Adeno-associated viral vector delivery of beta-sarcoglycan and treatment of muscular dystrophy

Abstract

To provide adeno-associated viral vector delivery of β-sarcoglycan and treatment of muscular dystrophy.SOLUTION: Disclosed herein are: a method for treating muscular dystrophy comprising administering a recombinant AAV(rAAV)scAAVrh74.MHCK7.hSGCB vector; a method for expressing beta-sarcoglycan gene in a patient; a pharmaceutical composition comprising rAAV; and a method for producing rAAV. Also disclosed herein are: a gene therapeutic vector (e.g., AAV) for expressing β-sarcoglycan; a method for alleviating and / or preventing fibrosis by delivering β-sarcoglycan to muscles; and / or increasing muscular strength; and / or a method for treating a mammal subject suffering from muscular dystrophy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 810,917, filed February 26, 2019, U.S. Provisional Application No. 62 / 834,012, filed April 15, 2019, U.S. Provisional Application No. 62 / 858,644, filed June 7, 2019, U.S. Provisional Application No. 62 / 881,901, filed August 1, 2019, U.S. Provisional Application No. 62 / 909,564, filed October 2, 2019, and U.S. Provisional Application No. 62 / 910,779, filed October 4, 2019, all of which are incorporated herein by reference in their entireties.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and is identified as follows: Filename: 54016_Seqlisting.txt, Size: 33,466 bytes, Created: February 12, 2020.

[0003] Described herein are therapeutic vectors, such as AAV vectors that express β-sarcoglycan, and methods of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy. [Background technology]

[0004] Limb-girdle muscular dystrophy (LGMD) type 2E (LGMD2E) is an autosomal recessive disorder resulting from mutations in the gene encoding β-sarcoglycan (SGCB), resulting in loss of functional protein. LGMD2E represents a relatively common and severe form of LGMD in the United States, with a reported incidence of 1 / 200,000–1 / 350,000 worldwide. (2) The absence of β-sarcoglycan leads to progressive dystrophy accompanied by chronic muscle fiber loss, inflammation, fatty replacement, and fibrosis, all of which lead to deterioration of muscle strength and function. (3, 4) As complexes, sarcoglycans (α-, β-, γ-, and δ-), ranging in size from 35 to 50 kD, (5) are all transmembrane proteins that provide stability to the sarcolemma, protecting it from mechanical stress during muscle activity. (3) Loss of β-sarcoglycan in LGMD2E typically leads to a concomitant loss of other sarcoglycan proteins to varying degrees, contributing to muscle membrane fragility, leading to muscle fiber loss. 1 The clinical phenotype of LGMD2E varies widely, but diagnosis is typically made by age 10 years, with loss of ambulation occurring by the mid- to late teens. Patients present with elevated serum creatine kinase (CK), proximal muscle weakness, difficulty arising from bed rest, and progressive loss of ambulation. Cardiac involvement occurs in 50% of cases.

[0005] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is presented in GenBank Accession No. NC_002077, the complete genome of AAV-2 is presented in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 {1983), the complete genome of AAV-3 is presented in GenBank Accession No. NC_1829, the complete genome of AAV-4 is presented in GenBank Accession No. NC_001829, the AAV-5 genome is presented in GenBank Accession No. AF085716, the complete genome of AAV-6 is presented in GenBank Accession No. NC_001862, at least portions of the AAV-7 and AAV-8 genomes are presented in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is presented in Gao et al. The AAV-10 genome is presented in Mol. Ther., 13(1):67-76(2006), and the AAV-11 genome is presented in Virology, 330(2):375-383(2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within 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.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), drive the rep gene to produce four rep proteins (rep78, rep68, rep52, and rep40). The rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites contribute to the production of the 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).

[0006] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, enabling the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA within a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. This makes AAV less susceptible to cryopreservation, as it readily withstands the conditions used to inactivate adenovirus (56°C–65°C for several hours). AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0007] Several studies have demonstrated long-term (more than 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 Sci 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). Furthermore, because muscle is highly vascularized, Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci As described in J. Immunol., 94:13921-13926 (1997), recombinant AAV transduction results in the appearance of transgene products in the systemic circulation after intramuscular injection. Furthermore, Lewis et al., J. Virol., 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutics. An emerging form of therapy for LGMD2E is viral-mediated gene delivery to restore wild-type proteins to affected muscles, resulting in recovery of muscle function. Given that a subset of patients may develop cardiomyopathy (8, 9, 10, 13), this should be considered in the long-term care of these patients. Previous reports have well characterized Sgcb null mice. Araishi et al.3 developed β-sarcoglycan-deficient mice, which exhibit loss of not only sarcospan but all sarcoglycans, with at least minor preservation of merosin, dystroglycan, and dystrophin, recapitulating the clinical picture seen in LGMD2E. The histological changes in this animal model, including overt skeletal muscle fibrosis, were also a prototype of the clinical counterpart. (14) Dressman et al. (25) injected the transversus abdominis muscle with rAAV2.CMV.SGCB. Getut expression persisted for 21 months, and muscle fibers were protected from recurrent necrosis. The use of self-complementary AAVs to enhance transgene expression, 16 muscle-specific promoters to better target skeletal muscle, (20, 26) and optimization of the human β-sarcoglycan gene (hSGCB) have also been described. Both gene restoration and reduction of fibrosis are necessary to improve function in patients with LGMD and other muscular dystrophies. There is a need for methods to reduce fibrosis that can be repaired with gene restoration methods for more effective treatment of LGMD and other muscular dystrophies. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 9,434,928 [Non-patent literature]

[0009] [Non-Patent Document 1] Srivastava et al., J. Virol., 45:555-564{1983) [Non-patent document 2] Gao et al., J.Virol., 78:6381 - 6388(2004)

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Summary of the Invention

Means for Solving the Problems

[0010] Described herein are gene therapy vectors (e.g., AAV) that express the β-sarcoglycan gene, and methods for delivering β-sarcoglycan to muscle to reduce and / or prevent fibrosis, and / or increase muscle strength, and / or treat mammalian subjects suffering from muscular dystrophy.

[0011] In one aspect, a method of treating muscular dystrophy in a subject in need thereof includes administering to a subject in need thereof recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV delivers approximately 1.0 x 10 mAb to a subject in need thereof, based on a supercoiled plasmid as a quantification standard, using a systemic route of administration. 12 vg / kg ~ approx. 5.0×10 14 at a dose of 1.0 × 10 vg / kg or based on linearized plasmid as a quantification standard 13 vg / kg ~ approx. 1.0×10 14 vg / kg, wherein the serum creatine kinase (CK) level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV.

[0012] In another aspect, a method is provided for treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the level of beta-sarcoglycan gene expression in the subject's cells increases after administration of the rAAV compared to the level of beta-sarcoglycan gene expression before administration of the rAAV, the number of beta-sarcoglycan-positive fibers in the subject's muscle tissue increases after administration of the rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of the rAAV, or motor function is improved in the subject compared to the subject's motor function before administration of the rAAV, wherein the motor function is determined by a timed 100-meter walk test.

[0013] In another aspect, the present disclosure provides a method of treating limb-girdle muscular dystrophy in a subject in need thereof, comprising administering to a subject a dose of about 5.0×10 ATP based on supercoiled plasmid as a quantitative standard. 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or approximately 7.41 x 10 13 The present disclosure provides a method of expressing a beta-sarcoglycan gene in cells of a subject, the method comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct comprising a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 19. In one aspect, the present disclosure provides a method of increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, the method comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 19.

[0014] In one aspect, described herein is a method of increasing alpha-sarcoglycan expression in a subject in need thereof, comprising administering to the subject an rAAV comprising an scAAVrh74.MHCK7.hSGCB construct having a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO: 19. In another aspect, described herein is a method of increasing alpha-sarcoglycan localization to the cell membrane in a subject in need thereof, comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO: 19. In another aspect, provided is a method of increasing sarcoglycan expression in muscle tissue or improving muscle function in a subject, comprising administering to the subject an rAAV comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:19. In another aspect, the present disclosure provides a method for increasing sarcoglycan expression in muscle tissue of a subject, the method comprising administering to the subject a construct comprising a nucleotide sequence encoding a first sarcoglycan, and detecting increased expression of at least a second sarcoglycan in the cell membrane of a cell expressing the first sarcoglycan.

[0015] In another aspect, a composition is described comprising a rAAV scAAVrh74.MHCK7.hSGCB vector, a buffering agent, an ionic strength agent, and a surfactant. In another aspect, a pharmaceutical composition is described herein comprising a recombinant AAV (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the scAAVrh74.MHCK7.hSGCB comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 19.

[0016] In another aspect, a method of producing a recombinant AAV scAAVrh74.MHCK7.hSGCB is provided, comprising transfecting a plasmid into a cell, wherein the plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 24. In particular, the plasmid comprises the nucleotide sequence of SEQ ID NO: 24. In another embodiment, the plasmid comprises the nucleotide sequence of SEQ ID NO: 19.

[0017] In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding β-sarcoglycan. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1, for example, and encodes a protein that retains β-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan consists of the nucleotide sequence set forth in SEQ ID NO: 1.

[0018] In another aspect, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding a β-sarcoglycan that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2, and the protein retains β-sarcoglycan activity.

[0019] In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding a functional β-sarcoglycan comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 1, or its complement.

[0020] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as 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 (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, but the rate of hybridization will be affected. Where deoxyoligonucleotide hybridization is involved, examples of additional stringent hybridization conditions include washing in 6×SSC, 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0021] When ranges are used herein for physical properties such as molecular weight, concentration, or dosage, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is approximate within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary, for example, by 1% to 15% of the stated numerical value or numerical range.

[0022] Other agents can be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization. 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, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate; other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4, although the rate of hybridization is largely independent of pH under typical ionic strength conditions. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art to take these variables into account and allow DNAs of different sequence similarities to form hybrids.

[0023] In another embodiment, the recombinant AAV vectors described herein can be operably linked to a muscle-specific regulatory element, such as a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor MEF, a muscle creatine kinase (MCK), a tMCK (a truncated MCK), a myosin heavy chain (MHC), a MHCK7 (a hybrid version of MHC and MCK), a C5-12 (a synthetic promoter), a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0024] In some embodiments, the muscle-specific promoter is MHCK7 (SEQ ID NO:4). An exemplary rAAV described herein is pAAV.MHCK7.hSCGB, which comprises the nucleotide sequence of SEQ ID NO:3. Within the nucleotide sequence of SEQ ID NO:3, the MCHK7 promoter spans nucleotides 130-921, the SV40 chimeric intron (SEQ ID NO:20) spans nucleotides 931-1078, the β-sarcoglycan sequence (SEQ ID NO:1) spans nucleotides 1091-2047, and polyA (SEQ ID NO:21) spans nucleotides 2054-2106. In some embodiments, the rAAV pAAV.MHCK7.hSCGB comprises the nucleotide sequence of SEQ ID NO:19. Within the nucleotide sequence of SEQ ID NO: 19, the MCHK7 promoter spans nucleotides 128-919, the SV40 chimeric intron spans nucleotides 929-1076, the β-sarcoglycan sequence spans nucleotides 1086-2042, and poly A spans nucleotides 2049-2101.

[0025] In some embodiments, the rAAV pAAV.MHCK7.hSCGB is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, identical to the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:19. 99% identical, or at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:2.

[0026] In one embodiment, the polynucleotide sequence encodes a protein that retains sarcoglycan activity, including beta- and / or alpha-sarcoglycan activity, hi another embodiment, the polynucleotide sequence encodes a protein that retains beta-sarcoglycan activity.

[0027] In some embodiments, the muscle-specific promoter is tMCK (SEQ ID NO: 6). An exemplary rAAV described herein is pAAV.tMCK.hSCGB, which comprises the nucleotide sequence of SEQ ID NO: 5. Within the nucleotide sequence of SEQ ID NO: 5, the tMCK promoter spans nucleotides 141-854, the SV40 chimeric intron spans nucleotides 886-1018, the β-sarcoglycan sequence spans nucleotides 1058-2014, and polyA spans nucleotides 2021-2073. In some embodiments, the polynucleotide sequence encoding pAAV.tMCK.hSCGB comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to the nucleotide sequence set forth, for example, in SEQ ID NO:5, and the polynucleotide sequence encodes a protein that retains sarcoglycan activity, including, but not limited to, beta- and / or alpha-sarcoglycan activity.

[0028] AAV can be any serotype, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, and AAVrh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0029] Compositions comprising any of the rAAV vectors described herein are also contemplated.

[0030] In some embodiments, the present disclosure includes a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:19. or a nucleotide sequence encoding a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, and more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 2. Additionally, the present disclosure provides compositions or pharmaceutical compositions comprising scAAVrh74.MHCK7.hSCGB rAAV vectors that include the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 19, or that encode a polypeptide that includes the amino acid sequence of SEQ ID NO: 2.

[0031] 1. A method of treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV is administered in an amount of about 1.0 x 10 12 vg / kg ~ approx. 5.0×10 14 vg / kg.

[0032] 1. A composition for treating muscular dystrophy, comprising about 1.0×10 12 vg / kg ~ approx. 5.0×10 14 Also provided is a composition comprising the recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGC at a dose of 1000 mg / kg, wherein the composition is formulated for systemic administration.

[0033] Additionally, there is provided a use of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGC for the preparation of a medicament for treating muscular dystrophy, wherein the medicament comprises about 1.0 x 10 12 vg / kg ~ approx. 5.0×10 14 The use is provided wherein the medicament comprises scAAVrh74.MHCK7.hSGC at a dose of 1000 mg / kg or less, and the medicament is formulated for systemic administration.

[0034] In any of the provided methods, compositions and uses, the level of beta-sarcoglycan gene expression in the subject's cells increases after administration of rAAV compared to the level of beta-sarcoglycan gene expression before administration of rAAV, the serum creatine kinase (CK) level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV, and / or the number of beta-sarcoglycan-positive fibers in the subject's muscle tissue increases after administration of rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of rAAV.

[0035] In another embodiment, in any of the provided methods, compositions and uses, motor function is improved in the subject compared to the motor function of the subject before administration of rAAV, and motor function is measured by a 100-meter timed walk test.For example, motor function is improved by at least 5% one month or 30 days after gene transfer, at least 10% two months or 60 days after gene transfer, or at least 15% three months or 90 days after gene transfer.In some embodiments, motor function is improved by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50%.

[0036] For example, in any of the provided methods, compositions, and uses, the systemic administration route is an intravenous route. For example, rAAV is administered using an intravenous route, and the dose of rAAV administered is about 1.85 x 10 based on a linearized plasmid as a quantitative standard. 13 vg / kg or approximately 7.41 × 1013 The dose of rAAV administered is approximately 5 x 10 vg / kg, based on supercoiled plasmid as a quantification standard. 13 vg / kg or approximately 2 × 10 14 vg / kg.

[0037] In some embodiments, the dose of rAAV administered using the intravenous route is about 1.0 x 10, based on supercoiled plasmid as a quantification standard. 13 vg / kg ~ approx. 5×10 14 , or approximately 1.0 x 10 based on linearized plasmid as a quantification standard. 13 vg / kg ~ approx. 1.0×10 14 vg / kg.

[0038] In addition, the dose of rAAV administered is approximately 1.5 × 10 13 vg ~ approx. 2×10 16 vg, or 1.5 × 10 13 vg~1×10 16 vg, or approximately 1.5 × 10 13 vg ~ approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 15 vg. Additionally, in any of the methods, provided methods, compositions, and uses, the dose of rAAV is administered at a concentration of about 10 mL / kg. In any of the provided methods, provided methods, compositions, and uses, the muscular dystrophy is limb-girdle muscular dystrophy.

