vectors described herein results in restoration of gamma-sarcoglycan expression in knockout mice. Administration of the rAAV vectors described herein will result in reversal of dystrophic features, including fewer degenerated fibers, reduced inflammation, and improved functional recovery by preventing eccentric contractions with increased force generation. The present disclosure includes treatment of limb-girdle muscular dystrophy in a subject (e.g., a human subject) by administering the rAAV vectors described herein.

[0047] In this specification, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and its fraction (such as one tenth and one hundredth of an integer) when appropriate, unless otherwise specified. It should be understood that, unless otherwise indicated, the terms "one" and "a" used herein refer to "one or more" of the listed components. The use of alternatives (such as "or") should be understood to refer to one of the alternatives, both, or any combination thereof. As used herein, the terms "include" and "comprising" are synonyms. As used herein, "plurality" can refer to one or more components (such as one or more miRNA target sequences). In this application, unless otherwise indicated, the use of "or" means "and / or".

[0048] As used in this application, the terms "approximately" and "approximately" are used as equivalents. Any numeral with or without about / approximately used in this application is intended to cover any normal fluctuations understood by those of ordinary skill in the relevant art. In certain embodiments, the terms "approximately" or "approximately" refer to any direction (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or the range of values ​​in the less range of the reference value of the statement, unless otherwise stated or can be clearly seen from the context (unless the numeral exceeds 100% of the possible value).

[0049] "Decrease" or "reduction" refers to a decrease or reduction of at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%) of a particular value compared to a reference value. A decrease or reduction of a particular value can also be expressed as a fold change in the value compared to a reference value, such as a decrease of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 times or more compared to a reference value.

[0050] "Increase" refers to an increase of at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, 500% or more) of a particular value compared to a reference value. An increase in a particular value can also be expressed as a fold change in the value compared to a reference value, such as an increase of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 times or more compared to a reference value.

[0051] "Complementary" refers to the ability to pair between two sequences including naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or their analogs by base stacking and specific hydrogen bonding. For example, if a base at a position of a nucleic acid can hydrogen bond with a base at a corresponding position of a target, the base is considered to be complementary to each other at that position. Nucleic acids can include universal bases or inert abasic spacers that do not provide positive or negative contributions to hydrogen bonding. Base pairing can include canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It should be understood that for complementary base pairing, adenosine bases (A) are complementary to thymidine bases (T) or uracil bases (U), cytosine bases (C) are complementary to guanosine bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary. Nichols et al., Nature, 1994; 369: 492-493 and Loakes et al., Nucleic Acids Res., 1994; 22: 4039-4043. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33(19): 6258-6267.

[0052] The term "subject" includes animals, such as mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is livestock, such as cattle, sheep, goats, cows, pigs, etc.; or domesticated animals, such as dogs and cats. In some embodiments (e.g., particularly in a research context), the subject is a rodent (e.g., mouse, rat, hamster), rabbit, primate, or pig (e.g., inbred pig), etc. The terms "subject" and "patient" are used interchangeably herein.

[0053] "Administering" herein refers to the introduction of an agent or composition into a subject.

[0054] As used herein, "treatment" refers to the delivery of an agent or composition to a subject to affect a physiological outcome. In some embodiments, treatment refers to the treatment of a disease in a subject (e.g., in a human), including (a) inhibiting the disease, e.g., preventing the development of the disease or preventing the progression of the disease; (b) alleviating the disease, e.g., causing regression of the disease state; (c) curing the disease; and (d) preventing the onset of the disease, e.g., preventing the development of the disease in an asymptomatic subject identified as a carrier of a genetic defect. In one aspect, treatment does not include prevention or prophylaxis.

[0055] When the disease is muscular dystrophy, the following clinical endpoints are non-limiting examples of treatment: reduction in specific force, increase in resistance to injury, increase in muscle strength, increase in muscle endurance, increase in muscle mass, reduction in contraction-induced damage, reduction in fatty infiltration, reduction in central nucleation, reduction in degenerated or necrotic fibers, reduction in inflammation, increase in creatine kinase levels, reduction in muscle fiber atrophy and hypertrophy and / or reduction in dystrophic calcification.

[0056] When the disease is fibrosis, the following clinical endpoints are non-limiting examples of treatment: reduction in fibrotic tissue, reduction in inflammation, reduction in fibroblastic lesions, reduction in activated fibroblast proliferation, reduction in myofibroblast occurrence, reduction in the rate of decline in forced vital capacity (FVC) (where FVC is the total amount of air exhaled during a pulmonary function test), absolute and relative increases in FVC from baseline, absolute increase in FVC from baseline (percent predicted), increase in progression-free survival, reduction from baseline in the St. George's Respiratory Questionnaire (SGRQ) total score (where SGRQ is a health-related quality of life questionnaire with 3 sections: symptoms, activities, and impacts, with the total score (total weight) ranging from 0 to 100, with lower scores indicating better health status), and relative reduction from baseline in the high-resolution computed tomography (HRCT) quantitative lung fibrosis (QLF) score (where QLF scores range from 0 to 100%, with higher values ​​indicating greater amounts of lung fibrosis and considered to be worse health status).Non-limiting exemplary clinical endpoints for the treatment of fibrosis and tests that can be performed to measure the clinical endpoints are described in the following clinical trials: NCT03733444 (clinicaltrials.gov / ct2 / show / NCT03733444) (last accessed January 9, 2019), NCT00287729 (clinicaltrials.gov / ct2 / show / NCT00287729) (last accessed January 9, 2019), NCT00287716 (clinicaltrials.gov / ct2 / show / NCT00287717) (last accessed January 9, 2019), altrials.gov / ct2 / show / NCT00287716) (last accessed January 9, 2019), NCT02503657 (clinicaltrials.gov / ct2 / show / NCT02503657) (last accessed January 9, 2019), NCT00047645 (clinicaltrials.gov / ct2 / show / NCT00047645) (last accessed January 9, 2019), NCT02802345 ( clinicaltrials.gov / ct2 / show / NCT02802345) (last accessed January 9, 2019), NCT01979952 (clinicaltrials.gov / ct2 / show / NCT01979952) (last accessed January 9, 2019), NCT00650091 (clinicaltrials.gov / ct2 / show / NCT00650091) (last accessed January 9, 2019), NCT013 35464 (clinicaltrials.gov / ct2 / show / NCT01335464) (last accessed on January 9, 2019), NCT01335477 (clinicaltrials.gov / ct2 / show / NCT01335477) (last accessed on January 9, 2019), NCT01366209 (clinicaltrials.gov / ct2 / show / NCT01366209) (last accessed on January 9, 2019). Other non-limiting examples of treatments for fibrosis and tests that can be performed to measure the clinical endpoints are described in King et al., (2014) N Engl J Med. May 29; 370(22): 2083-92 and Richeldi et al., (2014) N Engl J Med. May 29; 370(22): 2071-82.

[0057] The term "effective amount" or "therapeutically effective amount" refers to the minimum amount of an agent or composition required to produce a specific physiological effect (e.g., the amount required to increase, activate, or enhance a specific physiological effect). The effective amount or therapeutically effective amount of a particular agent can be expressed in a variety of ways depending on the nature of the agent, such as mass / volume, # of cells / volume, particles / volume, (mass of agent) / (mass of subject), # of cells / (mass of subject), or particles / (mass of subject). The effective amount or therapeutically effective amount of a particular agent can also be expressed as a half maximal effective concentration (EC 50 ), which refers to the concentration of an agent that causes a specific physiological response that is intermediate between the reference level and the maximum response level.

[0058] A "population" of cells refers to any number of cells greater than 1, but preferably at least 1 x 10 3 cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, at least 1x10 8 cells, at least 1x10 9 cells, at least 1x10 10 A cell population may refer to an in vitro population (e.g., a cell population in culture) or an in vivo population (e.g., a cell population located in a specific tissue).

[0059] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, and with a reasonable benefit / risk ratio.

[0060] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspension stabilizer, isotonic agent, solvent, surfactant or emulsifier approved by the U.S. Food and Drug Administration for use in humans and / or livestock.

[0061] As used herein, "vector" refers to a nucleic acid molecule that can transfer or transport a nucleic acid molecule to a cell together with one or more viral proteins (in a viral vector) (e.g., a viral capsid for encapsulated viruses). The transferred nucleic acid is typically connected to (e.g., inserted into) a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication or reverse transcription in the cell, or may contain sequences sufficient to allow integration into host cell DNA. "Vector" includes gene therapy vectors. As used herein, the term "gene therapy vector" refers to a vector that can be used to perform gene therapy (e.g., deliver a polynucleotide sequence encoding a therapeutic polypeptide to a subject). A gene therapy vector may contain a polynucleotide ("transgene") encoding a protein (e.g., γ-sarcoglycan).

[0062] As used herein, the term "expression cassette" refers to a DNA fragment integrated in the expression cassette that is capable of driving the expression of a polynucleotide (e.g., a transgene) encoding a protein (e.g., γ-sarcoglycan) under appropriate conditions. When introduced into a host cell, the expression cassette is particularly capable of directing the cell's mechanism to transcribe the transgene into RNA, which is then generally further processed and finally translated into a polypeptide with therapeutic activity. The gene therapy vector may comprise an expression cassette or consist essentially of an expression cassette. The term expression cassette does not include the polynucleotide sequences of 5' to 5' ITR and 3' to 3' ITR. Provided herein is a host cell comprising the rAAV vector of the present disclosure or consisting essentially of the vector or consisting of the vector. The cell may be of any suitable species, such as a mammalian cell.