[0039] Additionally, there is provided a method of treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV is administered in an amount of about 1.0 x 10 12 vg / kg ~ approx. 5.0×10 14vg / kg, wherein the level of beta-sarcoglycan gene expression in the subject's cells is increased after administration of the rAAV compared to the level of beta-sarcoglycan gene expression before administration of the rAAV, the serum CK level in the subject is decreased after administration of the rAAV compared to the serum CK level before administration of the rAAV, or the number of beta-sarcoglycan-positive fibers in the subject's muscle tissue is increased after administration of the rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of the rAAV. For example, in any of the provided methods, the systemic administration route is intravenous, and the administered dose of rAAV is about 5.0 x 10 13 In another embodiment, the dose of rAAV administered is about 2.0 x 10 vg / kg, based on supercoiled plasmid as a quantification standard. 14 In another embodiment, the dose of rAAV administered is about 7.41 x 10 vg / kg, based on linearized plasmid as a quantification standard. 13 In another embodiment, the dose of rAAV administered is about 1.85 x 10 vg / kg, based on linearized plasmid as a quantification standard. 13 In addition, the administered dose of rAAV is approximately 1.5×10 13 vg ~ approx. 2×10 16 vg, or 1.5 × 10 13 vg~1×10 16 vg, or approximately 1.5 × 10 13 vg ~ approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 15 vg. Additionally, in any of the methods, the dose of rAAV is administered at a concentration of about 10 mL / kg. In any of the provided methods, the muscular dystrophy is limb-girdle muscular dystrophy.

[0040] In some embodiments, the disclosure includes a method of treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein motor function is demonstrably improved in the subject compared to the subject's motor function before administration of the rAAV, as determined by a timed 100-meter walk test. In some aspects, motor function improves by at least 5% at 1 month or 30 days after gene transfer, at least 10% at 2 months or 60 days after gene transfer, or at least 15% at 3 months or 90 days after gene transfer. In some aspects, motor function improves by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50%.

[0041] Methods for increasing alpha-sarcoglycan levels in a subject in need thereof are provided, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19. Additionally, compositions for increasing alpha-sarcoglycan levels in a subject in need thereof are provided, the compositions comprising a scAAVrh74.MHCK7.hSGCB construct comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19. Also provided is the use of a scAAVrh74.MHCK7.hSGCB construct comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19 for the preparation of a medicament for increasing alpha-sarcoglycan levels in a subject in need thereof. In some embodiments, alpha-sarcoglycan co-localizes to the membrane of cells expressing beta-sarcoglycan encoded by scAAVrh74.MHCK7.hSGCB.

[0042] In some embodiments, the scAAVrh74.MHCK7.hSGCB construct comprises an intron sequence. In one embodiment, the intron sequence comprises the nucleotide sequence of SEQ ID NO:20. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a polyA sequence. In one embodiment, the polyA sequence comprises the nucleotide sequence of SEQ ID NO:21. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a 5' inverted terminal repeat (ITR) sequence. In one embodiment, the 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO:22. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a 3' inverted terminal repeat (ITR) sequence. In one embodiment, the 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:23.

[0043] Also provided is a method for increasing sarcoglycan expression in muscle tissue of a subject, comprising administering to the subject a construct comprising a nucleotide sequence encoding a first sarcoglycan and detecting increased expression of at least a second sarcoglycan in the cell membrane of cells expressing the first sarcoglycan. In some embodiments, the first sarcoglycan is β-sarcoglycan (SGCB), and the second sarcoglycan is α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD).

[0044] In any of the provided methods, uses, and compositions for treating muscular dystrophy, the subject is 4 to 15 years old, has confirmed beta-sarcoglycan (SGCB) mutations in both alleles, is negative for AAVrh74 antibodies, and / or has a 100-meter walk test score of greater than 40% or normal. In any of the provided methods, uses, and compositions for treating muscular dystrophy, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject, e.g., a subject in the 1 to 10 year old range. In some embodiments, the subject is 4 to 15 years old. In one embodiment, the subject is an adolescent subject, e.g., a subject in the 10 to 19 year old range. Additionally, in one embodiment, the subject is a young adult subject, e.g., a subject in their late teens or early twenties, e.g., a subject may be in the 15 to 29 year old range. In some embodiments, the subject is a middle-aged adult or an elderly subject, such that a middle-aged adult may be in the 25 to 55 year old range and an elderly subject may be over 50 years old.

[0045] In some embodiments, rAAV is administered by injection, infusion, or implantation. For example, rAAV is administered by infusion over approximately 1 to 2 hours. Additionally, rAAV is administered intravenously via a peripheral limb vein.

[0046] In a method of treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, the rAAV is administered using a systemic route to a concentration of about 1.0 x 10, based on supercoiled plasmid as a quantification standard. 12 vg / kg ~ approx. 5.0×10 14The rAAV is administered at a dose of 1000 mg / kg, and the rAAV comprises the human β-sarcoglycan nucleotide sequence of SEQ ID NO: 1. In addition, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 4. In some embodiments, the rAAV is of the AAVrh.74 serotype. In addition, the rAAV comprises the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 19.

[0047] In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises an intron sequence. In one embodiment, the intron sequence comprises the nucleotide sequence of SEQ ID NO:20. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a polyA sequence. In one embodiment, the polyA sequence comprises the nucleotide sequence of SEQ ID NO:21. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a 5' inverted terminal repeat (ITR) sequence. In one embodiment, the 5' ITR sequence comprises the nucleotide sequence of SEQ ID NO:22. In another embodiment, the scAAVrh74.MHCK7.hSGCB construct comprises a 3' inverted terminal repeat (ITR) sequence. In one embodiment, the 3' ITR sequence comprises the nucleotide sequence of SEQ ID NO:23.

[0048] In an exemplary embodiment, a method of treating muscular dystrophy in a subject in need thereof includes administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV is administered in a dose of about 1.0 x 10 12 vg / kg ~ approx. 5.0×10 14 The subjects were afflicted with limb-girdle muscular dystrophy, and the rAAV was administered at a dose of approximately 5.0 x 10 vg / kg, based on supercoiled plasmid as a quantification standard. 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or 7.41 x 10 13The rAAV is administered by intravenous infusion over approximately 1-2 hours at a dose of 1000 mg / kg, and the rAAV comprises the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0049] Additionally, the present disclosure provides a composition for treating limb-girdle muscular dystrophy, comprising a dose of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV comprises the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 19, and wherein the composition is administered by intravenous infusion over approximately 1-2 hours to a patient in need thereof, in an amount of approximately 5.0 x 10 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or 7.41 x 10 13 The compositions are formulated to deliver a dose of 100 mg / kg.

[0050] The present disclosure also provides a use of a dose of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB for the preparation of a medicament for treating limb-girdle muscular dystrophy, the dose being approximately 5.0 x 10 based on a supercoiled plasmid as a dose quantification standard for rAAV. 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or 7.41 x 10 13 vg / kg, wherein the medicament is formulated to deliver the dose by intravenous infusion over approximately 1 to 2 hours.

[0051] The present disclosure further provides a method of improving motor function in a subject, the method comprising administering to the subject a construct comprising a nucleotide sequence having at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1, 3, 5, or 19. Additionally, a composition for improving motor function in a subject is provided, the composition comprising a construct comprising a nucleotide sequence having at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1, 3, 5, or 19. Also provided is the use of a construct comprising a nucleotide sequence having at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1, 3, 5, or 19 for the preparation of a medicament for improving motor function in a subject.

[0052] In any of the provided methods, uses, or compositions, the subject has a genetic mutation in a gene encoding a sarcoglycan or a muscular dystrophy. In some embodiments, the sarcoglycan is β-sarcoglycan (SGCB), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD). In some embodiments, the sarcoglycan is β-sarcoglycan or α-sarcoglycan.

[0053] In any of the provided methods, uses, or compositions, the level of beta-sarcoglycan gene expression in the cells of the subject is increased after administration of rAAV compared to the level of beta-sarcoglycan gene expression before administration of rAAV.

[0054] Additionally, in any of the provided methods, uses or compositions, expression of the beta-sarcoglycan gene in cells is detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration.

[0055] In any of the provided methods, uses, or compositions, the level of beta-sarcoglycan protein is at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or or at least 45%, or at least 46%, or at least 47%, or at least 48%, or at least 49%, or at least 50%, or at least 51%, or at least 52%, or at least 53%, or at least 54%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59%, or at least 60%, or at least 63%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% increase. For example, beta-sarcoglycan protein levels are increased by at least 33% when detected by measuring beta-sarcoglycan protein levels by Western blot in muscle biopsies before and after rAAV administration, or beta-sarcoglycan protein levels are increased by at least 38% or at least 39% when detected by measuring beta-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after rAAV administration.

[0056] In any of the methods, uses, or compositions provided herein, serum CK levels in a subject are reduced after administration of rAAV compared to serum CK levels before administration of rAAV. For example, the serum CK level in the subject is reduced by at least 50%, or at least 51%, or at least 52%, or at least 53%, or at least 54%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59%, or at least 60%, or at least 63%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 95%, or at least 98% 60 to 90 days after administration of the rAAV, 60 days, or 90 days after administration of the rAAV, compared to the serum CK level before administration of the rAAV.

[0057] In any of the methods, uses, or compositions provided herein, the number of beta-sarcoglycan-positive fibers in the muscle tissue of a subject is increased after administration of rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of rAAV. For example, the number of beta-sarcoglycan-positive fibers is detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration. For example, the number of beta-sarcoglycan-positive fibers in the muscle tissue of a subject is at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 45% after administration of rAAV. or at least 46%, or at least 47%, or at least 48%, or at least 49%, or at least 50%, or at least 51%, or at least 52%, or at least 53%, or at least 54%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59%, or at least 60%, or at least 63%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% increase.

[0058] In any of the methods, compositions and uses provided herein, the level of alpha-sarcoglycan in a subject is increased after administration of rAAV compared to the level of alpha-sarcoglycan before administration of rAAV. The level of alpha-sarcoglycan is detected by measuring the level of alpha-sarcoglycan protein by immunohistochemistry or Western blot in muscle biopsies before and after administration of rAAV.

[0059] Another embodiment provides a method for expressing a beta-sarcoglycan gene in a cell, comprising administering to a subject a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0060] Also provided is a composition for expressing a beta-sarcoglycan gene in a cell, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0061] The present disclosure also provides use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for expressing a beta-sarcoglycan gene in a cell, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0062] In any of the provided methods, uses, or compositions for expressing a beta-sarcoglycan gene in cells, expression of the beta-sarcoglycan gene in cells is detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.MHCK7.hSGCB construct. For example, the cells have an AAV viral copy number greater than 1. In addition, the beta-sarcoglycan gene is measured in a subject by detecting greater than 1 rAAV vector genome copy per nucleus.

[0063] Also provided is a composition for reducing serum CK levels in a subject in need thereof, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0064] The present disclosure also provides use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for reducing serum CK levels in a subject in need thereof, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0065] In any of these methods, uses and compositions, serum CK levels in the subject are reduced by at least 82% 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV.

[0066] A method for increasing beta-sarcoglycan-positive fibers in muscle tissue of a subject is provided, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 19.

[0067] Also provided is a composition for increasing beta-sarcoglycan-positive fibers in the muscle tissue of a subject, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:19, or that includes the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0068] The present disclosure also provides use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for increasing beta-sarcoglycan-positive fibers in the muscle tissue of a subject, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0069] In any of these methods, uses, and compositions, the number of beta-sarcoglycan-positive fibers is detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration. Additionally, in any of the methods, uses, and compositions, the number of beta-sarcoglycan-positive fibers is measured by detecting more than one rAAV vector genome copy per nucleus.

[0070] Another embodiment provides a method for increasing expression of alpha-sarcoglycan in a subject in need thereof, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:19, or comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0071] Also provided is a composition for increasing the expression of alpha-sarcoglycan in a subject in need thereof, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0072] The present disclosure also provides use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for increasing expression of alpha-sarcoglycan in a subject, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0073] Also provided is a method for increasing the localization of alpha-sarcoglycan to the cell membrane in a subject in need thereof, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0074] Also provided is a composition for increasing the localization of alpha-sarcoglycan to the cell membrane in a subject in need thereof, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0075] The present disclosure also provides the use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for increasing alpha-sarcoglycan localization to the cell membrane in a subject in need thereof, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0076] In any of these methods, uses, and compositions, alpha-sarcoglycan levels are detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration. Additionally, in any of the provided methods, uses, and compositions, alpha-sarcoglycan co-localizes to the membrane of cells expressing beta-sarcoglycan encoded by scAAVrh74.MHCK7.hSGCB.

[0077] Another embodiment provides a method for increasing sarcoglycan expression in muscle tissue of a subject in need thereof, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:19, or comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:19.

[0078] Also provided is a composition for increasing sarcoglycan expression in muscle tissue of a subject in need thereof, the composition comprising a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to, or includes, the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:19.

[0079] The present disclosure also provides use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for increasing sarcoglycan expression in muscle tissue of a subject in need thereof, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to, or comprises, the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:19.

[0080] In any of these methods, uses, and compositions for increasing sarcoglycan expression in muscle tissue, the subject has a genetic mutation in a gene encoding sarcoglycan or muscular dystrophy. For example, in any of these methods, uses, or compositions, the sarcoglycan is β-sarcoglycan (SGCB), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD).

[0081] Also provided are methods for producing recombinant AAV vector particles, comprising culturing cells transfected with a plasmid described herein and recovering recombinant AAV particles from the supernatant of the transfected cells. Viral particles comprising any of the recombinant AAV vectors described herein are also contemplated. In one embodiment, a method for generating rAAV comprises transferring an AAV vector plasmid into a host cell. In another embodiment, the plasmid comprises a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 24. In another aspect, the present disclosure provides a cell comprising an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 24. The cells described herein include insect cells, such as Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells), or mammalian cells (preferably human cells, e.g., human primary cells or established cell lines). In one embodiment, the mammalian cells comprise 293 cells, COS cells, HeLa cells, or KB cells.

[0082] In another embodiment, the plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, 3, 5, or 19. In some embodiments, the vector plasmid comprises the nucleotide sequence of any one of SEQ ID NO: 1, 3, 5, or 19. In some embodiments, the AAV vector plasmid is stably expressed in a host cell. Host cells stably harboring the AAV vector plasmid can be used to produce rAAV. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCB.KAN plasmid.

[0083] The methods of producing recombinant AAV vector particles provided herein can further include the step of transfecting a packaging plasmid and / or a helper virus into a host cell. For example, the method can further include the step of the packaging cell containing a stably integrated AAVcap gene and / or the step of the packaging cell containing a stably integrated AAVrep gene. The present invention also provides a cell containing a plasmid containing a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO:24, or a plasmid containing the nucleotide sequence of SEQ ID NO:24. Cells containing the nucleotide sequence of SEQ ID NO:1, 3, 5, or 19 are also provided.

[0084] Also provided is a method for reducing fibrosis in a mammalian subject in need thereof. In this regard, the method comprises administering a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein) to the mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In some embodiments, administering the AAV vector described herein (or a composition comprising an AAV vector described herein) reduces fibrosis in the subject's skeletal or cardiac muscle.

[0085] The term "muscular dystrophy" as used herein refers to a disorder that gradually reduces strength and muscle mass.Non-limiting examples of muscular dystrophy include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanopathy, congenital muscular dystrophy such as partial LAMA2 deficiency congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, type 1D congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, limb-girdle type 1A muscular dystrophy, limb-girdle type 2A muscular dystrophy, limb-girdle type 2B muscular dystrophy, limb-girdle type 2C muscular dystrophy. Dystrophies can include limb-girdle muscular dystrophy type 2D, limb-girdle muscular dystrophy type 2E, limb-girdle muscular dystrophy type 2F, limb-girdle muscular dystrophy type 2G, limb-girdle muscular dystrophy type 2H, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2J, limb-girdle muscular dystrophy type 2K, limb-girdle IC muscular dystrophy, ankylosing spinal muscular dystrophy with epidermolysis bullosa simplex, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy and Ullrich scleroatonic muscular dystrophy.In some embodiments, the subject suffers from limb-girdle muscular dystrophy type 2E (LGMD2E).

[0086] As used herein, the term "fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues after injury, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include collagen (e.g., collagen 1, collagen 2, or collagen 3) and fibronectin.

[0087] In another aspect, described herein is a method of increasing muscle strength and / or muscle mass in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein). In one embodiment, the subject is a human.

[0088] In any of the methods of the invention, the subject may be suffering from a muscular dystrophy, such as limb-girdle muscular dystrophy or any other dystrophin-associated muscular dystrophy.

[0089] Also provided are methods for treating muscular dystrophy in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein). In some embodiments, the muscular dystrophy is limb-girdle muscular dystrophy.

[0090] In any of the methods of the invention, the rAAV is administered by intramuscular or intravenous injection. In addition, in any of the methods of the invention, the rAAV is administered systemically, such as parenterally by injection, infusion, or implantation.

[0091] The compositions of the present invention are formulated for intramuscular or intravenous injection. In addition, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation.

[0092] In addition, any of the compositions is formulated for administration to a subject suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-associated muscular dystrophy). In some embodiments, the composition may further comprise a second recombinant AAV vector comprising the polynucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:8.

[0093] In any of the uses of the present invention, the medicament is formulated for intramuscular injection or intravenous injection.In addition, in any of the uses of the present invention, the medicament is formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.In addition, any of the medicaments can be prepared for administration to the subject suffering from muscular dystrophy (for example, limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy).In some embodiments, the medicament can further comprise a second recombinant AAV vector comprising the polynucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:8.

[0094] The present invention also provides formulations or compositions comprising rAAV virions comprising capsids derived from AAVrh74, a buffer, an ionic strength agent, and a detergent. In the provided formulations or compositions, the rAAV is present in a concentration of about 1.0 x 10 12 vg / ml ~ approx. 5.0x10 14 vg / ml, or approximately 5.0x10 12 vg / ml ~ approx. 1.0x10 14 In addition, the rAAV was at a concentration of approximately 2.0x10 13 vg / ml, 4x10 13 vg / ml, or 5x10 13 The concentration is 1000 mg / ml. In the provided formulations or compositions, the rAAV may be a scAAVrh74.MHCK7.hSGCB viral particle or a scAAVrh74.MHCK7.hSGCB vector. For example, in any of the provided formulations or compositions, the scAAVrh74.MHCK7.hSGCB comprises the nucleotide sequence of SEQ ID NO: 19.

[0095] In the provided formulations or compositions, the buffer comprises one or more of Tris, Tricine, Bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. For example, the buffer comprises Tris at a concentration of about 5 mM to about 40 mM, pH 8.0, or the buffer comprises Tris at about 20 mM, pH 8.0.

[0096] In any of the provided formulations or compositions, the ionic strength agent comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NHCl), ammonium acetate, magnesium chloride (MgCl), magnesium acetate, magnesium sulfate, manganese chloride (MnCl), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. For example, the ionic strength agent comprises MgCl at a concentration of about 0.2 mM to about 4 mM, or the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM, or the ionic strength agent comprises MgCl at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM, or the ionic strength agent comprises MgCl at a concentration of about 1 mM and NaCl at a concentration of about 200 mM.

[0097] In any of the provided formulations or compositions, the surfactant includes one or more of sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. For example, the poloxamers include one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. The poloxamers may be present at a concentration of about 0.00001% to about 1%. An exemplary surfactant is poloxamer 188 at a concentration of about 0.001%.

[0098] The preceding paragraphs are not intended to define all aspects of the invention; additional 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 combinations of features are not found together in the same sentence, paragraph, or section of this document. The invention includes, as an additional aspect, all embodiments of the invention that are somewhat narrower in scope than the variations defined in the particular paragraphs above. For example, if a particular aspect of the invention is described as a genus, it should be understood that each member of the genus is individually an aspect of the invention. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating muscular dystrophy in a subject in need thereof, comprising administering to the subject recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB; The rAAV was administered using a systemic route to deliver approximately 1.0 x 10 12 vg / kg ~ approx. 5.0×10 14 vg / kg, wherein the serum creatine kinase (CK) level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV. (Item 2) 1. A method of treating muscular dystrophy in a subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB; the level of beta-sarcoglycan gene expression in the cells of the subject is increased after administration of the rAAV compared to the level of beta-sarcoglycan gene expression before administration of the rAAV; the number of beta-sarcoglycan-positive fibers in the muscle tissue of the subject is increased after administration of the rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of the rAAV; or The method, wherein motor function is improved in the subject compared to the subject's motor function before administration of the rAAV, and the motor function is determined by a 100 meter timed walk test. (Item 3) 3. The method of claim 2, wherein the motor function improves by at least 5% at 1 month or 30 days after gene transfer, at least 10% at 2 months or 60 days after gene transfer, or at least 15% at 3 months or 90 days after gene transfer. (Item 4) 4. The method of item 2 or 3, wherein the motor function is improved by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50%. (Item 5) 5. The method of any one of items 1 to 4, wherein the rAAV is administered using an intravenous route. (Item 6) The rAAV was found to be approximately 5.0 x 10 based on supercoiled plasmid as a quantification standard. 13 vg / kg or approximately 2.0 × 10 14 vg / kg or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or 7.41 x 10 13 6. The method of any one of items 1 to 5, wherein the antibody is administered in an amount of 0.1 mg / kg. (Item 7) 7. The method of any one of items 1 to 6, wherein the rAAV is administered at a concentration of about 10 mL / kg. (Item 8) 8. The method of any one of items 1 to 7, wherein the rAAV is administered by injection, infusion, or implantation. (Item 9) 9. The method of any one of items 1 to 8, wherein the rAAV is administered by infusion over a period of about 1 to 2 hours. (Item 10) 9. The method of any one of items 1 to 8, wherein the rAAV is administered by an intravenous route via a peripheral limb vein. (Item 11) 11. The method of any one of items 1 to 10, wherein the rAAV comprises the human β-sarcoglycan nucleotide sequence of SEQ ID NO: 1. (Item 12) 12. The method of any one of items 1 to 11, wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 4. (Item 13) 13. The method of any one of items 1 to 12, wherein the rAAV is an rAAV of the serotype AAVrh.74. (Item 14) 14. The method of any one of items 1 to 13, wherein the rAAV comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 19. (Item 15) 15. The method of any one of items 1 to 14, wherein the rAAV comprises an intron sequence of SEQ ID NO: 20. (Item 16) 16. The method of any one of items 1 to 15, wherein the rAAV comprises a polyA sequence of SEQ ID NO: 21. (Item 17) 17. The method of any one of items 1 to 16, wherein the rAAV comprises a 5' inverted terminal repeat (ITR) sequence of SEQ ID NO: 22. (Item 18) 18. The method of any one of items 1 to 17, wherein the rAAV comprises a 3' inverted terminal repeat (ITR) sequence of SEQ ID NO: 23. (Item 19) 19. The method according to any one of items 1 to 18, wherein the muscular dystrophy is limb-girdle muscular dystrophy. (Item 20) 19. The method according to any one of items 1 to 18, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2E. (Item 21) 1. A method for treating limb-girdle muscular dystrophy in a subject in need thereof, comprising administering about 5.0 x 10 β-glucan monophosphate (GMD) to a subject in need thereof, based on supercoiled plasmid as a quantitative standard. 13 vg / kg or approximately 2.0 × 10 14vg / kg, or approximately 1.85 × 10 based on linearized plasmid as a quantification standard. 13 vg / kg or 7.41 x 10 13 administering to the subject an intravenous infusion of rAAV at a dose of 1000 mg / kg over approximately 1 to 2 hours, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 19. (Item 22) A method for expressing a beta-sarcoglycan gene in cells of a subject, comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 19. (Item 23) A method for increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in a subject's muscle tissue, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 19. (Item 24) The method of item 22 or 23, wherein the expression of the beta-sarcoglycan gene or the number of positive beta-sarcoglycan-positive fibers is detected by measuring beta-sarcoglycan protein levels by Western blot in muscle biopsies before and after administration of the rAAV. (Item 25) 23. The method of claim 22, wherein the expression of the beta-sarcoglycan protein is increased by at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40% after administration of rAAV. (Item 26) 26. The method of any one of items 22 to 25, wherein the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan-positive muscle fibers is detected by measuring the beta-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 27) 23. The method of claim 22, wherein the expression of the beta-sarcoglycan protein is increased by at least 39% after rAAV administration. (Item 28) The method of claim 23, wherein the number of beta-sarcoglycan-positive fibers in the muscle tissue of the subject is increased by at least 40, 41, or 42% after administration of the rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of the rAAV. (Item 29) 23. The method of claim 22, wherein the cells have an AAV viral copy number greater than 1. (Item 30) 30. The method of any one of items 22 to 29, wherein the serum CK level in the subject is decreased after administration of the rAAV compared to the serum CK level before said administration of the rAAV. (Item 31) 31. The method of claim 30, wherein the serum CK level in the subject is reduced by at least 82, 83, 84, 85, 86, 87, 88, 89, or 90% 60 to 90 days, 60 days, or 90 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. (Item 32) 32. The method of any one of items 1 to 31, wherein the level of alpha-sarcoglycan in the subject is increased after administration of the rAAV compared to the level of alpha-sarcoglycan before administration of the rAAV. (Item 33) A method for increasing the expression of alpha-sarcoglycan in a subject in need thereof, comprising administering to the subject an rAAV containing the scAAVrh74.MHCK7.hSGCB construct having a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 19. (Item 34) A method for increasing the localization of alpha-sarcoglycan to the cell membrane in a subject in need thereof, comprising administering to the subject a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 19. (Item 35) 35. The method of claim 33 or 34, wherein the alpha-sarcoglycan is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 36) 35. The method of claim 33 or 34, wherein the alpha-sarcoglycan is detected by measuring alpha-sarcoglycan protein levels by Western blot in muscle biopsies before and after administration of the rAAV. (Item 37) 37. The method of any one of paragraphs 34 to 36, wherein the alpha-sarcoglycan is co-localized to the membrane of cells expressing the beta-sarcoglycan encoded by scAAVrh74.MHCK7.hSGCB. (Item 38) A method for increasing sarcoglycan expression in a subject's muscle tissue or improving muscle function, comprising administering to the subject an rAAV comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 19. (Item 39) 39. The method of claim 38, wherein the subject has a genetic mutation in a gene encoding a sarcoglycan or suffers from muscular dystrophy. (Item 40) 39. The method of claim 38, wherein the sarcoglycan is β-sarcoglycan (SGCB), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD). (Item 41) 39. The method of claim 38, wherein the nucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 19. (Item 42) A method for increasing sarcoglycan expression in muscle tissue of a subject, comprising administering to the subject a construct comprising a nucleotide sequence encoding a first sarcoglycan, and detecting increased expression of at least a second sarcoglycan in the cell membrane of cells expressing the first sarcoglycan. (Item 43) Item 43. The method of item 42, wherein the first sarcoglycan is β-sarcoglycan (SGCB) and the second sarcoglycan is α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), and / or δ-sarcoglycan (SGCD). (Item 44) 44. The method of any one of items 1 to 43, wherein the subject is a human subject aged 4 to 15 years. (Item 45) 44. The method of any one of items 1 to 43, wherein the subject is a pediatric subject, an adolescent subject or a young adult subject. (Item 46) 44. The method of any one of items 1 to 43, wherein the subject is a human subject aged 4 to 15 years, has confirmed beta-sarcoglycan (SGCB) mutations in both alleles, is negative for AAVrh74 antibodies, and / or has a 100 meter walk test score of greater than 40% or normal. (Item 47) 44. The method of any one of items 1 to 43, wherein the subject is a middle-aged adult or an elderly subject. (Item 48) 44. The method of any one of items 1 to 43, wherein the subject is a human subject aged 25 to 55. (Item 49) 44. The method of any one of items 1 to 43, wherein the subject is a human subject over 50 years of age. (Item 50) 1. A composition comprising: rAAV scAAVrh74.MHCK7.hSGCB vector, A buffering agent; an ionic strength agent; A composition comprising: a surfactant. (Item 51) The rAAV is about 1.0 x 10 12 vg / ml ~ approx. 5.0x10 14 vg / ml, or approximately 5.0x10 12 vg / ml ~ approx. 1.0x10 14 51. The composition of item 50, having a concentration of 0.015 mg / ml. (Item 52) The rAAV is about 2.0 x 10 13 vg / ml, 4 × 10 13 vg / ml, 5 × 10 13 51. The composition of item 50, having a concentration of 0.015 mg / ml. (Item 53) 51. The composition of claim 50, wherein the buffer comprises one or more of Tris, Tricine, Bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. (Item 54) 54. The composition according to item 53, wherein the buffer comprises Tris at a concentration of about 5 mM to about 40 mM and a pH of 8.0. (Item 55) 54. The composition of claim 53, wherein the buffer comprises Tris at about 20 mM and pH 8.0. (Item 56) 51. The composition of claim 50, wherein the ionic strength agent comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. (Item 57) 51. The composition of claim 50, wherein the ionic strength agent comprises MgCl at a concentration of about 0.2 mM to about 4 mM. (Item 58) 51. The composition of claim 50, wherein the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM. (Item 59) 51. The composition of claim 50, wherein the ionic strength agent comprises MgCl at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM. (Item 60) 51. The composition of claim 50, wherein the ionic strength agent comprises MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM. (Item 61) 51. The composition of claim 50, wherein the surfactant comprises one or more of a sulfonate, sulfate, phosphonate, phosphate, poloxamer, and cationic surfactant. (Item 62) 62. The composition of claim 61, wherein the poloxamer comprises one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. (Item 63) 62. The composition of claim 61, wherein the surfactant comprises the poloxamer at a concentration of about 0.00001% to about 1%. (Item 64) 62. The composition of claim 61, wherein the surfactant comprises poloxamer 188 at a concentration of about 0.001%. (Item 65) A pharmaceutical composition comprising recombinant AAV (rAAV) scAAVrh74.MHCK7.hSGCB, wherein said scAAVrh74.MHCK7.hSGCB comprises a nucleotide sequence that is at least 95% or 99% identical to SEQ ID NO:19. (Item 66) 66. The pharmaceutical composition of claim 65, wherein the scAAVrh74.MHCK7.hSGCB comprises the nucleotide sequence of SEQ ID NO: 19. (Item 67) A method for producing a recombinant AAV scAAVrh74.MHCK7.hSGCB, comprising transfecting a plasmid into a cell, wherein the plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:24. (Item 68) 84. The method of claim 83, wherein the plasmid comprises the nucleotide sequence of SEQ ID NO: 24. (Item 69) 68. The method of claim 67, wherein the plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, 3, or 19. (Item 70) 70. The method of any one of items 67 to 69, wherein the plasmid comprises the nucleotide sequence of SEQ ID NO: 19. (Item 71) 71. The method of any one of items 67 to 70, further comprising transfecting the cells with a packaging plasmid and / or a helper virus. (Item 72) 71. The method of any one of items 67 to 70, wherein the cells contain a stably integrated AAVcap gene. (Item 73) 71. The method of any one of items 67 to 70, wherein the cells contain a stably integrated AAVrep gene. (Item 74) A cell, comprising a plasmid comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:24. (Item 75) 75. The cell of item 74, wherein the plasmid comprises the nucleotide sequence of SEQ ID NO: 24. (Item 76) 76. The cell of item 74 or 75, comprising the nucleotide sequence of SEQ ID NO: 19. (Item 77) 77. The cell of any one of items 74 to 76, wherein the cell is an insect cell, a mosquito cell, or a mammalian cell. [Brief explanation of the drawings]