[0063] As used herein, the phrases "operably linked" or "under transcriptional control" with respect to a polynucleotide interchangeably refer to a configuration of a promoter or muscle-specific control element and a polynucleotide that enables the polynucleotide to be transcribed by a polymerase capable of binding to the promoter. In one aspect, the muscle-specific control element is to restrict expression to muscle. Non-limiting examples of muscle-specific control elements are human skeletal actin gene element (GenBank Accession No. NG_006672.1), cardiac actin gene element (GenBank Accession No. NG_007553.1), myocyte-specific enhancer binding factor MEF (GenBank Accession No. NG_016443.2), muscle creatine kinase (MCK) (GenBank Accession No. AF188002.1), tMCK (truncated MCK), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK), C5-12 (a synthetic promoter), mouse creatine kinase enhancer element, skeletal fast-twitch troponin C gene element, slow-twitch cardiac troponin C gene element, slow-twitch troponin I gene element, hypoxia-inducible nuclear factor, steroid-inducible element, or glucocorticoid response element (GRE).

[0064] General methods in molecular and cellular biochemistry can be found in the following standard textbooks: Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference.

[0065] 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 co-infection with a helper virus. General information and reviews about AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these evaluations will apply to other AAV serotypes characterized after the date of publication of the evaluation, because it is well known that various serotypes have close relationships in structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JRPattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently display very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The extent of the relatedness is further indicated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments corresponding to "inverted terminal repeats" (ITRs) at the ends. Similar patterns of infectivity also suggest that replication functions in each serotype are under similar regulatory control.

[0066] As used herein, "AAV vector" refers to a vector comprising one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in host cells that have been transfected with vectors encoding and expressing rep and cap gene products.

[0067] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the 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 usually referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of AAV vector particles necessarily includes the production of AAV vectors, because such vectors are contained within the AAV vector particles.

[0068] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs). AAV has multiple serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 {1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least a portion of the genome of AAV-7 and AAV-8 is provided in GenBank Accession Nos. AX753246 and AX753249, respectively; and at least a portion of the genome of AAV-7 and AAV-8 is provided in GenBank Accession Nos. AX753246 and AX753249, respectively. The AAV-9 genome is provided in et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Pat. No. 9,434,928, which is incorporated herein by reference. The cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained in the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) 9), plus differential splicing of a single AAV intron (nucleotides 2107 and 2227), results in the production of four rep proteins (rep78, rep 68, rep 52, and rep 40). The Rep proteins have a variety of enzymatic properties and are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-shared translation start sites are responsible for the production of three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0069] AAV has unique characteristics that make it attractive as a vector for delivering exogenous DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells, making it possible to target many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells and can persist as transcriptionally active nuclear episomes (extrachromosomal elements) during the life cycle of these cells. The AAV proviral genome is inserted into a plasmid as cloned DNA, which makes the construction of recombinant genomes possible. In addition, since the signals for directing AAV replication and genome encapsidation are contained in the ITRs of the AAV genome, part or all of the approximately 4.3 kb inside the genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with exogenous DNA. To produce AAV vectors, rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a very stable and exciting virus. It can easily withstand the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigeration of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to superinfection.

[0070] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers have the necessary cytokines for proper antibody glycosylation, folding, and secretion, indicating that muscle is capable of stably expressing secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present disclosure comprises a nucleic acid molecule encoding γ-sarcoglycan (e.g., SEQ ID NO: 1) and one or more AAV ITRs flanking the nucleic acid molecule, or consists essentially of, or consists of, the sequence. The AAV DNA in the rAAV genome can be from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh74. The production of pseudotyped rAAVs is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, can also be considered. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art. In order to promote skeletal muscle-specific expression, AAV1, AAV5, AAV6, AAV8 or AAV9 can be used. Thus, in one aspect, described herein is a recombinant AAV vector comprising, or consisting essentially of, a polynucleotide sequence encoding γ-sarcoglycan under the transcriptional control of a promoter and / or muscle-specific control elements.In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises, consists essentially of, or consists of a sequence having, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the codon-optimized nucleotide sequence of human γ-sarcoglycan set forth in SEQ ID NO: 1 (see Table 1), and encodes a protein that retains γ-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a sequence encoding a protein that retains γ-sarcoglycan activity and is, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0071] The term "sequence identity" refers to the percentage of bases or amino acids that are identical and in the same relative position between two polynucleotide or polypeptide sequences. Therefore, compared with another polynucleotide or polypeptide sequence, a polynucleotide or polypeptide sequence has a certain percentage of sequence identity. In order to compare sequences, a sequence is usually used as a reference sequence to compare it with a test sequence. The term "reference sequence" refers to a molecule compared with a test sequence. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with a reference sequence means that, after alignment, the base (or amino acid) at each position in the test sequence of this percentage is the same as the base (or amino acid) at the same position in the reference sequence. This comparison and homology or sequence identity percentage can be determined using software programs known in the art, such as the software described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the following Internet address: ncbi.nlm.nih.gov / blast / Blast.cgi. An "equivalent" of a polypeptide or protein is a polypeptide or protein that has a certain sequence identity to a reference polypeptide or protein (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity compared to a reference) and retains a similar activity or function compared to the reference polypeptide or protein.

[0072] "Comprising" or "including" is intended to indicate compositions (e.g., media) and methods that include the listed elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements that have any substantial significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" means excluding other ingredients and substantial method steps beyond trace elements. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0073] In one aspect, the gene expression cassette of the rAAV vector of the present disclosure is flanked by one or more AAV inverted terminal repeats. In another aspect, the polynucleotide sequence encoding γ-sarcoglycan of the rAAV vector comprises, consists essentially of, or consists of a nucleotide sequence at least 95% identical to SEQ ID NO:1 and / or the nucleotide sequence shown in SEQ ID NO:1, and encodes a protein that retains γ-sarcoglycan activity. In another aspect, the polynucleotide sequence encoding γ-sarcoglycan of the rAAV vector encodes an amino acid sequence that is at least 95% identical, at least 99% identical, or 100% identical to SEQ ID NO:2, and encodes a protein that retains γ-sarcoglycan activity.

[0074] In some embodiments, the rAAV vector disclosed herein is a vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh74. In other embodiments, the genome of the rAAV vector comprises or consists essentially of muscle-specific control elements, and wherein the muscle-specific control elements are operably linked to the polynucleotide sequence. Non-limiting examples of muscle-specific control elements are human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C5-12, mouse creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin I gene element, hypoxia-inducible nuclear factor, steroid-inducible element, and glucocorticoid response element (gre). In one aspect, the muscle-specific control element of the rAAV vector is a truncated MCK (tMCK). In another aspect, the promoter and / or muscle-specific control element of the rAAV vector is the MHCK7 promoter. In another aspect, the MHCK promoter comprises the nucleotide sequence shown in SEQ ID NO:3 or its equivalent, or consists essentially of the sequence, or consists of the sequence, and provides promoter function. In one embodiment, the genome of the rAAV vector disclosed herein comprises an intron containing the nucleotide sequence shown in SEQ ID NO:5, or consists essentially of the sequence, or consists of the sequence.

[0075] In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan consists of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide sequence encoding γ-sarcoglycan that has at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93%, or 94%, even more usually at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and retains γ-sarcoglycan activity.

[0076] In another aspect, the recombinant AAV vectors described herein comprise, or essentially consist of, a polynucleotide sequence encoding a γ-sarcoglycan having at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93%, or 94%, even more usually at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of human γ-sarcoglycan (as shown in SEQ ID NO: 2 (see Table 1)), and the protein retains γ-sarcoglycan activity.

[0077] Table 1: Non-limiting examples of protein and nucleotide sequences

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] On the other hand, a recombinant AAV vector is described herein, comprising a polynucleotide sequence encoding a functional γ-sarcoglycan, comprising a nucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 or its complementary sequence under stringent conditions, or substantially consisting of the sequence, or consisting of the sequence. Functional γ-sarcoglycan refers to a γ-sarcoglycan polypeptide that retains γ-sarcoglycan activity. γ-sarcoglycan activity is essential for muscle function. γ-sarcoglycan is one of several sarcolemmal transmembrane glycoproteins that interact with dystrophin and form a dystrophin-glycoprotein complex, which spans the sarcolemma and is composed of dystrophin, interstitial protein, α-troponin and β-troponin, and sarcoglycans including γ-sarcoglycan. The dystrophin-glycoprotein complex provides a structural connection between the subsarcolemmal cytoskeleton and the extracellular matrix of the muscle cell. Non-limiting examples of muscle cells include heart, diaphragm, leg, pelvic girdle, shoulder and arm muscle cells. Other non-limiting examples of γ-sarcoglycan activity and consequences of γ-sarcoglycanopathy are described in Blake et al. (2002) Physiol Rev.; 82(2):291-329 and Tarakci et al. (2016) Front Biosci (Landmark Ed); 21:744-56.

[0085] The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents (e.g., formamide). Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (e.g., higher temperatures, lower ionic strengths, higher concentrations of formamide or other denaturing agents) may also be used, but the hybridization rate will be affected. In the case of deoxyoligonucleotide hybridization, other exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37°C (for 14 base oligonucleotides), 48°C (for 17 base oligonucleotides), 55°C (for 20 base oligonucleotides), and 60°C (for 23 base oligonucleotides).

[0086] To reduce non-specific and / or background hybridization, other reagents may be included in the hybridization and washing buffers. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, ultrasonically treated salmon sperm DNA (or other non-complementary DNA) and dextran, although other suitable reagents may also be used. The concentration and type of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8-7.4, however, under typical ionic strength conditions, the hybridization rate is almost independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions may be adjusted by those skilled in the art to accommodate these variables and to allow DNA of different sequence associations to form hybrids.

[0087] In another aspect, the recombinant AAV vectors described herein may comprise, consist essentially of, or consist of a polynucleotide sequence encoding γ-sarcoglycan operably linked to a promoter and / or a muscle-specific control element. For example, the muscle-specific control element is 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 skeletal fast-twitch 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 steroid-inducible element, or a glucocorticoid response element (GRE). In one embodiment, the rAAV vector comprises the MHCK7 promoter (SEQ ID NO: 4).