[0099] [Figure 1]This figure provides a schematic diagram of the therapeutic β-sarcoglycan transgene cassette. This self-complementary AAV vector contains the codon-optimized human β-sarcoglycan gene (hSGCB). The muscle-specific MHCK7 promoter drives expression. The cassette also contains a chimeric intron that enhances processing and polyadenylation signals for stability. [Figure 2A] Human β-sarcoglycan expression in skeletal muscle. A) Immunofluorescence imaging of skeletal muscle, diaphragm, and heart from SGCB- / - mice intravenously injected with a total dose of 3e12vg scAAVrh.74.MHCK7.hSGCB. Representative images show all muscles transduced to >98%. 20x magnification images are shown. B) Western blotting showing expression of the hSGCB transgene (43 kDa) in muscles treated with the clinical dose (#716) and high doses (#785, #786). N=6 per treatment group; 100 kDa corresponds to the α-actinin loading control. [Figure 2B] Same as above. [Figure 3-1] Effect of systemic treatment with high-dose scAAVrh74.MHCK7.hSGCB on muscle pathology. (A) H&E staining of quadriceps and diaphragm muscles from C57BL / 6 WT, SGCB- / -, and scAAVrh.74.MHCK7.hSGCB-treated mice. (B) Quantification of the reduction in centrally nucleated fibers, (C) normalization of fiber distribution, and (D) increase in mean fiber size. N=6 per group. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4] Figure 1 shows correction of force deficits in the diaphragm of SGCB- / - mice. After 24 weeks of treatment, diaphragm strips were harvested from the mice to measure force production after stimulation. Treatment restored force to WT levels and provided greater recovery compared to the previously studied dose (1e12vg total dose) (WT: n=5; KO: n=4; low dose: n=6; high dose: n=6). **=p<0.01. [Figure 5]Overall gait in the x and y planes was significantly reduced in KO mice and slightly improved in MHCK7-treated mice. Vertical rearing of the hind limbs was slightly improved in MHCK7-treated mice (n=6). [Figure 6] Western biodistribution plots are shown for muscle and organs from SGCB- / - mice systemically injected with two high doses (2.0 x 1014 vg / kg) of scAAVrh.74.MHCK7.hSGCB. 43 kDa corresponds to the β-sarcoglycan protein. 125 kDa corresponds to the vinculin loading control. [Figure 7] 1 provides β-sarcoglycan protein expression in muscle biopsies of human subjects following systemic administration of 5.0×10 13 vg / kg of scAAVrh.74.MHCK7.hSGCB, as detected and quantified by immunohistochemistry. [Figure 8] 1 provides β-sarcoglycan protein expression in muscle biopsies of human subjects 90 days after systemic administration of 5.0×10 13 vg / kg of scAAVrh.74.MHCK7.hSGCB as detected and quantified by Western blot. [Figure 9] Figure 1 shows that β-sarcoglycan protein expression upregulated the expression of the sarcoglycan complex as shown by detection and quantification of alpha-sarcoglycan by immunohistochemistry. [Figure 10] 1 shows restoration of β-sarcoglycan, α-sarcoglycan expression in the membrane of patient 3 during the study, as well as co-localization of β-sarcoglycan and α-sarcoglycan. [Figure 11] 1 shows the mean percent change from baseline and improvement in the 100-meter time test in three subjects over the first three months after gene transfer with scAAVrh.74.MHCK7.hSGCB. [Figure 12A]The changes from baseline in North Star Assessment of Limb-Girdle Muscular Dystrophy (NSAD) for natural growth control subjects (FIG. 12A) and study subjects (FIG. 12B) after administration of 5.0×10 vg / kg of scAAVrh.74.MHCK7.hSGCB are provided. NSAD data for six individual natural growth control patients are shown in FIG. 12C. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 13] 1 provides a schematic map of the pAAV.MHCK7.hSGCB.KAN AAV vector plasmid. DETAILED DESCRIPTION OF THE INVENTION

[0100] The present disclosure is based on the discovery that administration of an AAV vector containing a polynucleotide expressing β-sarcoglycan reduces or completely reverses muscle fibrosis in an animal model of limb-girdle muscular dystrophy. As shown in the examples, administration of the AAV vector described herein reverses dystrophic features, including a reduction in degenerated fibers, reduced inflammation, and improved functional recovery due to protection against eccentric contractions through increased force generation.

[0101] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and by heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0102] As used herein, "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0103] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, since such a vector is contained within the AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0104] AAV The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus 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 rh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. AAV1, AAV5, AAV6, AAV8, or AAV9 can be used to promote skeletal muscle-specific expression.

[0105] The DNA plasmid of the present invention contains an rAAV genome. The DNA plasmid is introduced into a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for incorporation of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be of any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype different from the 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. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0106] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAVrep and cap genes, the AAVrep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0107] 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 approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988), Samulski et al. al. (1989, J. Virol., 63:3822-3828), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. (1993) Human Gene Therapy 4:609-615, Clark et 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 incorporated herein by reference in their entireties, with particular emphasis on the portions of the documents relating to rAAV production.

[0108] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 cells). In another embodiment, the packaging cells are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0109] Recombinant AAV (i.e., infectious, encapsidated rAAV particles) of the invention comprise an rAAV genome. Embodiments include, but are not limited to, an rAAV designated pAAV.MHCK7.hSCGB, which comprises the polynucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:19, and pAAV.tMCK.hSCGB, which comprises the polynucleotide sequence set forth in SEQ ID NO:5.

[0110] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0111] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The compositions described herein comprise the rAAV in a pharmaceutically acceptable carrier. The composition may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients and are preferably inert at the dosages and concentrations used, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; 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; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0112] The titer of the rAAV administered in the methods of the invention will vary depending, for example, on the particular rAAV, the method of administration, the treatment goal, the individual, and the cell type being targeted, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 The dosage may range from 1000 to 10 ...

[0113] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated by the present invention. In vivo methods include administering an effective dose or effective doses of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state to be treated, slows or prevents progression to the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present invention is muscular dystrophy, e.g., limb-girdle muscular dystrophy. Accordingly, provided is a method for transducing target cells with rAAV scAAVrh74.MHCK7.hSGCB, comprising the nucleotide sequence of SEQ ID NO: 3 or 19.

[0114] Combination therapy is also contemplated by the present invention. As used herein, combination therapy includes simultaneous or sequential therapy. Combination of the methods of the present invention with standard medical treatments (e.g., steroids, corticosteroids, and / or glucocorticoids, including but not limited to, one or more of prednisone, prednisolone, and deflazacort) is specifically contemplated, as is combination with novel therapies. In this regard, this combination includes administering to a subject one or more steroids, corticosteroids, and / or glucocorticoids, including but not limited to, one or more of prednisone, prednisolone, and deflazacort, before, simultaneously with, or after administering to a subject the rAAV of the methods of the present invention.

[0115] In related embodiments of the combination therapies contemplated by the present invention, glucocorticoids include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0116] It is recognized that antigen-specific T cell responses can occur in subjects administered rAAV vectors. This response is expected 2–4 weeks after gene transfer. One possible consequence of such antigen-specific T cell responses is clearance of transduced cells and loss of transgene expression. To attenuate the host's immune response to rAAV-based therapy, subjects can be initiated on prophylactic prednisone or an equivalent glucocorticoid orally at approximately 1 mg / kg / day, up to a maximum dose of 60 mg / day, prior to treatment, e.g., 24 hours before the treatment procedure. If necessary, an equivalent glucocorticoid can also be administered intravenously at approximately 1 mg / kg / day. Treatment lasts approximately one month. A tapering protocol of prednisone or an equivalent glucocorticoid can be implemented based on the individual subject's immune response to gene transfer and can be assessed by ELISpot assay and liver function monitoring with GGT.

[0117] A therapeutically effective amount of a rAAV vector is from about 1e13vg / kg to about 5e14vg / kg, or from about 1e13vg / kg to about 2e13vg / kg, or from about 1e13vg / kg to about 3e13vg / kg, or from about 1e13vg / kg to about 4e13vg / kg, or from about 1e13vg / kg to about 5e13vg / kg, or from about 1e13vg / kg to about 6e13vg / kg, or from about 1e13vg / kg to about 7e13vg / kg, or from about 1e13vg / kg to about 8e13vg / kg, or from about 1e13vg / kg to about 9e13vg / kg, or 1e13vg / kg to about 1e14vg / kg, or about 1e13vg / kg to about 2e14vg / kg, or 1e13vg / kg to about 3e14vg / kg, or about 1e13 to about 4e14vg / kg, or about 3e13vg / kg to about 4e13vg / kg, or about 3e13vg / kg to about 5e13vg / kg, or about 3e13vg / kg to about 6e13vg / kg, or about 3e13vg / kg to about 7e13vg / kg, or about 3e13vg / kg to about 8e13vg / kg, or about 3e13vg / kg to about 9e13vg / kg, or about 3e13vg / kg to about 1e14vg / kg, or about 3e13vg / kg to about 2e14vg / kg, or 3e13vg / kg to about 3e14vg / kg, or about 3e13 to about 4e14vg / kg, or about 3e13vg / kg to about 5e14vg / kg, or about 5e13vg / kg to about 6e13vg / kg, or about 5e13vg / kg to about 7e13vg / kg, or about 5e13vg / kg to about 8e13vg / kg, or about 5e13vg / kg to about 9e13vg / kg, or about 5e13vg / kg to about 1e14vg / kg, is a dose of rAAV in the range of about 5e13vg / kg to about 2e14vg / kg, or 5e13vg / kg to about 3e14vg / kg, or about 5e13 to about 4e14vg / kg, or about 5e13vg / kg to about 5e14vg / kg, or about 1e14vg / kg to about 2e14vg / kg, or 1e14vg / kg to about 3e14vg / kg, or about 1e14 to about 4e14vg / kg, or about 1e14vg / kg to about 5e14vg / kg, 6e14vg / kg, 7e14vg / kg, 8e14vg / kg, or 9e14vg / kg.The present invention also includes compositions comprising these ranges of rAAV vectors.

[0118] For example, the therapeutically effective amount of rAAV vector is 1e13vg / kg, about 2e13vg / kg, about 3e13vg / kg, about 4e13vg / kg, about 5e13vg / kg, about 6e13vg / kg, about 7e13vg / kg, about 7.4e13vg / kg, about 8e13vg / kg, about 9e13vg / kg, about 1e14vg / kg, about 2e14vg / kg, about 3e14vg / kg, about 4e14vg / kg and 5e14vg / kg. The titer or dosage of AAV vector may vary based on the physical form of plasmid DNA as a quantitative standard. For example, the titer or dosage value may vary based on supercoiled standard qPCR titration or linear standard qPCR titration. In one embodiment, the therapeutically effective amount of rAAV is a 5e13 vg / kg dose based on a supercoiled plasmid as the quantification standard, or a 1.85e13 vg / kg dose based on a linearized plasmid as the quantification standard, hi another embodiment, the therapeutically effective amount of rAAV is a 2e14 vg / kg dose based on a supercoiled plasmid as the quantification standard, or a 7.41e13 vg / kg dose based on a linearized plasmid as the quantification standard. In another embodiment, the therapeutically effective amount of scAAVrh74.MHCK7.hSGCB is from about 1e13 vg / kg to about 5e14 vg / kg, or from about 1e13 vg / kg to about 2e13 vg / kg, or from about 1e13 vg / kg to about 3e13 vg / kg, or from about 1e13 vg / kg to about 4e13 vg / kg, or from about 1e13 vg / kg to about 5e13 vg / kg, or from about 1e13 vg / kg to about 6e13 vg / kg, or from about 1e13 vg / kg to about 7e13 vg / kg, or 1e13vg / kg to about 8e13vg / kg, or about 1e13vg / kg to about 9e13vg / kg, or about 1e13vg / kg to about 1e14vg / kg, or about 1e13vg / kg to about 2e14vg / kg, or 1e13vg / kg to about 3e14vg / kg, or about 1e13 to about 4e14vg / kg, or about 3e13vg / kg to about 4e13vg / kg, or about 3e13vg / kg to about 5e13vg / kg, or about 3e13vg / kg to about 6e13vg / kg, or about 3e13vg / kg to about 7e13vg / kg,or about 3e13vg / kg to about 8e13vg / kg, or about 3e13vg / kg to about 9e13vg / kg, or about 3e13vg / kg to about 1e14vg / kg, or about 3e13vg / kg to about 2e14vg / kg, or 3e13vg / kg to about 3e14vg / kg, or about 3e13 to about 4e14vg / kg, or about 3e13vg / kg to about 5e14vg / kg, or about 5e13vg / kg to about 6e13vg / kg, or about 5e13vg / kg to about 7e13vg / kg, or about 5e13vg / kg to about 8e13vg / kg, or about 5e13vg / kg to about 9e13vg / kg g, or about 5e13 vg / kg to about 1e14 vg / kg, or about 5e13 vg / kg to about 2e14 vg / kg, or 5e13 vg / kg to about 3e14 vg / kg, or about 5e13 to about 4e14 vg / kg, or about 5e13 vg / kg to about 5e14 vg / kg, or about 1e14 vg / kg to about 2e14 vg / kg, or 1e14 vg / kg to about 3e14 vg / kg, or about 1e14 to about 4e14 vg / kg, or about 1e14 vg / kg to about 5e14 vg / kg, 6e14 vg / kg, 7e14 vg / kg, 8e14 vg / kg, or 9e14 vg / kg. The present invention also includes compositions comprising these doses of rAAV vectors.

[0119] Administration of an effective dose of the composition can be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV components of the rAAV of the present invention (specifically, the AAV ITRs and capsid proteins) can be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state to be treated and the target cells / tissues that express β-sarcoglycan.

[0120] The present invention provides for local administration and systemic administration of effective doses of the rAAV and compositions of the present invention.For example, systemic administration refers to administration into the circulatory system so that the entire body is affected.Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration by injection, infusion, or implantation.

[0121] In particular, the actual administration of the rAAV of the present invention can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, injection into the bloodstream, and / or direct injection into the liver. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual way with rAAV). The capsid protein of rAAV may be modified to target rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference.

[0122] The pharmaceutical composition can be prepared as an injectable formulation or as a local formulation delivered to the muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been developed and can be used in the practice of the present invention. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. Thus, in another aspect, the present application relates to a formulation comprising an rAAV comprising a capsid derived from AAVrh74, a buffer, an ionic strength agent, and a surfactant. In one embodiment, the rAAV is administered at a concentration of about 1.0x10 12 vg / ml ~ approx. 5.0×10 14 In another embodiment, the rAAV is at a concentration of about 5.0 x 10 vg / ml, based on a supercoiled plasmid as a quantification standard. 12 vg / ml ~ approx. 1.0×10 14 In another embodiment, the rAAV is at a concentration of about 2.0 x 10 vg / ml, based on a supercoiled plasmid as a quantification standard. 13In one embodiment, the rAAV is a scAAVrh74.MHCK7.hSGCB vector. In one embodiment, the concentration of rAAV in the composition or formulation is 1 x 10 vg / ml, based on supercoiled plasmid as a quantification standard. 13 vg / ml ~ 2 × 10 14 In another embodiment, the concentration is 2×10 vg / ml, based on supercoiled plasmid as a quantification standard. 13 vg / ml, 4 × 10 13 vg / ml, or 5 × 10 13The concentration is vg / ml. In one embodiment, the buffering agent comprises one or more of Tris, Tricine, bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In another embodiment, the buffering agent comprises Tris at a pH of 8.0 at a concentration of about 5 mM to about 40 mM. In one embodiment, the buffering agent comprises Tris at a pH of 8.0 at about 20 mM. In one embodiment, the ionic strength agent comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NHCl), ammonium acetate, magnesium chloride (MgCl), magnesium acetate, magnesium sulfate, manganese chloride (MnCl), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. In one embodiment, the ionic strength agent comprises MgCl at a concentration of about 0.2 mM to about 4 mM. In another embodiment, the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM. In one embodiment, the surfactant comprises one or more of a sulfonate, sulfate, phosphonate, phosphate, poloxamer, and cationic surfactant. In one embodiment, the poloxamer comprises one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. In one embodiment, the surfactant comprises a poloxamer at a concentration of about 0.00001% to about 1%. In another embodiment, the surfactant comprises poloxamer 188 at a concentration of about 0.001%. For intramuscular injection purposes, solutions in adjuvants such as sesame or peanut oil or in aqueous propylene glycol, and sterile aqueous solutions can be employed. Such aqueous solutions can be buffered if necessary, and the liquid diluent first rendered isotonic with saline or glucose.Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0123] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0124] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent, and then sterilizing by filtration, as necessary, with various other components listed above.Generally, dispersion is prepared by mixing sterilized active ingredient into a sterile vehicle that contains a basic dispersion medium and other required components listed above.For the preparation of sterile powder for sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0125] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and then reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not generate an inappropriate immune response in the subject.

[0126] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in an appropriate medium and screening for cells carrying the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by a variety of techniques, including intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0127] Transduction of cells with the rAAV of the present invention results in sustained expression of β-sarcoglycan. Thus, the present invention provides methods for administering / delivering rAAVs expressing β-sarcoglycan to mammalian subjects, preferably humans. These methods involve transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction can be performed with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present invention may be used in combination with, but is not limited to, the actin and myosin gene families, such as those from the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], the muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], the human skeletal muscle actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], the cardiac actin gene, and the muscle creatine kinase sequence element [Johnson et al. al., Mol. Cell. Biol., 9:3393-3399 (1989)], and regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene; promoters containing hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), a steroid-inducible element, and a glucocorticoid response element (GRE) (Mader and The present invention provides methods for transducing muscle cells and muscle tissue that are driven by muscle-specific regulatory elements, including those described herein (see, for example, White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)), as well as other regulatory elements.

[0128] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention contemplates sustained expression of miRNAs from transduced myofibrils.

[0129] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived 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 cardiomyoblasts.

[0130] The term "transduction" is used to refer to the administration / delivery of a polynucleotide of interest (e.g., a polynucleotide sequence encoding β-sarcoglycan) to a recipient cell either in vivo or in vitro via the described replication-deficient rAAV, resulting in expression of β-sarcoglycan by the recipient cell.

[0131] Thus, also described herein is a method of administering an effective dose (or doses administered essentially simultaneously or at an interval) of a rAAV encoding β-sarcoglycan to a mammalian subject in need thereof.

[0132] All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0133] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0134] In another embodiment, the present disclosure provides a method for generating rAAV pAAV.MHCK7.hSCGB, the method comprising introducing an AAV vector plasmid into a host cell. Methods for introducing DNA into a host cell are known in the art and include, but are not limited to, transfection, infection, transformation, electroporation, and transduction. In one embodiment, the vector plasmid comprises a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO:24. In another embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO:24. In another aspect, the disclosure provides a host cell comprising an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO:24. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. Host cells stably harboring the AAV vector plasmid can be used to produce rAAV. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCB.KAN plasmid. The pAAV.MHCK7.hSGCB.KAN plasmid is shown in Figure 11.

[0135] In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, 3, 5, or 19. In one embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 19. The method of producing rAAV, in one embodiment, further comprises transfecting a packaging plasmid and / or a helper virus into a host cell. The packaging plasmid, in some embodiments, comprises an AAVrep and / or cap gene operably linked to a promoter. In one embodiment, the promoter is an AAV transcription promoter. In one embodiment, the host cell is a packaging cell. In one embodiment, the packaging cell comprises a stably integrated AAVcap gene. In another embodiment, the packaging cell comprises a stably integrated AAVrep gene.

[0136] As used herein, the term "host cell" refers to a cell that can be used to express an exogenous DNA sequence. Non-limiting examples of host cells include microorganisms, yeast cells, insect cells, and / or mammalian cells. Host cells can be used as recipients of AAV helper constructs, packaging plasmids, AAV vector plasmids, accessory function vectors, or other DNA. As used herein, the term encompasses the progeny of the original host cell after expression of an exogenous DNA sequence in the original host cell. Non-limiting examples of host cells for AAV production include Sf9 insect cells and HEK293T cells. In one embodiment, the cells described herein include insect cells, such as Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells), or mammalian cells (preferably human cells, e.g., human primary cells or established cell lines). In one embodiment, mammalian cells include 293 cells, COS cells, HeLa cells, or KB cells. The AAV vector plasmid can be introduced into host cells (e.g., Sf9 or 293T) by infection (virus or baculovirus), transient transfection using reagents (e.g., liposomes, calcium phosphate), or physical means (e.g., electroporation), or other means known in the art. In another embodiment, the host cell line is stably integrated with the rAAV plasmid into its genome. Such stable cell lines can be established by incorporating a selectable marker into the vector plasmid.

[0137] In one embodiment, the host cell is a packaging cell for producing AAV viral particles. Accordingly, in another aspect, the present disclosure provides a host cell comprising an AAV vector plasmid comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 24. In one embodiment, the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 24. In another embodiment, the host cell comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 19. [Example]

[0138] Preclinical studies using scAAVrh74.MHCK7.hSGCB are described in WO2017 / 180976, which is incorporated herein by reference in its entirety.

[0139] Example 1 material and method Animal models - All procedures were approved by the Research Institute at Nationwide Children's Hospital Institutional Animal Care and Use Committee (protocol AR12-00040). tm1Kcam / 1J Heterozygous mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA, strain #006832). - / - Mice were generated by breeding heterozygous mice. KO mice were bred and maintained as homozygous animals under standard conditions at the Animal Resources Core of the Research Institute at Nationwide Children's Hospital. Mice were maintained on a Teklad Global Rodent Diet (3.8z5 dietary fiber, 18.8% protein, 5% fat diet) with a 12:12 h dark:light cycle. SGCB - / - Mice were identified by PCR-based genotyping. All animals were housed in standard mouse cages and allowed free access to food and water.

[0140] Beta-sarcoglycan gene construction. Full-length human beta-sarcoglycan cDNA (GenBank accession number NM_0034994.3) was codon-optimized and constructed using GenScript The cDNA sequences were synthesized by GenScript (www.genscript.com), a company based in Piscataway, NJ, USA. Codon optimization by GenScript uses an algorithm that considers parameters including transcription, mRNA processing and stability, translation, and protein folding to design cDNA sequences that result in maximal expression in muscle tissue.

[0141] For the pAAV.tMCK.hSGCB construct, the cDNA was then cloned into a plasmid containing the AAV2 ITRs. The cassette contained a consensus Kozak sequence (CCACC), an SV40 chimeric intron, and a synthetic polyadenylation site (53 bp). The recombinant tMCK promoter was a gift from Dr. Xiao Xiao (University of North Carolina). It is a modification of the previously described CK6 promoter,27 containing modifications in the enhancer upstream of the promoter region containing transcription factor binding sites. The enhancer is composed of two E-boxes (right and left). The tMCK promoter modification includes a mutation that converts the left E-box to a right E-box (2R modification) and inserts 6 bp (S5 modification). The pAAV.tMCK.hSGCB vector was constructed by ligation of a 1040 bp KpnI / XbaI fragment from pUC57-BSG (Genscript Inc.) into the KpnI / XbaI sites of pAAV.tMCK.hSGCA.26.

[0142] The pAAV.MHCK7.hSGCB vector was constructed by removing the tMCK promoter and SV40 chimeric intron with NotI / KpnI sites and inserting a PCR-amplified fragment containing the MHCK7 promoter and the same SV40 chimeric intron with NotI / KpnI sites. MHCK7 is an MCK-based promoter that utilizes a 206-bp enhancer taken from approximately 1.2 kb 5' of the transcription start site within the endogenous muscle creatine kinase gene (enh358MCK, 584 bp) with a proximal promoter. 3、12The MCK7 promoter itself contains this modified CK7 cassette from the MCK family of genes linked to a 188-bp α-MyHC (α-myosin heavy chain) enhancer 5' of the CK portion to enhance cardiac expression. 12 The creatine kinase portion of the promoter (CK) is 96% identical between tMCK and MHCK7. Finally, the pAAV.MHCK7.hSGCB vector was constructed by ligating the 960 bp NotI / KpnI MHCK7 + intron fragment from pAAV.MHCK7.DYSF5'DV44 into the NotI / KpnI sites of pAAV.tMCK.hSGCB (Pozgai et al., Gene Ther. 23:57-66, 2016).

[0143] rAAV Production. rAAV vectors were produced using a modified cross-packaging approach previously reported by Rodino-Klapac et al. (J. Trans. Med. 5:45, 2007). Here, a triple transfection method with CaPO4 precipitation in HEK293 cells allows packaging of AAV2 ITRs into different AAV capsid serotypes. (28, 29) The production plasmids were (i) pAAV.tMCK.hSGCB or pAAV.MHCK7.hSGCB, (ii) the rep2-caprh.74 modified AAV helper plasmid encoding the cap serotype 8-like isolate rh.74, and (iii) the adenovirus type 5 helper plasmid (pAdhelper) expressing the adenovirus E2A, E4 ORF6, and VA I / II RNA genes. The vector was purified, and the encapsidated vg titer was determined as previously described (using a Prism 7500 Taqman detector system, PE Applied Biosystems, Carlsbad, CA, USA). The primers and fluorescent probe targeted the tMCK promoter and were as follows: tMCK forward primer, 5'-ACC CGA GAT GCC TGG TTA TAA TT-3' (SEQ ID NO: 10), tMCK reverse primer, 5'-TCC ATG GTG TAC AGA GCC TAA GAC-3' (SEQ ID NO: 11), and tMCK probe, 5'-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C-TAMRA-3' (SEQ ID NO: 12). Primers and fluorescent probes targeted the MHCK7 promoter and were as follows: MHCK7 forward primer, 5'-CCA ACA CCT GCT GCC TCT AAA-3' (SEQ ID NO: 16), MHCK7 reverse primer, 5'-GTC CCC CAC AGC CTT GTT C-3' (SEQ ID NO: 17), and MHCK7 probe, 5'-FAM-TGG ATC CCC-Zen-TGC ATG CGA AGA TC-3IABKFQ-3' (SEQ ID NO: 18).

[0144] Systemic gene delivery: Systemic delivery is - / - This was achieved by injecting the vector into the tail vein of the mouse. Using a 30-gauge ultra-fine insulin syringe, 3 x 10 insulin diluted in saline was administered. 12 vg scAAVrh.74.MHCK7.hSGCB (2.0 × 10 14 Mice were injected with a dose of 100 mg / kg (vg / kg). The mice were restrained in a holding tube, their tails placed in the tail slot, and warmed to dilate the blood vessels for easier injection. After positioning the artery below the midline of the tail, the injection was made into one of the purple / blue lateral veins that run alongside the tail artery. All treated mice were injected at 4-5 weeks of age and euthanized 6 months after injection.

[0145] Immunofluorescence. Cryostat sections (12 μm) were incubated with monoclonal human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK, catalog number NCL-Lb-SARC) at a dilution of 1:50 in blocking buffer (1× TBS, 10% goat serum, 0.1% Tween) for 1 hour at room temperature in a humidified chamber. Sections were then washed three times with TBS for 20 minutes each and reblocked for 30 minutes. AlexaFluor 594-conjugated goat anti-mouse secondary IgG1 antibody (Life Technologies, Grand Island, NY, USA, catalog number A21125) was applied at a dilution of 1:250 for 45 minutes. Sections were washed three times with TBS for 20 minutes each and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Four random 20x images covering four different quadrants of the muscle sections were taken using a Zeiss AxioCam MRC5 camera. The percentage of fibers positive for beta-sarcoglycan staining (450% myofascial staining intensity) was determined for each image and averaged for each muscle.

[0146] Western blot analysis. Tissue sections or muscle biopsies were collected in a microcentrifuge and homogenized in 100 μl of homogenization buffer (125 mM Tris-HCl, 4% SDS, 4 M urea) in the presence of one protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). After homogenization, samples were centrifuged at 10,000 rpm for 10 minutes at 4°C. Protein was quantified using a NanoDrop (Thermo Scientific, Waltham, MA, USA). Protein samples (20 μg) were electrophoresed on a 3-8% polyacrylamide Tris-acetate gel (NuPage, Invitrogen, Carlsbad, CA, USA) at 150 V for 1 hour and 5 minutes, then transferred to a PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) at 35 V for 1 hour and 15 minutes. The membrane was blocked for 1 hour in 5% nonfat dry milk in TBST and then incubated with a rabbit polyclonal human beta-sarcoglycan antibody (Novus Biologicals, Littleton, CO, USA, catalog number NBP-1-90300, 1:100 or 1:250 dilution) and a monoclonal mouse gamma-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA, catalog number T6557) at 1:5000 or a monoclonal mouse α-actinin antibody (Sigma-Aldrich, St. Louis, MO, USA, catalog number A7811) at 1:500 dilution. A rabbit polyclonal mouse cardiac troponin I antibody (Abcam, Cambridge, MA, catalog number ab47003) at 1:500 dilution and a rabbit monoclonal mouse vinculin antibody (Invitrogen, Frederick, MD, catalog number 70062) at 1:1000 dilution were used. Anti-mouse (Millipore, Billerica, MA, USA, Cat. No. AP308P) and anti-rabbit (Life Technologies, Cat. No. 656120) secondary-HRP antibodies were used for ECL immunodetection.

[0147] Biodistribution qPCR analysis. TaqMan quantitative PCR was performed to quantify the number of vector genome copies present in the targeted and nontargeted contralateral muscles as previously described. (18, 30) A vector-specific primer-probe set was used to amplify sequences in the intron region immediately downstream from the tMCK promoter, which is unique and located within the scAAVrh.74.tMCK.hSGCB transgene cassette. The following primers and probes were used in this study: tMCK and MHCK7 intron forward primer 5'-GTG AGG CAC TGG GCA GGT AA-3' (SEQ ID NO: 13), tMCK and MHCK7 intron reverse primer 5'-ACC TGT GGA GAG AAA GGC AAA G-3' (SEQ ID NO: 14), and tMCK and MHCK7 intron probe 5'-6FAM-ATC AAG GTT ACA AGA CAG-GTT TAA GGA GAC CAA TAG AAA -tamra-3' (IDT) (SEQ ID NO: 15). Copy numbers are reported as vector genomes per microgram of genomic DNA. Immunohistochemistry for immunocytostaining. Immunohistochemistry was used to identify immune cells. Frozen tissue sections on Fisherbrand Superfrost charged microscope slides were probed with an anti-rat Ig HRP detection kit (BD The tissue was incubated with rat anti-mouse monoclonal antibodies (Pharmagen, San Jose, CA, USA, Cat: 551013): CD3 (Cat: 555273), CD4 (Cat: 550280), CD8 (Cat: 550281), and Mac-3 (Cat: 550292) for macrophages. All primary antibodies were diluted 1:20 in phosphate-buffered saline. Positive immunostaining was visualized using streptavidin-HRP peroxidase ectastain ABC peroxidase-containing DAB chromogen diluted in DAB buffer. Ten random 40x magnification images were taken for each muscle and each corresponding stain. The number of mononuclear cells was counted and analyzed at 1 mm. 2 The results were expressed as the total number of hits.