[0088] An exemplary rAAV vector described herein is pAAV.MHCK7.hSCGC, which comprises the nucleotide sequence of SEQ ID NO:3, wherein the MCHK7 promoter spans nucleotides 136-927 (SEQ ID NO:4), the intron spans nucleotides 937-1084 (SEQ ID NO:5), the γ-sarcoglycan sequence spans nucleotides 1094-1969 (SEQ ID NO:1), and the polyA spans nucleotides 1976-2028 (SEQ ID NO:6). See Figure 1 In some cases, the only viral sequences included in the rAAV vector are inverted terminal repeats, which are required for viral DNA replication and packaging. In some cases, the intron spanning nucleotides 7-116 (SEQ ID NO:5) and the 5'UTR spanning nucleotides 2128-2231 (SEQ ID NO:7) are derived from the plasmid pCMVβ (Clontech). In some cases, the 3'UTR comprises the sequence listed in SEQ ID NO:8. In some cases, pAAV.MHCK7.hSCGC is packaged in an AAV rh.74 capsid.

[0089] The DNA plasmid of the present disclosure comprises the rAAV genome. The DNA plasmid is transferred to cells that allow infection by the helper virus of AAV (e.g., adenovirus, adenovirus or herpes virus deleted by E1) to assemble the rAAV genome into infectious viral particles. The technology for producing rAAV particles is a standard technology in the art, in which the AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells. The production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus function. AAV rep and cap genes can be from any AAV serotype from which recombinant viruses can be derived, and can be from AAV serotypes different from rAAV genome ITRs, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. In some embodiments, the rAAV vector comprises the reverse terminal repeat (ITR) sequence of AAV2. The production of pseudotyped rAAV is disclosed in, for example, WO01 / 83692, which is incorporated herein by reference in its entirety. In some aspects, the rAAV vector comprises the reverse ITR sequence of AAV2 and is encapsidated in the capsid of AAVrh.74. In some cases, the genome of the rAAV vector comprises the polynucleotide sequence shown in SEQ ID NO:11. In some cases, the AAV rh.74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 10. In some cases, the rAAV vector comprises a polynucleotide comprising, consisting essentially of, or consisting of a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence set forth in SEQ ID NO: 11, and encoding the capsid proteins VP1, VP2, and VP3 of rAAV.In some embodiments, the rAAV vector comprises a polypeptide comprising, consisting essentially of, or consisting of a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of AAV rh.74VP3 set forth in SEQ ID NO:10.

[0090] The method for producing packaging cell lines is to produce cell lines that stably express all the necessary components for producing AAV particles. For example, a plasmid (or plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and a selection marker (e.g., neomycin resistance gene) is integrated into the genome of the cell. The AAV genome has been introduced into bacterial plasmids by the following methods: for example, GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73), or by direct blunt end connection (Senapathy & Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line is then infected with a helper virus (e.g., adenovirus). The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells. In some cases, the genome of the rAAV vector comprises the polynucleotide sequence shown in SEQ ID NO: 11. In some cases, the AAVrh.74 capsid comprises the amino acid sequence shown in SEQ ID NO: 10. In some cases, the rAAV vector comprises a polynucleotide comprising, or consisting essentially of, or consisting of a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence shown in SEQ ID NO:11, and encoding the capsid proteins VP1, VP2 and VP3 of rAAV. In some embodiments, the rAAV vector comprises a polypeptide comprising, consisting essentially of, or consisting of a sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of AAV rh.74VP3 set forth in SEQ ID NO:10.

[0091] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul etal. (1993) Human Gene Therapy 4:609-615; Clark et al. al.(1996)Gene Therapy 3: 1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on the portions of the documents related to rAAV production.

[0092] Therefore, the present disclosure provides packaging cells for producing infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (homologous 293 cell lines). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus monkey fetal lung cells).

[0093] The recombinant AAV of the present invention (ie, the infectious encapsidated rAAV particle) comprises the rAAV genome. Embodiments include, but are not limited to, the rAAV named pAAV.MHCK7.hSCGC, which comprises the polynucleotide sequence shown in SEQ ID NO:3.

[0094] rAAV can be purified by methods standard in the art (e.g., by column chromatography or cesium chloride gradients). Methods for purifying rAAV vectors from helper viruses are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0095] In another embodiment, the present invention encompasses a composition comprising or consisting essentially of a rAAV of the present invention. The compositions described herein comprise or consist essentially of a rAAV in a pharmaceutically acceptable carrier. In a specific embodiment, the compositions of the present disclosure comprise or consist essentially of lactated Ringer's Solution (LRS). The compositions may also include other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to the recipient, are preferably inert at the dosages and concentrations employed, and include: buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions in the form of salts such as sodium; and / or nonionic surfactants such as Tweens, pluronics, or polyethylene glycols (PEG). The disclosed compositions can be used for one or more of the following: treating gamma-sarcoglycanopathy in a subject in need thereof; increasing muscle strength, muscle endurance, and / or muscle mass; reducing fibrosis; reducing contraction-induced damage; reducing fatty infiltration; and / or reducing central nucleation, and / or treating muscular dystrophy in a subject with muscular dystrophy to reduce degenerated or necrotic fibers; reducing inflammation; increasing creatine kinase levels; treating muscle fiber atrophy and hypertrophy, and / or reducing dystrophic calcification.

[0096] The titer of the rAAV to be administered in the methods of the present disclosure will vary depending on, for example, the specific rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type being targeted, and can be determined by standard methods in the art. The titer of the rAAV can be about 1×10 6 , about lxl0 7 , about 1x10 8 , about lxl0 9 , about lxl0 10 , about lxl0 11 , about lxl0 12 , about lxl0 13 To about lxl0 14 The dosage may also be expressed in units of viral genomes (vg).

[0097] The present disclosure contemplates methods for transducing target cells with rAAV in vivo or in vitro. The term "transduction" is used to refer to the administration / delivery of a polynucleotide of interest (e.g., a polynucleotide sequence encoding γ-sarcoglycan) to a recipient cell in vivo or in vitro by a described replication-deficient rAAV, resulting in the recipient cell expressing γ-sarcoglycan.

[0098] In one aspect, provided herein are methods for one or more of: treating gamma-sarcoglycanopathy in a subject in need thereof; increasing muscle strength, muscle endurance and / or muscle mass; reducing fibrosis; reducing contraction-induced damage; reducing fatty infiltration; and / or reducing central nucleation, and / or treating muscular dystrophy in a subject with muscular dystrophy to reduce degenerated or necrotic fibers; reducing inflammation; increasing creatine kinase levels; treating muscle fiber atrophy and hypertrophy and / or reducing dystrophic calcification, the method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector, or consisting essentially of, or consisting of a gene expression cassette comprising, or consisting essentially of, or consisting of a polynucleotide sequence encoding gamma-sarcoglycan under the transcriptional control of a promoter, the cassette being flanked by one or more AAV inverted terminal repeats. In some embodiments, the promoter is a muscle-specific control element. In one embodiment, the methods disclosed herein increase muscle strength, muscle endurance, and / or muscle mass of one or more muscles of a subject. Non-limiting examples of muscles include heart, diaphragm, thigh, calf, pelvic girdle shoulder, and arm muscles. In a specific embodiment, muscle strength, muscle endurance, and / or muscle mass increase by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 50%, or at least about 80% compared to an untreated control subject.

[0099] In a specific aspect, the subject suffers from limb-girdle muscular dystrophy. In another aspect, the limb-girdle muscular dystrophy suffered by the subject is limb-girdle muscular dystrophy type 2C.

[0100] The term "administering" or "dosing" involving the delivery of a polynucleotide to a subject includes any route by which a polynucleotide is introduced or delivered to a subject to perform a desired function. Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), intracranial, or topical administration. Other routes of administration include intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal or transtracheal. Administration includes self-administration and administration by others.

[0101] In one aspect, the methods disclosed herein comprise, consist essentially of, or consist of the following steps: administering a composition comprising, consisting essentially of, or consisting of a rAAV vector and a pharmaceutically acceptable carrier. In another aspect, the methods disclosed herein comprise, consist essentially of, or consist of the following steps: administering a rAAV vector or composition comprising, consisting essentially of, or consisting of a rAAV vector and a pharmaceutically acceptable carrier by intramuscular or intravenous injection. In yet another aspect, the methods disclosed herein comprise, consist essentially of, or consist of the following steps: administering a rAAV vector or composition comprising, consisting essentially of, or consisting of a rAAV vector and a pharmaceutically acceptable carrier systemically. In a specific aspect, the methods disclosed herein comprise, consist essentially of, or consist of the following steps: administering a rAAV vector or composition comprising, consisting essentially of, or consisting of a rAAV vector and a pharmaceutically acceptable carrier by injection, infusion, or implantation.

[0102] In one aspect, the polynucleotide sequence encoding γ-sarcoglycan of the rAAV vector used in the methods described herein comprises the nucleotide sequence shown in SEQ ID NO: 1, or consists essentially of the sequence, or consists of the sequence. On the other hand, the polynucleotide sequence encoding γ-sarcoglycan of the rAAV vector encodes the amino acid sequence of SEQ ID NO: 2. On the other hand, the rAAV vector used in the methods disclosed herein comprises a self-complementary AAV vector genome, or consists essentially of the genome, or consists of the genome. In a specific embodiment, the rAAV vector comprises a genome lacking AAV rep and cap DNA, or consists essentially of the genome, or consists of the genome. In another embodiment, the rAAV vector is a serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh74 vector. In another aspect, the rAAV vector is a serotype AAV rh74 vector, and the rAAV vector comprises, consists essentially of, or consists of an AAV rh.74 capsid. In another aspect, the AAV rh.74 capsid of the rAAV vector comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 10 or an equivalent thereof.

[0103] The rAAV vector used in the method disclosed herein may further include or be substantially composed of a promoter and / or a muscle-specific control element, and wherein the muscle-specific control element is operably connected to a polynucleotide encoding γ-sarcoglycan. Non-limiting examples Some muscle-specific control elements are human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C5-12, mouse creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin I gene element, hypoxia-inducible nuclear factor, steroid inducibility element, and glucocorticoid response element (gre). On the one hand, the muscle-specific control element of the rAAV vector is truncated MCK (tMCK). On the other hand, the promoter and / or muscle-specific control element of the rAAV vector is the MHCK7 promoter. In another aspect, the MHCK promoter comprises, consists essentially of, or consists of the nucleotide sequence shown in SEQ ID NO: 3 or an equivalent thereof.

[0104] In one embodiment, the genome of the rAAV vector disclosed herein comprises an intron comprising the nucleotide sequence shown in SEQ ID NO:5.

[0105] The in vivo method comprises the following steps, or consists essentially of the following steps, or consists of the following steps: administering an effective dose or effective multiple doses of a composition to an animal (including a human) in need, the composition comprising the rAAV of the present disclosure, or consisting essentially of the rAAV, or consisting of the rAAV. If the dose is administered before the development of the condition / disease, the administration is preventive. If the dose is administered after the condition / disease occurs, the administration is therapeutic. In an embodiment of the present disclosure, an effective dose is a dose that alleviates (eliminates or alleviates) at least one symptom associated with the condition / disease state being treated, slows or prevents progression to the condition / disease state, slows or prevents progression of the condition / disease state, alleviates the extent of the disease, causes the disease to be alleviated (partially or completely), and / or prolongs survival. An example of a disease that is expected to be prevented or treated with the methods of the present disclosure is muscular dystrophy, such as limb-girdle muscular dystrophy. In some embodiments, the disease that is expected to be prevented or treated with the methods of the present disclosure is limb-girdle muscular dystrophy type 2C (LGMD2C).

[0106] As used herein, the term "muscular dystrophy" refers to a disease in which strength and muscle volume gradually decrease. Non-limiting examples of muscular dystrophy diseases can include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanosis, congenital muscular dystrophy (e.g., congenital muscular dystrophy caused by partial deficiency of LAMA2), congenital muscular dystrophy lacking ferritin, ID congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, limb-girdle type 1A muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy type 2C (LGMD2C). In some embodiments, the subject suffers from limb-girdle muscular dystrophy type 2C (LGMD2C).

[0107] There are at least nineteen forms of LGMD, and they are classified according to their associated genetic defect.

[0108]

[0109]

[0110] In some aspects, the present disclosure relates to a method of treating muscular dystrophy (e.g., LGMD2C) in a subject, comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding γ-sarcoglycan as described herein, or a composition comprising or consisting essentially of such a rAAV vector.

[0111] In some aspects, the present disclosure relates to a method of increasing muscle strength, muscle endurance and / or muscle mass in a subject with muscular dystrophy (e.g., LGMD2C), the method comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding γ-sarcoglycan as described herein, or a composition comprising or consisting essentially of such a rAAV vector.

[0112] In some aspects, the disclosure includes a method of reducing contraction-induced damage in a subject with a muscular dystrophy (e.g., LGMD2C), the method comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding γ-sarcoglycan as described herein, or a composition comprising or consisting essentially of such a rAAV vector.

[0113] In some aspects, the present disclosure includes a method of treating γ-sarcoglycanopathy in a subject, comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding γ-sarcoglycan as described herein, or a composition comprising or consisting essentially of such a rAAV vector.

[0114] In some aspects, the present disclosure also includes a method of reducing fibrosis in a subject with muscular dystrophy (e.g., LGMD2C), the method comprising, consisting essentially of, or consisting of the following steps: administering to the subject a therapeutically effective amount of a rAAV vector encoding γ-sarcoglycan as described herein, or a composition comprising or consisting essentially of such a rAAV vector. As used herein, the term "fibrosis" refers to excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues (including skeletal muscle, myocardium, liver, lungs, kidneys, and pancreas) after injury. The deposited ECM components include collagen (e.g., collagen 1, collagen 2, or collagen 3) and fibronectin.

[0115] In certain embodiments, the object treated by the methods described herein can be a mammal. In some cases, the object is a human, non-human primate, pig, horse, cow, dog, cat, rabbit, mouse or rat. The object can be a human female or a human male. In some cases, the object is a human object aged between 1-7 years old, 7-15 years old, 16-25 years old, 26-50 years old, 50-70 years old or greater than 70 years old. It is expected that other age ranges include but are not limited to 5-10 years old, 10-15 years old, 15-20 years old, 20-25 years old, 25-30 years old, 30-40 years old, 40-50 years old, 60-70 years old or greater than 70 years old and any range included in the foregoing.

[0116] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder, or condition that can be treated or ameliorated by an rAAV comprising a nucleic acid sequence encoding γ-sarcoglycan, or a composition comprising or consisting essentially of such rAAV as provided herein. A patient or subject in need may, for example, be a patient or subject diagnosed with a disease associated with dysfunction of γ-sarcoglycan (e.g., LGMD2C). A subject may have a mutation or dysfunction in a γ-sarcoglycan gene or protein. "Subject" and "patient" are used interchangeably herein.

[0117] The present disclosure also contemplates combination therapy, which comprises one or more compositions disclosed herein and corticosteroids, or consists essentially of, or consists of. Combinations / combinations used herein include simultaneous treatment or sequential treatment. Combinations of the methods disclosed herein with standard drug treatments (e.g., corticosteroids), as well as combinations with novel therapies are particularly contemplated. In some embodiments, subjects may be treated with steroids (e.g., prednisone, prednisolone, deflazacort) to prevent or reduce an immune response to the administration of rAAV described herein. In some cases, if the subject expresses antibodies to rAAV described herein, the subject may receive apheresis or another immunomodulator.

[0118] In some embodiments, the therapeutically effective amount of the rAAV vector is a dose of rAAV in the range of about 1113 vg / kg to about 5114 vg / kg, or about 1113 vg / kg to about 2113 vg / kg, or about 1113 vg / kg to about 3113 vg / kg, or about 1113 vg / kg to about 4113 vg / kg, or about 1113 vg / kg to about 5113 vg / kg, or about 1113 vg / kg to about 6113 vg / kg, or about 1113 vg / kg to about 7113 vg / kg, or about 1113 vg / kg to about 8113 vg / kg, or about 1113 vg / kg to about 9113 vg / kg, or about 1113 vg / kg to about 1114 vg / kg, or about 1113 vg / kg to about 2114 vg / kg, or about 1113 vg / kg to about 3114 vg / kg, or about 1x13 to about 4el4 vg / kg, or about 3el3 vg / kg to about 4el3 vg / kg, or about 3el3 vg / kg to about 5el3 vg / kg, or about 3el3 vg / kg to about 6el3 vg / kg, or about 3el3 vg / kg to about 7el3 vg / kg, or about 3el3 vg / kg to about 8el3 vg / kg, or about 3el3 vg / kg to about 9el3 vg / kg, or about 3el3 vg / kg to about leel4 vg / kg, or about 3el3 vg / kg to about 2el4 vg / kg, or 3el3 vg / kg to about 3el4 vg / kg, or about 3el3 to about 4el4 vg / kg, or about 3el3 vg / kg to about 5el4 vg / kg, or about 5el3 vg / kg to about 6el3 vg / kg, or about 5el3 vg / kg vg / kg to about 7el3 vg / kg, or about 5el3 vg / kg to about 8el3 vg / kg, or about 5el3 vg / kg to about 9el3 vg / kg, or about 5el3 vg / kg to about le14 vg / kg, or about 5el3 vg / kg to about 2el4 vg / kg, or 5el3 vg / kg to about 3el4 vg / kg, or about 5el3 to about 4el4 vg / kg, or about 5el3 vg / kg to about 5el4 vg / kg, or about le14 vg / kg to about 2el4 vg / kg, or le14 vg / kg to about 3el4 vg / kg, or about le14 to about 4el4 vg / kg, or about le14 vg / kg to about 5el4 vg / kg. The present disclosure also encompasses, or consists essentially of, or consists of, a composition comprising, or consisting essentially of, or consisting of a rAAV vector within these ranges.

[0119] For example, the dosage of the therapeutically effective amount of the rAAV vector is about 1113 vg / kg, about 2113 vg / kg, about 3113 vg / kg, about 4113 vg / kg, about 5113 vg / kg, about 6113 vg / kg, about 7113 vg / kg, about 8113 vg / kg, about 9113 vg / kg, about 1114 vg / kg, about 2114 vg / kg, about 3114 vg / kg, about 4114 vg / kg, and 5114 vg / kg. The present disclosure also encompasses, or consists essentially of, or consists of, a composition comprising, or consisting essentially of, or consisting of a rAAV vector within these ranges.