[0148] Immunofluorescence: Cryostat sections (12 μm) from the tibialis anterior (TA), gastrocnemius (GAS), quadriceps femoris (QUAD), psoas major (PSOAS), psoas (GLUT), triceps brachii (TRI), and diaphragm muscles, along with the heart, were immunofluorescently stained for the hSGCB transgene using our previously used protocol as described in Pozgai et al., Gene Therap. 23:57-66, 2016. Sections were incubated with mouse monoclonal human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK, catalog number NCL-Lb-SARC) at a dilution of 1:100. Four random 20x magnification images covering four different quadrants of the muscle sections were taken using a Zeiss AxioCam MRC5 camera. The percentage of fibers positive for beta-sarcoglycan staining (>50% muscle membrane staining) was determined for each image and averaged for each muscle.

[0149] Morphometric analysis: For analysis, 7-month-old C57BL6 WT mice (n=5), sgcb - / - Mice (n = 5), and sgcb treated with rAAV.MHCK7.hSGCB for 6 months - / - Hematoxylin and eosin (H&E) staining was performed on 12-µm-thick frozen sections of muscles from mice (n=5). The percentage of myofibers with central nuclei was determined in the TA, GAS, QUAD, PSOAS, GLUT, TRI, and diaphragm muscles. Additionally, myofiber diameter was measured in the GAS, PSOAS, and TRI muscles. Four random 20x magnification images per muscle from each animal were taken with a Zeiss AxioCam MRC5 camera. Centrally nucleated fibers were quantified using NIH ImageJ software, and fiber diameter was measured using Zeiss Axiovision LE4 software.

[0150] X-ray laser monitoring of open field cage activity: An open field activity chamber was used to determine the overall activity of experimental mice. 7-month-old mice from C57BL6 WT (n=6) and untreated sgcb mice were used. - / -(n=6) The control group was treated with rAAV.MHCK7.hSGCB for 6 months. - / - Mice (n = 6) were analyzed according to a previously described protocol with some modifications (Kobayashi et al., Nature 456:511-5, 2008; Beastrom et al., Am. J. Pahol. 179:2464-74, 2011). All mice were tested at the same time each day, from early morning until late evening, when mice are most active. All mice were tested in an isolation room under dim light and by the same caregiver each time. To reduce anxiety and minimize variable behaviors that could affect the mice's normal activity and, therefore, the results of the assay, tested mice were not housed individually (Voikar et al., Genes Brain Behav. 4:240-52, 2005). Mouse behavior was monitored using a Photobeam Activity System (San Diego Instruments, San Diego, CA). This system monitors mouse position and movement in the XYZ plane using a grid of invisible infrared light beams that traverse the front, back, left, and right sides of the animal chamber. Activity was recorded in 1-hour cycles with 5-minute intervals. Mice were acclimated to the activity testing room for 1-hour sessions several days before data acquisition began. Mice were tested in groups of four in individual chambers. Testing equipment was cleaned after each use to reduce reactive and variable mouse behavior, which could alter our results. Collected data was converted into a Microsoft Excel worksheet, and all calculations were performed within the Excel program. Individual light breaks for each mouse's movement in the X and Y planes were summed to represent total ambulation, and light breaks in the Z plane were summed to obtain vertical activity within the 1-hour interval.

[0151] Example 2 Construction of scAAVrh.74.MHCK7.hSGCB A transgene cassette containing the codon-optimized full-length human SCGB cDNA shown in Figure 1 was constructed. The cassette contains a consensus Kozak sequence (CCACC), an SV40 chimeric intron, a synthetic polyadenylation site, and the muscle-specific MHCK7 promoter used to drive expression of the cassette. This is an MCK-based promoter that utilizes a 206-bp enhancer taken from approximately 1.2 kb 5' of the transcription start site within the endogenous muscle creatine kinase gene (enh358MCK, 584 bp) with a proximal promoter. 3、12 The cassette was packaged into the self-complementary (sc) AAVrh.74 vector, which is 93% homologous to AAV8. AAVrh.74 has been shown to be safe and effective in mice and non-human primates, particularly in crossing the blood barrier when delivered to muscle via the circulation (17, 18, 21).

[0152] Example 3 Long-term efficacy of high-dose scAAVrh.74.MHCK7.hSGCB systemic delivery 1.0×10 12 vg total dose (5.0x10 13 Following the strong results of a previous study using a dose of scAAVrh.74.MHCK7.hSGCB (vg / kg), the vector was administered at a high dose of 3.0 × 10 12 vg total dose (2.0x10 14 vg / kg) in 6 SGCBs - / -The transgene expression and efficacy of the vector was evaluated when delivered systemically to mice via tail vein injection over a 24-week time period. Mice were injected at 4–5 weeks of age, and complete necropsies were performed on all six mice 24 weeks post-injection. The following muscles were extracted for analysis: TA, gastrocnemius, quadriceps, gluteal, PSOAS, triceps, diaphragm, and heart. Organs were also harvested for toxicology and biodistribution studies. In summary, hSGCB transgene expression was similarly high (98.77% in all muscles) after 24 weeks of treatment at this high dose compared to the previously studied dose (98.10% in all muscles), and all muscles in treated mice were again nearly completely transduced. This was accompanied by improved muscle histopathology and improved function.

[0153] β-sarcoglycan expression Immunofluorescence (IF) staining of human β-sarcoglycan was used to determine hSGCB transgene expression in six types of skeletal muscle, in addition to the diaphragm and heart, of all six KO mice given systemic injections of the hSGCB vector. These muscles included the TA, gastrocnemius (GAS), quadriceps (QUAD), gluteus maximus (GLUT), psoas major (PSOAS), and triceps brachii (TRI). For the purposes of expression analysis and transduction efficiency, images of muscles from six treated mice were utilized for quantification. Four 20x images of each muscle were taken, and the percentage of hSGCB-positive fibers was determined for each image to obtain the average transduction rate for each muscle from each mouse. These data are presented in Appendix C. The results shown in the following panel of Figure 2A are representative images from treated mice, again demonstrating greater than 98% transduction in all muscles analyzed, including the diaphragm and heart. Finally, the Western blot, also shown in Figure 2B, shows expression of hSGCB in TA and TRI muscles that is similar to that achieved after delivery of the initial clinical dose.

[0154] Histopathology of treated muscles As previously discussed, the SGCB - / -Muscles from mice exhibit extensive myopathy, with both skeletal and cardiac muscle exhibiting prominent muscle fiber atrophy and hypertrophy, accompanied by multiple focal areas of necrosis. There is also increased numbers of mononuclear cell inflammation (lymphocytes and macrophages, interspersed with neutrophils), dystrophic calcification, fatty infiltration, central nucleation, and fibrosis. Figure 3, below, shows hematoxylin and eosin staining of SGCB mice compared to normal wild-type mice. - / - We demonstrate this dystrophic phenotype in mice and the improvement of muscle pathology after treatment. Quantification of histological parameters demonstrates a reduction in central nuclei (CNs) in multiple different skeletal muscles as a result of β-sarcoglycan gene transfer. Detailed analysis of muscle histopathology reveals a normalization of fiber size distribution with an increase in mean fiber diameter in vector-treated affected mice in all three muscles examined (gastrocnemius, psoas, and triceps brachii) (Figure 3).

[0155] Functional evaluation of systemic delivery To determine whether high-dose hSGCB gene transfer would provide even greater functional benefit to affected muscles, we analyzed SGCB treated with high doses of scAAVrh.74.MHCK7.hSCGB. - / - Functional characterization of mouse diaphragm muscle. Histopathology shown, SGCB - / - Functional deficits were established in the diaphragm and heart of these mice. β-sarcoglycan KO diaphragms showed a 50.9% reduction in specific force output (116.24 mN / mm) compared to BL6 WT mice. 2 236.67mN / mm 2 Tail vein delivery of high-dose scAAVrh.74.MHCK7.hSGCB resulted in nearly 100% hSGCB expression in the diaphragm and restored diaphragm specific force output to 259.97 mN / mm 2 (n=6) (FIG. 4). These data indicate that high-dose hSGCB gene transfer provides greater functional benefit to affected muscles that are deficient in β-sarcoglycan.

[0156] High-dose AAV.hSGCB therapy has been shown to improve the survival of affected SGCB patients, as occurred with the delivery of our clinical dose.- / - Conferring overall functional benefits to mice, ultimately resulting in the development of SGCB - / - To determine whether the mouse phenotype improved, laser monitoring of open-field cage activity was performed on all groups of mice. The graphs in Figure 5 below show a significant 58.6% reduction in overall walking in the x and y planes, along with a 48.9% reduction in vertical rearing of the hindlimbs, in KO mice compared to WT. Mice treated with high-dose scAAVrh.74.MHCK7.hSGCB were overall more active compared to KO mice by qualitative observation, as shown by quantification of activity in MCHK7-treated mice (n=6 per group), which showed a 36.2% increase in overall walking and a 39.0% increase in vertical rearing of the hindlimbs (Figure 5).

[0157] A higher dose of 3.0 × 10 12 vg total dose (2.0 × 10 14 Intravenous injection of scAAVrh.74.MHCK7.hSGCB at 100 μg / kg resulted in nearly complete transduction and restoration of hSGCB expression (>98%) in limb skeletal muscle, diaphragm, and critical cardiac muscle (Figure 2). High levels of transduction were observed at a relatively low dose (5.0 × 10) due to the self-complementary AAV vector and the rh.74 serotype. 13 vg / kg and 2.0 × 10 14 The fact that this was achieved in all muscles throughout the body using a 2000 mg / kg (vg / kg) dose offers great promise for adapting this therapy to LGMD2E patients. In the absence of β-sarcoglycan, the severe dystrophic lesions seen in all muscles were significantly improved after treatment (Figure 3). These results translated into observed increases in specific force output at the diaphragm and increased open-field cage activity (Figures 4 and 5).

[0158] Example 4 Toxicology and vector biodistribution The objective of this study was to evaluate the efficacy and safety of male and female SGCB at 24 weeks after delivery of the test article scAAVrh.74.MHCK7.hSGCB. - / -The objective of this study was to evaluate potential toxicity or safety concerns of SGCB gene therapy in mice. The test article was administered to 4-5 week old SGCB mice in a total volume of 520 μL, divided into two 260 μL injections, each 5 hours apart, to achieve the desired dose. - / - Mice were administered a 3.0 × 10 vg total dose (2.0 × 10 14 To assess the safety of our vector, six SGCB mice treated with the vector were administered, along with two WT and two KO subjects injected with LRS. - / - Hematoxylin and eosin staining was performed on frozen sections of muscle tissue and all isolated organs taken from groups of mice (Table 1). [Table 1]

[0159] These sections were then formally reviewed for toxicity by a veterinary pathologist, and no adverse effects were detected in most samples from any mouse, except for a few focal areas of liver lesions in the livers of two treated mice (#789 and #790). Protein expression and vector biodistribution were also assessed using qPCR and Western blotting, and these data indicate no expression of the hSGCB transgene in any non-muscle tissue, except in the livers of #785 and #787.

[0160] Histopathological examination of vector-transduced tissues 2.0 x 10 using systemic delivery 14 To determine the safety and toxicity profile of 100 vg / kg of scAAVrh.74.MHCK7.hSGCB, various skeletal muscles, including the diaphragm, along with the heart and five other organs, were injected with the vector. - / - H&E sections of each tissue collected from the mouse and control groups were formally reviewed by an independent veterinary pathologist. Group details and study design are shown in Table 1.

[0161] Dosing cohorts for scAAVrh.74.MHCK7.hSGCB histopathology study. Two BL6 WT mice and two SGCB - / - Mice were injected with LRS to serve as appropriate age-matched controls. - / - were administered intravenously at a total dose of 3.0 x 1012 vg. Mice were euthanized 24 weeks after injection, with an endpoint age of 28 weeks.

[0162] In summary, intravenous injection of high-dose scAAVrh.74.MHCK7.hSGCB did not induce any microscopic changes in myofibers of any skeletal muscles examined. Any changes noted in muscle were seen in both treated and control mice and were considered incidental findings. In addition, most nonmuscle tissues evaluated histologically showed no treatment-related lesions, with only small focal liver lesions observed in the livers of mice #789 and #790.

[0163] To further evaluate clinical liver function, we assessed the levels of liver enzymes in the serum of these mice. We analyzed the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to determine whether they were elevated compared to normal levels in six scAAVrh.74.MHCK7.hSGCB-treated mice, as well as two untreated BL6 WT mice and two untreated SGCB-treated mice. - / - Table 2 below shows that untreated SGCB− / − mice exhibit elevated ALT and AST levels, averaging 288 U / L and 784.5 U / L, respectively, outside the normal range for healthy mice. However, SGCB− / − mice administered AAV do not exhibit elevated ALT and AST levels, respectively, exhibiting average ALT and AST levels within the normal range of 89.5 U / L and 330.75 U / L.

[0164] Taken together, these data indicate that the test material was well tolerated by the test subjects. Furthermore, two age-matched untreated SGCB - / -Independent histopathology review, compared with reference specimens from mice, demonstrated that administration of scAAVrh.74.MHCK7.hSGCB significantly reduced the risk of developing SGCB - / - The results show that the test article substantially reduced muscle fiber atrophy and destruction in mice, thus indicating that the test article may alleviate the severity of myopathy associated with a pronounced deficiency of SGCB. [Table 2]

[0165] Table 2 shows the SGCB treated with scAAVrh.74.MHCK7.hSGCB. - / - mice (n=6), along with untreated BL6 WT (n=2) and SGCB - / - Analysis of alanine aminotransferase and aspartate aminotransferase levels in serum from (n=2) cohorts is provided. The means reported in the two rightmost columns are for each of the three cohorts. Reported in units / L. N / A indicates the sample was too hemolyzed to be analyzed.

[0166] Vector genome biodistribution A real-time quantitative PCR assay (qPCR) was used to examine the presence of test article-specific DNA sequences. Biodistribution analysis was performed in four vector-administered SGCB mice. - / - This was performed on tissue samples collected from animals. A positive signal was defined as ≥100 single-stranded DNA copies per μg of genomic DNA detected. Tissues were collected at necropsy, and a vector-specific primer probe set specific for sequences in the MHCK7 promoter was used. Table 3 shows the results for the high dose (3.0 × 10 12 The vector genome copies detected in each tissue sample from mice (nos. 785, 786, 789, and 790) injected with scAAVrh.74.MHCK7.hSGCB at a total dose of 1.0 × 10 vg (total dose) were compared with the clinical dose (1.0 × 10 12 The figures are shown together with the vg copy numbers from the same tissue samples in mice (nos. 712 and 713) treated with 100 mg of vg total dose.

[0167] The scAAVrh.74.MHCK7.hSGCB transcript was detected at various levels in all collected tissues. As expected, the highest levels were found in skeletal muscle and heart. The lowest levels were detected in gonads, lung, kidney, and spleen. Of note, the original clinical dose (5.0 × 10 13 vg / kg) at this high dose (2.0x10 14 The vector genome copy number was similar in each tissue when compared to the 2000 (vg / kg) cohort. These data indicate that the test sample was efficiently delivered to all examined tissues of the vector-treated mice. [Table 3]

[0168] Table 3 shows the results of four high-dose treated SGCBs. - / - Vector genome copy numbers in mouse organs and muscles are provided. Values ​​are shown in vg / μg genomic DNA.

[0169] As the qPCR results above demonstrate, intravenous delivery of high-dose scAAVrh.74.MHCK7.hSGCB resulted in varying levels of vector transcript distribution in most tissues, with the highest levels occurring in muscle. Therefore, the purpose of this portion of the study was to determine protein expression of the human β-sarcoglycan transgene in these tissues and to ensure functionality of the muscle-specific MHCK7 promoter. Western blotting was used to detect β-sarcoglycan expression in tissue samples from four treated mice (no. 785, no. 787, no. 789, and no. 790).

[0170] β-Sarcoglycan protein expression was observed in varying amounts in all skeletal muscle and heart samples and was detected in the livers of mice 785 and 787 (Table 4, Figure 6). To further investigate liver expression, Western blotting for β-sarcoglycan protein expression was performed on liver tissue from all six treated mice (785, 786, 787, 788, 789, and 790). The results of this Western blot, shown in Figure 6, demonstrate that β-sarcoglycan protein expression was observed in four of the six livers of vector-treated mice (785, 786, 787, and 788). Table 5 below lists detailed β-sarcoglycan protein expression results from all six mice (785, 796, 787, 788, 789, and 790) for which complete biodistribution Western blots were performed. [Table 4]

[0171] Table 4 shows the 2.0 x 10 14

[0039] Figure 1 provides β-sarcoglycan protein expression in individual tissues from six SGCB- / - mice systemically treated with 1000 mg / kg scAAVrh.74.MHCK7.hSGCB. X indicates protein expression in the corresponding tissue. NA = assay not performed.

[0172] This cardiac expression using the MHCK7 promoter is very promising at clinically applicable dosing levels, and given the high incidence of cardiac disease in β-sarcoglycan-deficient LGMD2E patients, systemic delivery could provide significant clinical benefit to these patients. This proposed high dose of 3.0 × 10 12 vg total dose (2.0 × 10 14 SGCBs receiving intravenous tail vein injection of scAAVrh.74.MHCK7.hSGCB at 1000 mg / kg (vg / kg) - / -Mice were completely necropsied, and all extracted muscles and organs were stained by H&E and sent to an independent veterinary pathologist for review. Livers from four vector-treated mice (nos. 785, 786, 787, and 788) demonstrated expression of the β-sarcoglycan transgenic protein, as previously demonstrated with systemic administration (Salva et al., Mol Ther, 2007, 15(2):320-9). Two of the six treated animals (nos. 789 and 790) were reported to have minimal to mild focal liver lesions, but all other organs and muscles reviewed from the six treated mice showed no adverse effects. To assess clinical signs of mild liver lesions in livers from mice nos. 789 and 790, serum liver enzyme levels of alanine aminotransferase and aspartate aminotransferase were measured in all six treated mice. The results of this experiment, shown in Table 4, indicate that the mean AST and ALT levels from the six treated mice were within the normal range, indicating the absence of clinical liver enzyme abnormalities. Livers from animals 789 and 790 exhibited lower vg copy numbers (Table 3), in addition to the absence of expression of the β-sarcoglycan transgenic protein. Collectively, this data indicates that the transgenic β-sarcoglycan protein may have been cleared from the livers of these two animals. However, there was no effect on skeletal muscle expression or liver function.

[0173] High dose (2.0×10 14 At a dose of 5.0 × 10 vg / kg, sustained β-sarcoglycan expression in the liver was observed, but at a lower dose (5.0 × 10 13 No significant toxicity was observed in animals treated with the NOAEL dose (5.0 × 10 vg / kg). No overt toxicity was observed at any of the doses offered. This study was conducted at the NOAEL dose (5.0 × 10 13 Patients are closely monitored for hepatotoxicity, and elevated liver enzymes have been effectively managed with corticosteroids in another study of systemic delivery of AAV for spinal muscular atrophy (Mendell et al., N Engl J Med 2017;377:1713-1722).

[0174] Example 5 LGMD2E open-label study Recombinant AAVrh74 carrying the human SGCB gene under the control of the muscle-specific MHCK7 promoter (scAAVrh74.MHCK7.hSGCB) was delivered via a single systemic infusion via a peripheral vein. The vector was delivered in approximately 10 ml / kg lactated Ringer as needed, infused over approximately 1-2 hours. Patients experienced minimal adrenal suppression from glucocorticosteroids after 30 days. However, as a precaution, each patient's maximum dose was reduced by 50% for 1 week and then reduced by 50% again for 1 week before stopping.

[0175] Cohort 1 included three treated subjects aged 4–15 years with confirmed SGCB mutations in both alleles, negative for AAVrh74 antibodies, and a normal 100-meter walk test score of >40%. Each subject received 5 × 10 13 Subjects received a dose of 1 mg / kg of prednisone. Sixty days after dosing, muscle biopsies were performed under appropriate anesthesia in the tibialis anterior and biceps brachii muscles under the guidance of an anesthesiologist (or anesthesiologist). Biopsies may be performed under ultrasound guidance. Each subject received 1 mg / kg of prednisone one day before gene transfer, and the dose was recorded for 30 days.

[0176] If biopsies are read and 50% or more of the muscle fibers express SGCB in the TA and biceps brachii in all subjects in Cohort 1, then there will be no dose escalation in Cohort 2. If these criteria are not met, subjects in Cohort 2 will receive 2 x 10 14 vg / kg. Three of the patients in Cohort 2 will receive placebo lactated Ringer. These placebo subjects will be treated approximately one year later with the same dose as the subjects treated in their cohort.

[0177] Pre-injection baseline measurements (days -60 to -2) After obtaining informed consent and completing enrollment procedures, a baseline patient history was collected, including a record of all medications and supplements the patient was taking. Baseline functional testing to establish a stable baseline was compared with functional test results collected in previous natural growth studies for consistency of baseline testing. At the screening visit, the 100-meter timed test must be 40% or greater than that expected for age-, height-, and weight-matched healthy controls for inclusion. If a subject is not included in screening, they may continue to participate in the LGMD natural growth study. The following assessments will be performed to confirm a subject's eligibility for this study. Baseline testing, which must be completed before treatment administration, includes the following: Baseline From day -60 to day -2 before gene transfer Informed consent Medical history Physical exam / vital EKG Cardiac MRI (performed without anesthesia, but if the procedure is poorly tolerated and considering the importance of cardiac evaluation in this condition, we will discuss the option of performing anesthesia using a protocol acceptable to Nationwide Children's protocols) Skeletal muscle MRI without anesthesia Hepatitis B, Hepatitis C, HIV antibody (IgG, IgM) testing Safety Laboratory: Complete blood count (CBC) with differential and including platelets Total serum protein Serum gamma-glutamyltransferase (GGT) Use GGT to monitor liver enzymes rather than ALT or AST. Because these enzymes are sourced from damaged muscle, levels can reach 9-10 times the ULN. ALT and AST can vary 30-40% from day to day, making interpretation difficult. GGT is not affected by muscle disease. 22、23 . Serum total bilirubin Glucose Creatine kinase (CK) (CK levels are preferably collected only at the 2-day visit, but may be tested at the 1-day visit at the discretion of the PI) Creatinine / BUN Cystatin C Alkaline phosphatase Amylase ○AST ○ALT Prothrombin time (PT), partial thromboplastin time (PTT) ○Electrolytes (sodium, potassium, chloride, CO2 Urine tests Serum-binding antibodies against rAAVrh74 Serum-binding antibodies against β-sarcoglycan ELISpot assay for AAVrh74 capsid protein and β-sarcoglycan Pregnancy testing (if investigators determine there is a possibility of childbirth) Strength testing of knee and elbow flexors and extensors, hip adductors, and shoulder abductors (measured with a handheld dynamometer) PROMIS Survey Set up your device for activity monitoring Pulmonary function tests (PFTs) including spirometry Timed Functional Tests (100m timed test, climbing four steps, timed up and go) Workspace volume North Star Assessment for Limb-Girdle Muscular Dystrophy (NSAD) Baseline muscle biopsies of upper and lower extremity muscles may be performed using ultrasound-guided techniques. Selection will depend on clinical findings to target the appropriate muscles for analysis with the least risk to the patient. Placebo-delay subjects will not undergo a second baseline muscle biopsy. ·Chest X-ray Day -1 Physical examination and vital signs Start prednisone or a similar glucocorticoid Photos of potential injection sites Safety Laboratory: Complete blood count (CBC) with differential and including platelets Total serum protein Serum gamma-glutamyltransferase (GGT) Use GGT to monitor liver enzymes rather than ALT or AST. Because these enzymes are sourced from damaged muscle, levels can reach 9-10 times the ULN. ALT and AST can vary 30-40% from day to day, making interpretation difficult. GGT is not affected by muscle disease. 22、23 . Serum total bilirubin Glucose Creatine kinase (CK) (CK levels are preferably collected only at the 2-day visit, but may be tested at the 1-day visit at the discretion of the PI) Creatinine / BUN Cystatin C Alkaline phosphatase Amylase ○AST ○ALT Prothrombin time (PT), partial thromboplastin time (PTT) ○Electrolytes (sodium, potassium, chloride, CO2) Urine tests

[0178] Prednisone prophylaxis Antigen-specific T cell responses to the AAV vector were expected between 2 and 4 weeks after gene transfer. One possible consequence of such antigen-specific T cell responses was clearance of transduced cells and loss of transgene expression. To attenuate the host's immune response to AAV-based therapy, subjects were initiated on prophylactic oral prednisone or equivalent glucocorticoids at approximately 1 mg / kg / day 24 hours before the procedure, with a maximum dose of 60 mg / day. If necessary, equivalent glucocorticoids could also be administered intravenously at approximately 1 mg / kg / day. Treatment lasted approximately 1 month. A tapering protocol of prednisone or equivalent glucocorticoids was implemented based on each subject's immune response to gene transfer, assessed by ELISpot assay and liver function monitoring with GGT.

[0179] Gene transfer protocol The scAAVrh74.MHCK7.hSGCB gene vector was prepared according to the Manual of The vectors were prepared by the study physician according to the MOP Operating Procedures. Appropriate dilution of the test article was completed by the pharmacy immediately prior to transport to the clinical site. The vector was diluted using Lactobacillus Ringer and withdrawn into a sterile 60 ml polypropylene syringe. Documentation of the dilution was completed by the pharmacy according to standard pharmacy protocols.

[0180] Syringes containing the vector were transported at room temperature and administered to subjects within 24 hours of preparation. Handling of scAAVrh74.MHCK7.hSGCB followed compliance standards for Biosafety Level 1 vectors (NIH Guidelines for Research Involving recombinant or Synthetic Acid Molecules [NIH Guidelines], April 2016, Department of Health and Human Services, National Institutes of Health Office of Science Policy, Office of Biotechnology Activities.

[0181] Subjects were admitted to either the PICU or pulmonary PICU for gene transfer and examined by either the PI or Co-I the night before gene transfer (day -1). Subjects remained NPO after midnight the night before the gene transfer procedure. The procedure was performed in the patient's room under sterile conditions.

[0182] For vector delivery, an intravenous catheter with a heparin lock was placed in a peripheral vein. A second intravenous catheter was placed for use in the event of complications at the first site. Photographs of these sites were taken on the day of gene transfer. The vector was delivered intravenously while the patient was awake. If deemed necessary by the investigator, patients received conscious sedation according to protocol. Patients were medicated with scAAVrh74.MHCK7.hSGCB administered via a 60 mL polypropylene syringe over approximately 1-2 hours using a syringe pump. Patient vital signs were monitored every 15 minutes for 4 hours during the infusion and hourly for the remaining 24 hours after the infusion.

[0183] Post-transfer monitoring Patient vital signs were monitored every 15 minutes for 4 hours and hourly for the remaining 24 hours after the infusion. Safety laboratory and urinalysis were checked the day after the procedure. Concomitant medications and all adverse / serious adverse events were also monitored and documented after the injection. Subjects were discharged one day after gene transfer (if no safety-related side effects were observed). Subjects returned for follow-up visits at days 7, 14, 30, 60, 90, and 180, and at months 9, 12, 18, 24, 30, and 36. Toxicity monitoring at each of these dates included the following: Physical examination and vital signs Safety Laboratory: Complete blood count (CBC) with differential and including platelets Total serum protein Serum gamma-glutamyltransferase (GGT) * Use GGT to monitor liver enzymes rather than ALT or AST. Because these enzymes are sourced from damaged muscle, levels can reach 9-10 times the ULN. ALT and AST can vary 30-40% from day to day, making interpretation difficult. GGT is not affected by muscle disease. 22、23 Serum total bilirubin Glucose Creatine kinase (CK) (CK levels are preferably collected only at the 2-day visit, but may be tested at the 1-day visit at the discretion of the PI) Creatinine / BUN Cystatin C Alkaline phosphatase Amylase ○AST ○ALT Prothrombin time (PT), partial thromboplastin time (PTT) ○Electrolytes (sodium, potassium, chloride, CO2) Urine tests Immunological testing Physical therapy assessments beginning at day 30 (100-meter time test, strength testing, PROMIS questionnaire, North Star Assessment for Limb-Girdle Muscular Dystrophy (NSAD), 4-step stair climb, timed up-and-go, and workspace volume) Urine tests Photographs of the injection site (days -1, 0, 1, 7, 14, and 30) Adverse events (collected at all study visits) EKG (180 days, 12, 24, 36 months) Cardiac and skeletal muscle MRI (12, 24, and 36 months), Pulmonary function test (60th, 180th day, 12th, 24th, 36th month) Post-gene transfer muscle biopsy at day 60 for cohorts 1 and 2 and 2 years after treatment for all subjects. Muscle selection will be the same as the pre-treatment biopsy site. Post-treatment biopsy will preferably be on the same side unless risk dictates that the biopsy be performed on the contralateral limb.

[0184] Long-term monitoring Recent FDA guidelines will be followed regarding long-term subject follow-up after gene transfer. As discussed, based on previous experience with rAAV or transgenes, the likelihood of delayed adverse events related to gene transfer is very low. Short-term safety will be assessed over a 3-year period, incorporating the active phase of the protocol. If any newly identified risks are related to the product or if subjects suffer any adverse events during this period, long-term follow-up will be initiated in accordance with FDA guidelines.

[0185] CBER will be notified if there is any indication that the follow-up period needs to be extended. All subjects will be provided with written instructions on how to contact the investigator or study coordinator if they experience a serious adverse event thought to be related to their study treatment or participation. This information is included in the informed consent document. All subjects will be instructed to notify the investigator of any changes in their address or contact information.

[0186] Post-exam follow-up We will follow recent FDA guidance regarding long-term follow-up of subjects after gene transfer. As indicated in the guideline, the vector has a very low probability of delayed adverse events related to gene transfer. Safety will be evaluated for three years after administration, incorporating the active phase of the protocol. If any newly identified risks are related to our product or if the subject suffers any adverse events during this period, long-term follow-up will be initiated in accordance with FDA guidelines.