[0120] In some embodiments, the therapeutically effective amount of rAAV is a rAAV dose of about 1e14 vg / kg to about 1e15 vg / kg or about 1e15 vg / kg to about 1e16 vg / kg. In some embodiments, the present invention provides a method of administering to a subject a rAAV vector of the present invention at a dose of about 1el4 vg / kg, about 1.5el4 vg / kg, about 2el4 vg / kg, about 2.5el4 vg / kg, about 3el4 vg / kg, about 3.5el4 vg / kg, about 4el4 vg / kg, about 4.5el4 vg / kg, about 5el4 vg / kg, about 5.5el4 vg / kg, about 6el4 vg / kg, about 6.5el4 vg / kg, about 7el4 vg / kg, about 7.5el4 vg / kg, about 8el4 vg / kg, about 8.5el4 vg / kg, about 9el4 vg / kg, about 9.5el4 vg / kg, about 1el5 vg / kg, about 1.5el5 vg / kg, about 2el5 vg / kg, about 2.5el5 vg / kg, about 3el5 vg / kg, about 3.5el5 vg / kg, about 4el5 vg / kg, about 4.5el5 vg / kg, or about 5el5 vg / kg. In some embodiments, the present invention provides methods of administering to a subject a rAAV vector of the invention at a total dose of about 4.0el4 vg / kg, about 4.1el4 vg / kg, about 4.2el4 vg / kg, about 4.3el4 vg / kg, about 4.4el4 vg / kg, about 4.5el4 vg / kg, about 4.6el4 vg / kg, about 4.7el4 vg / kg, about 4.8el4 vg / kg, about 4.9el4 vg / kg, about 5.0el4 vg / kg, about 5.1el4 vg / kg, about 5.2el4 vg / kg, about 5.3el4 vg / kg, about 5.4el4 vg / kg, about 5.5el4 vg / kg, about 5.6el4 vg / kg, about 5.7el4 vg / kg, about 5.8el4 vg / kg, about 5.9el4 vg / kg, or about 6el4 vg / kg.

[0121] In various embodiments, the administering step may include administering the total dose in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate doses. For example, the total dose may be delivered to multiple sites of a subject or to subjects spaced apart within minutes, hours, or days by injection.

[0122] Administration of an effective dose of the composition can be performed by standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal. Taking into account the infection and / or disease state to be treated and the target cells / tissues to express γ-sarcoglycan, one skilled in the art can select and / or match the administration route and serotype of the AAV components (particularly AAV ITRs and capsid proteins) of the rAAV disclosed herein.

[0123] The present disclosure provides local and systemic administration of effective doses of rAAV and compositions of the present disclosure. For example, systemic administration refers to administration into the circulatory system so that the whole body is affected. Systemic administration includes enteral administration (e.g., absorption through the gastrointestinal tract) and parenteral administration by injection, infusion or implantation.

[0124] In particular, the actual administration of the rAAV disclosed herein can be accomplished by using any physical method that transports the rAAV recombinant vector to the target tissue of the animal. Administration according to the present invention includes, but is not limited to, injection into muscle, bloodstream, and / or directly into the liver. It has been demonstrated that simply resuspending the rAAV in phosphate-buffered saline is sufficient to provide a vehicle that can be used for muscle tissue expression, and there are no known limitations on vectors or other components that can be co-administered with rAAV (although components that degrade DNA should be avoided in conventional ways of using rAAV).

[0125] The capsid protein of rAAV can be modified so that rAAV is targeted to a specific target tissue, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation for delivery to muscle by transdermal transport. A variety of formulations for intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the present disclosure. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0126] For the purpose of intramuscular injection, solutions in adjuvants (such as sesame oil or peanut oil) or in propylene glycol aqueous solutions and sterile aqueous solutions can be used. If necessary, such aqueous solutions can be buffered, and the liquid diluent is first made isotonic with saline or glucose. As a solution of rAAV of a free acid (DNA contains an acidic phosphate group) or a pharmacologically acceptable salt, it can be prepared by mixing with a surfactant (such as hydroxypropyl cellulose) in water. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under conventional storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous medium used can be easily obtained by standard techniques well known to those skilled in the art.

[0127] The pharmaceutical form suitable for injection purposes includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and must have a certain fluidity to achieve the purpose of easy injection. It must remain stable under production and storage conditions and must be treated with preservatives to prevent contamination by microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. The extended absorption of injectable compositions can be achieved by using agents that delay absorption (such as aluminum monostearate and gelatin).

[0128] Sterile injectable solutions are prepared by incorporating the desired amount of rAAV with the various other ingredients described above in an appropriate solvent, followed by filter sterilization. Dispersions are generally prepared by incorporating the sterilized active ingredient into a sterile vehicle comprising a basic dispersion medium and the other ingredients required as listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any other desired ingredients from a previously sterile filtered solution thereof.

[0129] rAAV transduction can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, allogeneic or xenogeneic muscle cells can be used, where these cells do not produce an inappropriate immune response in the subject.

[0130] Suitable methods for transducing and reintroducing the transduced cells into a subject are known in the art. In one embodiment, the cells can be transduced in vitro by combining rAAV with muscle cells (e.g., in a suitable culture medium) and screening for cells with the DNA of interest using conventional techniques (e.g., Southern blot and / or PCR), or by using a selection marker. The transduced cells can then be formulated into a pharmaceutical composition and introduced into a subject by various techniques, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by using, for example, a catheter to inject into smooth muscle and myocardium.

[0131] Transduction of cells with the rAAV of the present invention results in sustained expression of γ-sarcoglycan. Thus, the present disclosure provides methods of administering / delivering rAAV expressing γ-sarcoglycan to mammalian subjects (preferably humans). These methods include transducing tissues (including but not limited to tissues (e.g., muscle), organs (e.g., liver and brain), and glands (e.g., salivary glands)) with one or more rAAVs of the present disclosure. Transduction can be performed with a gene cassette comprising tissue-specific control elements. For example, one embodiment of the present disclosure provides a method for transducing muscle cells and muscle tissue directed by muscle-specific control elements, including but not limited to elements derived from the actin and myosin gene families, such as elements derived from the myoD gene family [see Weintraub et al, Science, 251:761-766 (1991)], myocyte-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)], control elements derived from the human skeletal actin gene [Muscat et al, Mol Cell Biol, 7:4089-4099 (1987)], cardiac actin gene, muscle creatine kinase sequence elements [see Johnson et al, Mol Cell Biol, 11:4089-4099 (1987)], myosin gene ... Biol, 9:3393-3399 (1989)] and the mouse creatine kinase enhancer (mCK) element, control elements from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypoxia-inducible nuclear factor (Semenza et ah, Proc Natl Acad Sci USA, 88:5680-5684 (1991)), steroid-inducible elements, and promoters including glucocorticoid response elements (GREs) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)) and other control elements.

[0132] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present disclosure encompasses sustained expression of a transgene (eg, γ-sarcoglycan) from transduced myofibers.

[0133] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle, such as skeletal muscle and smooth muscle, such as cells from the digestive tract, bladder, blood vessels, or heart tissue. Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyocytes.

[0134] Thus, also described herein are methods of administering to a mammalian subject in need thereof an effective dose (or doses administered substantially simultaneously or doses administered at intervals) of a rAAV encoding γ-sarcoglycan.

[0135] Further provided herein are kits comprising, or consisting essentially of, or consisting of any of one or more embodiments disclosed herein and optional instructions for use. The kit may comprise, or consisting essentially of, or consisting of one or more compositions disclosed herein and one or more of a corticosteroid or a combination therapy provided herein and optional instructions for use.

[0136] It should be understood that the present disclosure is not limited to the specific aspects described, as they may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the present invention, as the scope of the present disclosure will be limited only by the appended claims.

[0137] A number of embodiments of the present disclosure have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present disclosure. Therefore, the following embodiments are intended to illustrate rather than limit the scope of the disclosure described in the claims.

[0138] In the absence of explicit statement and unless otherwise indicated, it can be inferred that when the present technology relates to polypeptides, proteins, polynucleotides or antibodies, their equivalents or biological equivalents should be within the scope of the present technology.

[0139] Citation of any patent, patent application, publication or any other document does not constitute an admission that any of the above is relevant prior art nor does it constitute an admission as to the contents or date of such publication or document.

[0140] All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, the disclosed features (e.g., antibodies) are an example of a genus of equivalent or similar features.

[0141] As used herein, unless the context clearly indicates otherwise, all numerical values ​​or numerical ranges include integers within the range and fractions of numerical values ​​or integers within the range. In addition, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Thus, for example, reference to 80% or more includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., and 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so on.

[0142] References to integers having numbers greater than (more than) or less than include any number greater than or less than the reference number, respectively. Thus, for example, references to numbers less than 100 include 99, 98, 97, etc., down to one (1); and references to numbers less than 10 include 9, 8, 7, etc., down to one (1).

[0143] As used herein, unless the context clearly indicates otherwise, all values ​​or ranges include fractions of the value and integers within the range and fractions of integers within the range. Thus, for illustration purposes, reference to a numerical range (e.g., 1-10) includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to and including 50, and 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0144] Reference to a series of ranges includes ranges combining the values ​​of the boundaries of the different ranges within the series. Thus, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,000, 3,000-4,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, 9,000-10,000-11,000, 9,000-12,000, 9,000-13,000, 9,000-14,000, 9,000-15,000, 9,000-1 - 9,000, including ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

[0145] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, but these modifications and improvements are within the scope and spirit of the present technology.

[0146] The techniques illustratively described herein may be suitably practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions that have been employed are used as terms of description, not limitation, and the use of such terms and expressions does not exclude any equivalents of the features shown and described, or fragments thereof, and various modifications may be made within the scope of the claimed technology.

[0147] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent was expressly and individually indicated to be incorporated by reference. In the event of a conflict, the present application (including any definitions herein) will prevail. However, the mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0148] In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer in the range, and its fraction (such as one tenth and one hundredth of an integer) when appropriate, unless otherwise specified. The term "about" refers to the number or numerical range plus or minus 10% when it is immediately before a number or value. It should be understood that, unless otherwise noted, the terms "one" and "a kind of" used herein refer to "one or more" in the listed components. The use of substitution (such as "or") should be understood to refer to one of the alternatives, both, or any combination thereof. The term "and / or" should be understood to refer to one or both of the alternatives. As used herein, the terms "include" and "comprising" are synonyms.

[0149] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0150] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims. Example

[0151] Example 1: Construction and vector titer of scAAVrh74.tMCK.hSGCB

[0152] Constructed as Figure 1 The SGCG AAV construct shown contains a codon-optimized full-length human γ-sarcoglycan (SCGB) cDNA (SEQ ID NO: 1). The SGCG AAV construct is configured to be packaged using a self-complementary AAV backbone to achieve more efficient transduction efficiency. The SGCG cDNA (969) is configured to be driven by the MHCK7 promoter (792 bp). Introns and 5'UTR are derived from the plasmid pCMVβ (Clontech). The SGCG AAV construct has a consensus Kozak immediately before the start ATG, and a small 53 bp synthetic polyA signal for mRNA termination. The cDNA is codon-optimized for human use and synthesized by GenScript (Piscataway, NJ). The only viral sequence contained in the vector is the inverted terminal repeat of AAV2, which is required for viral DNA replication and packaging.

[0153] The vector used for this study was produced using a triple transfection method of HEK293 cells under research-grade conditions. Post-production vector characterization included titer determination by qPCR using a supercoiled standard, endotoxin level determination (U / mL), and sterility assessment. The produced vector was analyzed by SDS-PAGE to verify the consistency of the banding pattern with the expected rAAV. The vector preparation was titrated with a linear plasmid standard and titrated again with a supercoiled plasmid standard. The vector was produced using a plasmid containing the following components: full-length human γ-sarcoglycan cDNA (NC_000013.11), a muscle-specific MHCK7 promoter to drive expression, a consensus Kozak sequence (CCACC), an SV40 chimeric intron, a synthetic polyadenylation site (53 bp) ( Figure 1 The SGCG expression cassette was cloned between the AAV2 ITRs packaged in a self-complementary (sc) AAVrh.74 vector to enhance transduction of cardiac tissue.

[0154] Table 2 provides an overview of the study design. Dose values ​​were determined by estimating the total number of vector genomes (vg) by qPCR. Inclusion of at least some partially intact AAV capsids in the vector preparation may cause the qPCR method to overestimate the dose. Therefore, efficacy measurements at a given dose (e.g., 5E+13) indicate that efficacy can be observed at lower qPCR-measured vector doses when the vector is purified to remove partially intact AAV capsids. The total dose (vg) and dose in units of vector genomes per kilogram of subject (vg / kg) listed in Table 2 and throughout the "Examples" section do not account for partially intact AAV capsids.

[0155] Table 2: Overview of the scAAVrh74.MHCK7.hSGCG study design

[0156]

[0157]

[0158] N / A: Not applicable

[0159] IF: immunofluorescence; H&E: hematoxylin & eosin staining; TA Phys: specific force measurements of TA muscle and resistance to ECC injury; Dia Phys: specific force measurements of diaphragm; Histopathology: formal histopathological review; '—': not injected.

[0160] All competent animals were treated at 4-8 weeks of age and necropsied 3 months after injection. SGCG- / - negative control mice were necropsied at 4 months of age.

[0161] The potency of the scAAVrh.74.MHCK7.hSGCG test article was determined by intramuscular and systemic injection of the vector into SGCG- / - mice. Wild-type mice injected with lactated Ringer's solution (LRS) were used as positive controls, and uninjected SGCG- / - mice were used as negative controls.

[0162] Tibialis anterior (TA) muscles from 8-week-old BL6 wild-type (WT) mice and γ-sarcoglycan knockout (γ-SG KO) mice were extracted and tissue sections were stained with hematoxylin and eosin (H&E) to observe the histology of each muscle. Even at this young age, γ-SGKO mice showed a disease phenotype in the muscles, with the presence of necrotic myofibers, inflammatory infiltration, and fibrotic tissue. ( Figure 2 ).

[0163] The SGCG AAV construct was packaged into AAV of the rh.74 serotype to produce a recombinant AAV (rAAV) called scAAVrh.74.MHCK7.hSGCG. A total of 3 mice were injected to determine the titer of scAAVrh.74.MHCK7.hSGCG. One C57BL / 6WT mouse injected with LRS and one uninjected SGCG- / - mouse served as positive and negative controls, respectively. The remaining three mice were SGCG- / - and were injected with scAAVrh.74.MHCK7.hSGCG by IM in LTA (n=2) or IV in the tail vein (n=1) to determine whether the vector batch was effective. Table 3 summarizes the study design.

[0164] Table 3: scAAVrh.74.MHCK7.hSGCG titer determination

[0165]

[0166] γ-SG KO mice were injected with scAAVrh.74.MHCK7.hSGCG at a total dose of 3e10 vg into the TA muscle by intramuscular (IM) injection at 4 weeks of age. Mice were euthanized at 4 weeks post-injection (8 weeks of age), and TA muscles were extracted and freshly frozen in liquid nitrogen-cooled methylbutane. Immunofluorescence (IF) staining of γ-sarcoglycan showed the absence of γ-sarcoglycan in the uninjected right TA (RTA) muscle and the almost complete restoration of membrane γ-sarcoglycan protein expression in the injected left TA (LTA) muscle ( Figure 3A ). Western blot of γ-sarcoglycan ( Figure 3B ) shows: expression of γ-sarcoglycan in two BL6 WT TA muscles; absence of protein in γ-SG KO TA muscles; restoration of γ-sarcoglycan protein expression in TA muscles of injected mice #794 and #795.

[0167] 3x10 11 Delivery of scAAVrh.74.MHCK7.hSGCG to SGCG- / - mice by IM at the indicated total dose of vg resulted in 93.03% expression of hSGCG in the injected LTA muscle, which is similar to the level of β-sarcoglycan (scAAVrh.74.MHCK7.hSGCB) vector we have previously studied. Immunofluorescence imaging of mice given the vector (animal ID: 794, 795) confirmed the expression of the hSGCG transgene ( Figure 3A ). 20X images were included to visualize the expression in injected muscle. As expected, C57BL / 6WT mice showed 100% expression of γ-sarcoglycan protein, while SGCG- / - mice completely lacked γ-sarcoglycan expression ( Figure 3C ).

[0168] Systemic injection into an SGCG- / - mouse (#797) via the tail vein resulted in high-level expression of the hSGCG transgene. Applicants were able to 13 Total dose of vg (5x10 14 vg / kg) scAAVrh.74.MHCK7.hSGCG treated mice of this titer completed ≥94.00% transduction in all skeletal muscles. The average expression percentage of the hSGCG transgene delivered by AAV was 95.98% in all skeletal muscles analyzed. Applicants were also able to achieve very high transduction levels in the heart when delivered systemically. Figure 7 Representative 20X immunofluorescence images of all skeletal muscles as well as the diaphragm and heart are shown, illustrating the widespread expression of hSGCG.

[0169] Example 2: Potency and toxicity of scAAVrh74.tMCK.hSGCB vector in BL6 WT mice

[0170] BL6 WT mice were injected with scAAVrh.74.MHCK7.hSGCG at a total dose of 3e10 vg into the TA muscle at 4 weeks of age by intramuscular (IM) injection. Mice were euthanized at 4 weeks post-injection (8 weeks of age), and TA muscles were extracted and freshly frozen in liquid nitrogen-cooled methylbutane. Immunofluorescence (IF) staining of γ-sarcoglycan showed membranous staining of γ-sarcoglycan in the uninjected right TA (RTA) muscle and intracellular staining, indicating overexpression of γ-sarcoglycan protein in the injected left TA (LTA) muscle ( Figure 4A ). Western blot of γ-sarcoglycan ( Figure 4B) shows overexpression of γ-sarcoglycan in injected LTA muscle. H&E staining of TA muscle showed no toxicity in either uninjected RTA or injected LTA, with a complete absence of any central nucleus, necrotic fibers, inflammatory infiltration, or fibrotic tissue. ( Figure 5 ).

[0171] Example 3: Gene expression after systemic delivery of scAAVrh.74.tMCK.hSGCB

[0172] γ-SG KO mice were injected intravenously into the tail vein at 4-5 weeks of age with a total dose of 1e12 vg (5e13 vg / kg). Mice were euthanized after 6 weeks of treatment. Immunofluorescence staining of the TA, gastrocnemius (GAS), quadriceps (QUAD), gluteal (GLUT), PSOAS, TRICEP, diaphragm, and myocardium confirmed the widespread expression of γ-sarcoglycan ( Figure 6 ).

[0173] By using a single dose (1x10 13 Total dose of vg, 5x10 14 The potency of the scAAVrh.74.MHCK7.hSGCG test article was determined by systemic injection of 100 mg / kg (vg / kg) in SGCG- / - mice (genotype: sgcgC57). 12 Total dose of vg (5x10 13 vg / kg)), medium dose (4x10 12 Total dose of vg (2x10 14 vg / kg)) and high doses (1x10 13 Total dose of vg (5x10 14 vg / kg)) was systemically injected into the tail vein of SGCG- / - mice, and euthanasia was performed 3 months after injection.

[0174] According to the results of our scAAVrh.74.MHCK7.hSGCG titer assay, the applicant injected the vector at 1x10 13 Total dose of vg (5x10 14vg / kg) was delivered to 5 SGCG- / - mice to evaluate the transgenic expression and efficacy of our vector when delivered systemically at an extended time point of 3 months. Mice were injected at 4 weeks of age, and a complete autopsy was performed 3 months after injection. All skeletal muscles discussed in the above titer determinations, as well as the diaphragm and heart, were extracted for analysis. Organs including the lungs, kidneys, liver, spleen, and gonads were also removed for toxicology and biodistribution studies. In short, after 3 months of treatment, hSGCG transgenic expression was still high, and all muscles from treated mice were highly transduced again. This was accompanied by improved muscle tissue pathology and improved TA and diaphragm muscle function. Systemic delivery of scAAVrh.74.MHCK7.hSGCG vectors did not cause any toxicity in muscles or organs.