[0187] Primary Outcome of Clinical Trials This is a Phase I clinical trial, with safety as the primary outcome. Demonstration of β-SG protein expression (≥20% above baseline) as determined by quantitative immunofluorescence or immunoblot analysis in muscle biopsies at 8 weeks. Exploratory outcomes For each participant, 3 years after introgression, their 100-meter time improved by 10% or more compared to baseline. Reduction of CK after gene therapy will serve as an exploratory outcome. CK levels will preferably be collected only at the 2-day visit, but may be tested at the 1-day visit at the discretion of the PI. Workspace volume Handheld dynamometer measurements of knee and elbow flexors and extensors, hip adductors, and shoulder abductors Improved ejection fraction measured by cMRI Skeletal MRI Pulmonary function tests (PFTs) including spirometry Time function test [Climbing four steps, Time Up and Go] North Star Assessment for Limb-Girdle Muscular Dystrophy (NSAD) ○ Activity levels determined by a Fitbit or similar activity monitoring device Patient-reported physical function using the PROMIS Upper Extremity and Mobility questionnaire

[0188] Cohort 1 results All subjects in Cohort 1 were doing well at the time of study (Subjects 1 and 2, examined 90 days after injection; Subject 3, examined 60 days after injection). All subjects progressed well through 9 months after injection. There was one serious adverse event in the study; one subject experienced elevated liver enzymes and bilirubin after steroid discontinuation. This event resolved with increased steroids. Two of the subjects had elevated liver enzymes that resolved with increased steroids, and these levels returned to baseline.

[0189] Muscle needle biopsies from the tibialis anterior and biceps brachii muscles were used to quantify transgene expression compared to baseline by day 60 in Cohort 1. The primary endpoint was ≥20% expression of SGCB protein. If SGCB expression was ≥50% above baseline in all treated subjects, no dose escalation was performed. If SGCB expression was <50% in all treated subjects, the dose for Cohort 2 and placebo subjects was increased to 2x10 14 The serum saturation level was increased to 1000 mg / kg. If possible, a biopsy 2 years after treatment was performed in the same muscle as the baseline biopsy. All biopsy samples were blinded and coded by the laboratory director using a computer-generated code. Expression was quantified using direct immunofluorescence and Western blot analysis of muscle biopsies. Bioquant® automated software was used to quantify the number of muscle fibers expressing SGCB. Baseline patient demographics are shown in Table 5. [Table 5]

[0190] Figure 7 provides representative images showing robust SGCB expression in the muscles of all three subjects 8 weeks after vector administration. Table 6 provides the mean intensity and percentage of SGCB-positive fibers in each subject. The mean intensity of immunohistochemical staining for the entire cohort was 47%, and the mean percentage of SGCB-positive fibers was 51%. Figure 8 provides Western blots showing the detection of β-sarcoglycan expression in three subjects 90 days after vector administration. The Western blot data demonstrate that gene transfer delivers full-length β-sarcoglycan. Quantification by Western blot is provided in Table 7 and shows that the mean expression of β-sarcoglycan protein was increased by approximately 36.1% compared to normal. [Table 6] [Table 7]

[0191] Collected muscle biopsies were examined for the presence of test article-specific DNA sequences using a real-time quantitative PCR assay (qPCR). A positive signal was defined as 100 or more single-stranded DNA copies detected per μg of genomic DNA. An average of 8.4E+04 vector copies per μg of DNA and 0.6 copies per nucleus were detected in muscle biopsies.

[0192] The presence of sarcoglycan complexes in each subject was also investigated. As determined by Western blot, the average microdystrophin expression was 36% of normal (n=3). In addition, the expression of alpha-sarcoglycan was quantified by immunohistochemistry. Figure 9 shows that the expression of beta-sarcoglycan in the subjects upregulated the sarcoglycan complex, as indicated by the expression of alpha-sarcoglycan.

[0193] The subjects were tested for creatine kinase (CK) levels in their blood, and as shown in Table 9, the subjects' CK levels were reduced by an average of about 82%. [Table 9]

[0194] Example 6 β-sarcoglycan gene transfer restores sarcoglycan complexes to the membrane. Treatment with scAAVrh74.MHCK7.hSGCB restored sarcoglycan complexes to the membrane (Figure 10). Figure 10 shows the restoration of β-sarcoglycan and α-sarcoglycan expression at the membrane of the patient under study, as well as the colocalization of β-sarcoglycan and α-sarcoglycan. The colocalization of β-sarcoglycan and α-sarcoglycan indicates that scAAVrh74.MHCK7.hSGCB restored the sarcoglycan complex.

[0195] Example 7 LGMD2E patients treated with β-sarcoglycan gene transfer showed improvement in the 100-meter time test 3 months after treatment. Treatment with scAAVrh74.MHCK7.hSGCB resulted in patients with a clear improvement in the timed 100-meter test just three months after gene transfer (Figure 11). Timed walking tests, such as the timed 100-meter test, are used to measure function in subjects with muscular dystrophy. The tests in this study measured patients' baseline performance compared to their performance after treatment. Figure 11 shows the mean percent change from baseline for three subjects over the first three months after gene transfer. The data show a mean increase of more than 15% over baseline after three months, indicating improvement in motor function after β-sarcoglycan gene transfer.

[0196] Example 8 LGMD2E patients treated with β-sarcoglycan gene transfer showed improved functional measures 9 months after treatment. Treatment with scAAVrh74.MHCK7.hSGCB resulted in clear improvements in patients 9 months after systemic administration of scAAVrh.74.MHCK7.hSGCB. Three patients participated in functional testing. For example, in the 100-m time test, at baseline (pre-treatment), one patient had limited hip extension and flexion when running 100 meters. However, 9 months after treatment, the same patient demonstrated improved hip extension and flexion while running, demonstrating increased speed. In addition, in the trunk control test, another patient demonstrated improvement in the time to stand up test 9 months after treatment. At baseline or pre-treatment, this subject had poor trunk control, which also improved 9 months after treatment. Also, in the sitting test, the patient was asked to stand up from a sitting position. For example, the remaining patient showed a reduction in time to stand up 9 months after treatment compared to pre-treatment. These data are summarized in Table 10. [Table 10]

[0197] An age-matched natural growth study compared the change from baseline in NSAD for untreated subjects (denoted natural growth subjects, see Table 11) and subjects administered scAAVrh74.MHCK7.hSGCB, herein referred to as the "North Star Assessment of Limb-Girdle Muscular Dystrophy." As shown in Figures 12A-C, natural growth subjects showed a stable reduction in NSAD change over 200 days, while treated subjects showed a stable improvement in NSAD change over 270 days (Figures 12A and 12C). Treated subjects showed a stable improvement in NSAD change over 270 days (Figure 12B). [Table 11]

[0198] Example 9 formulation scAAVrh74.MHCK7.hSGCB is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188. In one embodiment, formulation information is summarized in Table 12. [Table 12]

[0199] The drug product is stored frozen at temperatures below −60° C. The frozen drug product must be thawed prior to clinical administration.

[0200] scAAVrh74.MHCK7.hSGCB is stored at -60°C or below, at which temperature the material is stable under long-term storage conditions. scAAVrh74.MHCK7.hSGCB vials are thawed at room temperature (20°C–25°C). The thawed vector vials are wiped with alcohol and placed in a biosafety cabinet. scAAVrh74.MHCK7.hSGCB formulations are prepared aseptically in a Class II biosafety cabinet under sterile conditions.

[0201] scAAVrh74.MHCK7.hSGCB for intravenous (IV) infusion is supplied in vials (2 mL per vial). The total vg dose is calculated based on the patient's weight. The appropriate number of vials is 5 x 10 13 vg / kg or 2 × 10 14 vg / kg equivalent, and 2 × 10 13 vg / mL, 5 × 10 13 vg / mL, or 4 × 10 13 Product titer for scAAVrh74.MHCK7.hSGCB lot in vg / ml will be determined for each patient based on body weight.

[0202] scAAVrh74.MHCK7.hSGCB is administered as a single IV infusion delivered via a syringe pump into a peripheral limb vein over approximately 1-2 hours.

[0203] Example 10 Elderly patients and persistent Gene replacement via scAAVrh74.MHCK7.hSGCB has shown positive results in the treatment of LGMD-2E and other related diseases. This study was conducted to test the ability of scAAVrh74.MHCK7.hSGCB to treat older, more severely affected muscles and the long-term persistence of the AAV viral vector. First, 4-week-old sgcb mice were transfected with scAAVrh74.MHCK7.hSGCB for long-term persistence testing. - / - Mice were systemically treated with scAAVrh74.MHCK7.hSGCB. More than 24 months after treatment, high levels of vector genome copy number were detected by PCR in all transduced muscles. Furthermore, immunofluorescence staining of treated muscles showed no decrease in protein expression levels (>95%) in any muscle compared to previous time points, and hSGCB protein remained correctly localized in the soma.

[0204] Second, a mouse model of LGMD2E (β-sarcoglycan) will be treated at an older age (e.g., 12 months) with systemic delivery of the scAAVrh74.MHCK7.hSGCB vector. At an endpoint of 6 months post-treatment, muscles from these mice will be evaluated for protein expression, histological rescue, and functional improvement. Gene expression in muscles will be observed throughout the lower limbs, upper limbs, and proximal trunk, particularly the diaphragm and heart. Furthermore, the level of fibrosis will be compared to untreated controls. Further functional studies will include assessment of force output in the tibialis anterior (TA) and diaphragm (DIA) muscles, and resistance to contraction-induced injury in the TA muscle.

[0205] While the present disclosure has been described in terms of specific embodiments, those skilled in the art will recognize that variations and modifications thereof occur. Accordingly, only such limitations as appear in the claims should be placed on the present disclosure.

[0206] All documents referenced in this application are incorporated herein by reference in their entirety. References: 1 Bonnemann CG, Modi R, Noguchi S, Mizuno Y, Yoshida M, Gussoni E et al. Beta-sarcoglycan(A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995;11:266-273. 2 Moore SA, Shilling CJ, Westra S, Wall C, Wicklund MP, Stolle C et al. Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 2006;65:995-1003.3 Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E et al. Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in beta-sarcoglycan-deficient mice.Hum Mol Genet 1999;8:1589-1598. 4 Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL et al.Disruption of the beta-sarcoglycan gene reveals pathogenic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell 2000;5:141-151. 5 Bonnemann CG,Passos-Bueno MR,McNally EM,Vainzof M,de Sa Moreira E,Marie SK et al.Genomic screening for beta-sarcoglycan gene mutations:missense mutations may cause severe limb-girdle muscular dystrophy type 2E(LGMD 2E).Hum Mol Genet 1996;5:1953-1961. 6 Angelini C,Fanin M,Freda MP,Duggan DJ,Siciliano G,Hoffman EP.The clinical spectrum of sarcoglycanopathies.Neurology 1999;52:176-179. 7 Sandona D,Betto R.Sarcoglycanopathies:molecular pathogenesis and therapeutic prospects.Exp Rev Mol Med 2009;11:e28. 8 Fanin M,Melacini P,Boito C,Pegoraro E,Angelini C.LGMD2E patients risk developing dilated cardiomyopathy.Neuromusc Disord 2003;13:303-309. 9 Sveen ML,Thune JJ,Kober L,Vissing J.Cardiac involvement in patients with limb-girdle muscular dystrophy type 2 and Becker muscular dystrophy.Arch Neurol 2008;65:1196-1201. 10 Melacini P,Fanin M,Duggan DJ,Freda MP,Berardinelli A,Danieli GA et al.Heart involvement in muscular dystrophies due to sarcoglycan gene mutations.Muscle Nerve 1999;22:473-479. 11 Narayanaswami P,Weiss M,Selcen D,David W,Raynor E,Carter G et al.Evidence-based guideline summary:diagnosis and treatment of limb-girdle and distal dystrophies:report of the guideline development subcommittee of the American Academy of Neurology and the practice issues review panel of the American Association of Neuromuscular & Electrodiagnostic Medicine.Neurology 2014;83:1453-1463. 12 Wong-Kisiel LC,Kuntz NL.Two siblings with limb-girdle muscular dystrophy type 2E responsive to deflazacort.Neuromusc Disord 2010;20:122-124. 13 Barresi R,Di Blasi C,Negri T,Brugnoni R,Vitali A,Felisari G et al.Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.J Med Genet 2000;37:102-107.14 Gibertini S,Zanotti S,Savadori P,Curcio M,Saredi S,Salerno F et al.Fibrosis and inflammation are greater in muscles of beta-sarcoglycan-null mouse than mdx mouse.Cell Tissue Res 2014;356:427-443. 15 McCarty DM,Fu H,Monahan PE,Toulson CE,Naik P,Samulski RJ.Adeno-associated virus terminal repeat (TR)mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther 2003;10:2112-2118. 16 McCarty DM,Monahan PE,Samulski RJ.Self-complementary recombinant adeno-associated virus(scAAV)vectors promote efficient transduction independently of DNA synthesis.Gene Ther 2001;8:1248-1254. 17 Chicoine LG,Rodino-Klapac LR,Shao G,Xu R,Bremer WG,Camboni M et al.Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates.Mol Ther 2014;22:713-724. 18 Rodino-Klapac LR,Montgomery CL,Bremer WG,Shontz KM,Malik V,Davis N et al.Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.Mol Ther 2010;18:109-117. 19 Rodino-Klapac LR,Janssen PM,Montgomery CL,Coley BD,Chicoine LG,Clark KR et al.A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy.J Transl Med 2007;5:45. 20 Wang B,Li J,Fu FH,Chen C,Zhu X,Zhou L et al.Construction and analysis of compact muscle-specific promoters for AAV vectors.Gene Ther 2008;15:1489-1499. 21 Chicoine LG,Montgomery CL,Bremer WG,Shontz KM,Griffin DA,Heller KN et al.Plasmapheresis eliminates the negative impact of AAV antibodies on micro-dystrophin gene expression following vascular delivery.Mol Ther 2014;22:338-347. 22 Matsuda R,Nishikawa A,Tanaka H.Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue:evidence of apoptosis in dystrophin-deficient muscle.J Biochem 1995;118:959-964. 23 Straub V,Rafael JA,Chamberlain JS,Campbell KP.Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.J Cell Biol 1997;139:375-385. 24 Mendell JR,Sahenk Z,Malik V,Gomez AM,Flanigan KM,Lowes LP et al.A phase 1 / 2a follistatin gene therapy trial for becker muscular dystrophy.Mol Ther 2015;23:192-201. 25 Dressman D,Araishi K,Imamura M,Sasaoka T,Liu LA,Engvall E et al.Delivery of alpha-and beta-sarcoglycan by recombinant adeno-associated virus:efficient rescue of muscle,but differential toxicity.Hum Gene Ther 2002;13:1631-1646. 26 Rodino-Klapac LR,Lee JS,Mulligan RC,Clark KR,Mendell JR.Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D.Neurology 2008;71:240-247. 27 Shield MA,Haugen HS,Clegg CH,Hauschka SD.E-box sites and a proximal reg-ulatory region of the muscle creatine kinase gene differentially regulate expres￢sion in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol Cell Biol 1996;16:5058-5068. 28 Rabinowitz JE,Rolling F,Li C,Conrath H,Xiao W,Xiao X et al.Cross-packaging of a single adeno-associated virus (AAV)type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.J Virol 2002;76:791-801. 29 Grieger JC,Choi VW,Samulski RJ.Production and characterization of adeno-associated viral vectors.Nat Protoc 2006;1:1412-1428. 30 Clark KR,Liu X,McGrath JP,Johnson PR.Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.Hum Gene Ther 1999;10:1031-1039. 31 Liu M,Yue Y,Harper SQ,Grange RW,Chamberlain JS,Duan D.Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury.Mol Ther 2005;11:245-256. 32 Hakim CH,Grange RW,Duan D.The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2-to 20-mo-old 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Claims

[Claim 1] A method of treating muscular dystrophy in a subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB; The method comprises administering the rAAV at a dose of about 1.0 x 10 12 vg / kg to about 5.0 x 10 14 vg / kg using a systemic administration route, based on supercoiled plasmid as a quantitative standard, and wherein serum creatine kinase (CK) levels in the subject are reduced after administration of the rAAV compared to serum CK levels before administration of the rAAV.

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