[0175] γ-Sarcoglycan expression

[0176] Immunofluorescence staining of human gamma-sarcoglycan was used to determine the hSGCG transgene expression in six skeletal muscles on the left and right sides of all SGCG- / - mice injected with scAAVrh.74.MHCK7.hSGCG vectors, except the diaphragm and heart. These muscles include TA, GAS, QUAD, GLUT, PSOAS, TRI. For the purpose of expression analysis and transduction efficiency, images of the left and right muscles from 5 treated mice were used for quantification. Four 20X images were taken for each muscle, and the percentage of hSGCG positive fibers (positive expression fiber number / total number of fibers) was determined for each image, thereby obtaining the average transduction percentage of each muscle of each mouse. Fig. 8A Representative images from treated mice are shown and demonstrate high levels of expression averaging 92.26% across all muscles quantified including the diaphragm. Again Applicants also saw high levels of transduction in the myocardium in all vehicle treated mice. Figure 8B Western blots are shown demonstrating expression of the hSGCG transgene in all skeletal muscles and hearts of mice delivered intravenously with scAAVrh.74.MHCK7.hSGCG vectors. Table 4 lists the average percent expression from four 20X images per muscle from each mouse, as well as the average for each muscle from all five mice.

[0177] Table 4. Average percentage of γ-sarcoglycan transgene expression

[0178] Animal ID 5229 5230 5231 5232 5233 average muscle TA 97.79 97.79 99.54 99.13 96.59 98.17 GAS 90.88 90.88 74.14 97.67 93.49 89.41 QUAD 88.37 88.37 96.85 95.01 76.15 88.95 GLUT 96.19 96.19 91.12 100 92.83 95.27 PSOAS 97.55 97.55 99.01 49.88 97.92 88.38 TRI 97.32 97.32 99.31 94.98 93.31 96.45 DIA 93.02 93.02 75.56 97.14 87.22 89.19

[0179] Histopathology of processed muscles

[0180] Both skeletal and cardiac muscle from SGCG- / - mice exhibited widespread myopathy, including marked myofiber atrophy and hypertrophy with multiple areas of necrosis. There was also increasing mononuclear inflammation (lymphocytes and macrophages, with scattered neutrophils) and increased dystrophic calcification, fatty infiltration, central nucleation, and fibrosis. Fig.9A Hematoxylin and eosin staining in the Figure illustrates this dystrophic phenotype and the amelioration of muscle pathology after treatment in SGCG- / - mice compared to conventional WT mice. Quantification of histological parameters showed a significant increase in the number of centrally nucleated fibers in skeletal muscle of SGCG- / - mice, followed by a decrease in central nuclei in many different skeletal muscles as a result of γ-sarcoglycan gene transfer ( Fig. 9B A closer analysis of muscle histopathology revealed normalization of fiber size distribution and an increase in mean fiber diameter in diseased SGCG- / - mice treated with vehicle in all three muscles examined (GAS, PSOAS, and TRI). Figures 10A-10F ). The number of individual central nuclei and mean fiber diameter of each muscle were analyzed from each mouse.

[0181] Example 4: Physiological defects of γ-SG KO mice

[0182] Sirius red staining will be performed to quantify the amount of fibrotic tissue. γ-SG KO mice and BL6 WT mice will be tested at 4 months of age to assess whether skeletal muscle has strength deficits. The tibialis anterior (TA) muscle will be tested for significant reductions in specific force and resistance to injury compared to controls. The diaphragm will also be tested in a similar manner to detect any significant reductions. This measurable decrease will provide a functional outcome indicator for determining the efficacy of AAV.hSGCB therapy.

[0183] Example 5: Functional consequences of scAAVrh74.tMCK.hSGCB treatment

[0184] γ-SG KO mice will be injected with rolling injections for a three-month study to quantify efficacy and toxicity (Table 5). Mice will be subjected to activity cage analysis prior to euthanasia to determine the overall activity of treated mice compared to γ-SG KO controls. Physiological analysis of the TA and diaphragm will be performed to determine specific force output and resistance to injury / fatigue. γ-SG KO muscles will be compared to BL6 WT controls to establish functional outcome measures that will be used to determine the efficacy of the treatment in treated mice. IF (immunofluorescence) staining will be performed on all skeletal muscles to detect the expression of γ-sarcoglycan and H&E staining will be performed for histopathology. Quantitative polymerase chain reaction (qPCR) will be performed on muscles and organs of injected mice to determine the biodistribution of the vector genome.

[0185] Table 5

[0186] Mouse strains Test Items Human Dosage Sample size end C57 / BL6 LR* NA 6 12 weeks SGCG KO LR NA 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG <![CDATA[5x10 13 vg / kg]]> 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG <![CDATA[1x 10 14 vg / kg]]> 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG <![CDATA[2x 10 14 vg / kg]]> 6 12 weeks

[0187] Example 6: Functional Assessment of Systemic Delivery

[0188] To determine whether hSGCG gene transfer provides functional benefits to diseased muscles, the applicant evaluated the functional properties of TA and diaphragm muscles of SGCG- / - mice treated with scAAVrh.74.MHCK7.hSCGG. As outlined in Examples 1 to 5, the applicant first confirmed histopathology in the limb skeletal muscles and diaphragms of mice lacking γ-sarcoglycan. In situ analysis of TA muscles of untreated SGCG- / - mice showed a statistically significant decrease in normalized specific force generation by 37.68% compared to BL6 WT TA muscles (BL6 WT: 291.65 mN / mm 2 vs.SGCG- / -:181.77mN / mm 2 After treatment, specific force output increased significantly to normal WT levels compared to SGCG- / - muscles (SGCG- / -: 181.77 mN / mm 2 vs. after treatment: 266.02mN / mm 2 )( Fig.11A and Fig. 11C ). Another functional outcome measure to determine the functional benefit of hSGCG gene transfer was to assess resistance to contraction-induced damage in TA muscles following repeated eccentric contractions. TA muscles from normal BL6 WT mice and untreated SGCG- / - mice lost only 18% of force production after a single bout of 10 eccentric contractions, compared to a 37% loss of force in untreated SGCG- / - TA muscles. Vehicle-treated SGCG- / - muscles improved above WT levels, with only a 10% loss of force seen following an eccentric contraction (ECC) protocol ( Fig. 11B ).

[0189] To further test the potential functional benefits resulting from systemic delivery of the therapeutic hSGCG transgene and ultimately amelioration of the disease phenotype in SGCG- / - mice, laser monitoring of open-field cage activity was performed on all groups of mice. Fig.12The graphs in the Figures show that SGCG- / - mice had a 23.64% decrease in overall movement in the x and y planes compared to normal BL6 WT (BL6 WT: 7655.42 beam breaks / hour vs. SGCG- / -: 5846.00 beam breaks / hour). Qualitative observations showed that mice treated with scAAVrh.74.MHCK7.hSGCG were more active overall than SGCG- / - mice, and quantitative measurements of open field cage activity showed a 24.90% increase in movement (SGCG- / -: 5846.00 beam breaks / hour vs. treated: 7301.80 beam breaks / hour). Detailed values ​​for each parameter in individual mice were also measured.

[0190] Example 7: Toxicology and Vector Biodistribution

[0191] The purpose of this study was to evaluate any potential toxicity or safety issues of hSGCG gene therapy in SGCG- / - mice 3 months after delivery of the test article scAAVrh.74.MHCK7.hSGCG using the same animals described above. At 4 weeks of age, 1.0x10 13 Total dose of vg (5x10 14 vg / kg), the test article was administered to 5SGCG- / -. Six uninjected SGCG- / - mice were used as untreated sick controls, while five C57BL / 6WT mice were used as normal healthy controls (Table 6). A complete necropsy was performed on all mice to extract six skeletal muscles (TA, GAS, QUAD, GLUT, PSOAS and TRI) on the left and right sides, the diaphragm and heart, and internal organs including the lungs, kidneys, liver, spleen and gonads. In order to evaluate the safety of our vector, hematoxylin and eosin staining was performed on frozen sections of muscle tissue, and all harvested organs were fixed with formalin and stained with hematoxylin and eosin. These sections were then formally examined for toxicity by an independent veterinary pathologist, and no adverse reactions were found. The results are summarized in Table 7 below, and a detailed histopathology report was prepared. Quantitative PCR was performed to evaluate the biodistribution of the vector, and these results are shown in Tables 7 and Fig.13 middle.

[0192] A review of histopathology of vector-transduced tissues

[0193] To determine the safety and toxicological profile of scAAVrh.74.MHCK7.hSGCG using systemic delivery, all skeletal muscle (including diaphragm), heart and five other organs harvested from the SGCG- / - mouse group administered with the vector and controls from this preclinical study were stained with H&E, and sections of each tissue were formally examined by an independent veterinary pathologist. Details of the cohort and study design are shown in Table 6.

[0194] Table 6. Summary of the scAAVrh.74.MHCK7.hSGCG gene transfer histopathology study cohort

[0195]

[0196] In summary, IV injection of scAAVrh.74.MHCK7.hSGCG did not cause any microscopic changes in the myofibers of any skeletal muscle examined (Table 7). In addition, no treatment-related lesions were seen in any tissue evaluated histologically, indicating that the test article was well tolerated, see the full report in Appendix J (Report No. AAVrh74-SGCG-MOUSE-001.1). Any noted changes were observed in both treated and control mice and were considered incidental findings. In addition, independent review showed that administration of the test article scAAVrh.74.MHCK7.hSGCG significantly reduced muscle fiber atrophy, degeneration, and destruction relative to reference specimens of control mice, suggesting that this vector can improve the extent of myopathy associated with SGCG deficiency in diseased mice.

[0197] Table 7. Histopathological results of scAAVrh.74.MHCK7.hSGCG safety study in SGCG- / - mice

[0198]

[0199] Vector genome biodistribution

[0200] The presence of test article specific DNA sequences was examined using a real-time quantitative PCR assay (qPCR). Biodistribution analysis was performed on tissue samples collected from two vehicle-dosed SGCG- / - animals. A positive signal indicated equal to or greater than 100 single-stranded DNA copies / μg of genomic DNA detected. Tissues were harvested at necropsy and vector-specific primer probe sets specific for the MHCK7 promoter sequence were used. Table 8 and Fig.13 Depicted are the vector genome copy numbers detected in each tissue sample from mice injected with scAAVrh.74.MHCK7.hSGCG.

[0201] scAAVrh.74.MHCK7.hSGCG transcripts were detected at varying levels in all collected tissues. As expected, due to the nature of the intravenous delivery route, high levels of vector were detected in the liver, but the highest levels were found in skeletal muscle and heart. The lowest levels were detected in the lungs, kidneys, and spleen. These data indicate that the test article was effectively delivered to all studied tissues of the vector-dosed mice.

[0202] Table 8. Quantitative PCR results after systemic delivery of high-dose scAAVrh.74.MHCK7.hSGCG in SGCG- / - mice

[0203]

[0204] Serum chemistry analysis

[0205] To further assess liver function, the applicant assessed the levels of two liver enzymes, which are normal serum chemistry parameters, alkaline aminotransferase (ALT) and aspartate aminotransferase (AST). Elevations in any of these enzymes can indicate hepatocellular damage and impaired liver function. The applicant analyzed the serum of all 6 C57BL / 6WT mice, all 6 untreated SGCG- / - mice, and all 5 scAAVrh.74.MHCK7.hSGCG-administered mice. Fig.14A ALT in untreated SGCG- / - mice was shown to be elevated to twice the level observed in healthy BL6 WT mice (BL6 WT: 44.20 U / L vs. SGCG- / -: 89.00 U / L). IV delivery of scAAVrh.74.MHCK7.hSGCG to SGCG- / - mice resulted in a 32.02% reduction in ALT levels (SGCG- / -: 89.00 U / L vs. treated: 60.50 U / L). Fig. 14B AST levels in all three groups of mice showed a significant increase of 113.27% in untreated SGCG- / - mice (BL6 WT: 326.00 U / L vs. SGCG- / -: 695.25 U / L). These AST levels were reduced by 41.10% ( Fig. 14B ). Taken together, although liver enzymes, considered biomarkers of liver damage, were elevated in sick SGCG- / - mice, systemic hSGCG gene transfer normalized the levels of ALT and AST in sick SGCG- / - mice. Individual values ​​for each enzyme were determined for all mice.

[0206] In conclusion, systemic delivery of two different doses of AAV virus carrying the hSGCB transgene was shown to be safe and nontoxic. The doses tested included 1.2x10 13 Total dose of vg (6.0x10 14 vg / kg) and 1.0x10 13 Total dose of vg (5.0x10 14 vg / kg). In particular, high doses (1.0x10 13 Total VG dose – 5.0x10 14 vg / kg) of scAAVrh.74.MHCK7.hSGCG can safely and effectively restore the expression of γ-sarcoglycan and reverse the dystrophic histopathology of diseased muscle.

Claims

1. Use of rAAV vectors in the preparation of drugs for the following effects: a) treating gamma-sarcoglycanopathy in a subject; b) treating a subject with limb-girdle muscular dystrophy type 2C; or c) Treat muscle fiber atrophy and hypertrophy, The rAAV vector comprises a polynucleotide sequence encoding γ-sarcoglycan, wherein the polynucleotide sequence encodes the amino acid sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein the polynucleotide sequence encoding γ-sarcoglycan is codon optimized.

3. The use according to claim 1 or 2, wherein the polynucleotide sequence encoding γ-sarcoglycan comprises SEQ ID NO:

1.

4. The use according to any one of claims 1-3, wherein the rAAV vector is a self-complementary AAV vector genome, which comprises a genome lacking AAV rep and cap DNA, and is a serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh74 vector, or a variant thereof.

5. The use according to any one of claims 1-4, wherein the rAAV vector is a serotype AAV rh74 vector.

6. The use according to any one of claims 1-5, wherein the rAAV vector comprises an AAV rh.74 capsid.

7. The use according to claim 6, wherein the AAV rh.74 capsid comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The use according to any one of claims 1 to 7, wherein the polynucleotide sequence encoding γ-sarcoglycan is operably linked to a muscle-specific control element.

9. The use according to claim 8, wherein the muscle-specific control element is selected from the group consisting of human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor mef elements, muscle creatine kinase (MCK), truncated MCK (tMCK) promoter, myosin heavy chain (MHC) element, MHCK7 promoter, C5-12, mouse creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor, steroid-inducible element, and glucocorticoid response element (gre).

10. The use according to claim 8, wherein the muscle-specific control element is a truncated MCK (tMCK) promoter.

11. The use according to claim 8, wherein the muscle-specific control element is the MHCK7 promoter.

12. The use according to claim 11, wherein the MHCK7 promoter consists of the nucleotide sequence shown in SEQ ID NO:

4.

13. The use according to any one of claims 1-12, wherein the rAAV vector is formulated for intramuscular or intravenous use.

14. The use according to any one of claims 1-8, wherein the rAAV vector is formulated for systemic use.

15. The use according to any one of claims 1-8, wherein the rAAV vector is formulated for parenteral use.

16. The use according to any one of claims 1 to 15, wherein the amino acid sequence of SEQ ID NO: 2 encodes a protein having γ-sarcoglycan activity.

17. The use of claim 16, wherein the protein increases muscle strength, muscle endurance and / or muscle mass in one or more muscles of a subject suffering from muscular dystrophy.

18. Use according to claim 17, wherein the one or more muscles are selected from the group consisting of heart, diaphragm, thigh, calf, pelvic girdle and arm.

19. The use according to claim 16 or 17, wherein muscle strength, muscle endurance and / or muscle mass are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, or at least 80% compared to untreated control subjects.

20. The use according to claim 16, wherein the protein in a subject suffering from muscular dystrophy: Reduce degenerated fibers, necrotic fibers, or inflammation; Elevated creatine kinase levels; or Reduce malnutrition calcification.

21. A polynucleotide having a codon-optimized nucleotide sequence that is at least 95% identical to SEQ ID NO:

1.

22. The polynucleotide of claim 21, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:

1.

23. A recombinant adeno-associated virus (AAV) vector comprising the polynucleotide of claim 21 or 22.

24. The rAAV vector of claim 23, wherein the rAAV vector is a self-complementary AAV vector genome comprising a genome lacking AAV rep and cap DNA, and is a serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh74 vector, or a variant thereof.

25. The rAAV of claim 24, wherein the rAAV vector is a serotype AAV rh74 vector.

26. The rAAV of claim 24 or 25, wherein the rAAV vector comprises an AAV rh.74 capsid.

27. The rAAV of claim 26, wherein the AAV rh.74 capsid comprises the amino acid sequence shown in SEQ ID NO:

10.

28. The rAAV of any one of claims 23 to 27, wherein the genome of the rAAV vector further comprises a muscle-specific control element.

29. The rAAV of claim 28, wherein the muscle-specific control element is selected from the group consisting of a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor mef element, a muscle creatine kinase (MCK), a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) element, a MHCK7 promoter, C5-12, a rat creatine kinase enhancer element, a skeletal fast-twitch 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 steroid-inducible element, and a glucocorticoid response element (gre).

30. The rAAV according to any one of claims 23 to 29, wherein the genome of the rAAV vector comprises an intron, and the intron comprises the nucleotide sequence shown in SEQ ID NO:

5.

31. The rAAV according to any one of claims 23 to 28, further comprising a MHCK7 promoter.

32. The rAAV of claim 31, wherein the MHCK promoter comprises the nucleotide sequence shown in SEQ ID NO:

4.

33. The rAAV of any one of claims 23-32, wherein the polynucleotide comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

3.

34. The rAAV of any one of claims 23-33, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:

3.

35. The rAAV of any one of claims 23-34, for use in treating gamma-sarcoglycanopathy; increasing muscle strength, muscle endurance, and / or muscle mass; reducing fibrosis; or reducing contraction-induced damage in a subject.

36. The rAAV of any one of claims 23-34, for use in treating muscular dystrophy or muscle fiber atrophy and hypertrophy; reducing degenerated fibers, necrotic fibers, or inflammation; increasing creatine kinase levels; or reducing dystrophic calcification in a subject suffering from muscular dystrophy.

37. The rAAV of claim 36, wherein the muscular dystrophy is limb-girdle muscular dystrophy.

38. The rAAV of claim 37, wherein the limb-girdle muscular dystrophy is limb-girdle muscular dystrophy type 2C.

39. The rAAV of any one of claims 36-38, wherein the treatment comprises administering the rAAV vector or a composition comprising the rAAV vector and a pharmaceutically acceptable carrier by intramuscular injection or intravenous injection; systemically, or parenterally by injection, infusion or implantation.

40. The rAAV of any one of claims 36-39, wherein the use increases muscle strength, muscle endurance and / or muscle mass of one or more muscles of the subject.

41. The rAAV of claim 40, wherein the one or more muscles are selected from the group consisting of heart, diaphragm, thigh, calf, pelvic girdle, and arm.

42. The rAAV of claim 40 or 41, wherein muscle strength, muscle endurance, and / or muscle mass is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, or at least 80% compared to an untreated control subject.

43. Use of a polynucleotide comprising a codon-optimized nucleotide sequence that is at least 95% identical to SEQ ID NO: 1 in the preparation of a medicament for treating gamma-sarcoglycanopathy; increasing muscle strength, muscle endurance and / or muscle mass; or reducing fibrosis or reducing contraction-induced damage in a subject in need thereof.

44. Use of a codon-optimized polynucleotide comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1 in the preparation of a medicament for treating muscular dystrophy or muscle fiber atrophy and hypertrophy; reducing degenerated fibers, necrotic fibers, or inflammation; increasing creatine kinase levels; or reducing dystrophic calcification in a subject suffering from muscular dystrophy.

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