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 by reference in their entireties herein.

[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), causing 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) Absence of β-sarcoglycan causes progressive dystrophy with chronic muscle fiber loss, inflammation, fatty replacement, and fibrosis, all of which cause deterioration of muscle strength and function. (3, 4) As complexes, sarcoglycans (α-, β-, γ-, δ-), which range in size from 35–50 kD, (5) are all transmembrane proteins that provide stability to the sarcolemma, providing protection from mechanical stress during muscle activity. (3) Loss of 3-sarcoglycan in LGMD2E usually causes a concomitant loss of various degrees of other sarcoglycan proteins that contribute to muscle membrane brittleness leading to loss of muscle fibers. 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 teenage years. 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, containing two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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 in 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 rh.74 genome is presented in U.S. Pat. No. 6,371-6,383 (2004), 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. Pat. 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 ITR. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames that code for 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), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for 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 non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA within a plasmid making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, so that part or all of the internal approximately 4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) may 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. It easily survives the conditions used to inactivate adenovirus (56°C-65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not resist superinfection.

[0007] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle.See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad 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, muscles are highly vascularized, and therefore, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997), As described in USA, 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 required for correct 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 protein 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. In previous reports, Sgcb null mice have been well characterized. Araishi et al.3 developed β-sarcoglycan-deficient mice, with loss of all sarcoglycans, not just sarcospan, and with at least minor preservation of merosin, dystroglycan, and dystrophin, recapitulating the clinical picture seen in LGMD2E. The histological changes in this animal model were also prototypes of the clinical counterparts, including overt skeletal muscle fibrosis. (14) Dressman et al.(25) injected the transversus abdominis muscle with rAAV2.CMV.SGCB. Gettut 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,16 and optimization of the human β-sarcoglycan gene (hSGCB) have also been described. Both gene restoration and reduction of fibrosis are required for functional improvement in patients with LGMD and other muscular dystrophies. There is a need for a method 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. Pat. 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 of delivering β-sarcoglycan to muscle to reduce and / or prevent fibrosis, and / or increase muscle strength, and / or treat a mammalian subject suffering from muscular dystrophy.

[0011] In one aspect, a method of treating muscular dystrophy in a subject in need thereof includes administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB to a subject in need thereof, wherein the rAAV delivers approximately 1.0×10 mAb to a subject in need thereof using a systemic route of administration, based on a supercoiled plasmid as a quantification standard. 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, 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.

[0012] In another aspect, a method of treating muscular dystrophy in a subject in need of such treatment is provided, comprising administering a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, 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 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, or motor function is improved in the subject compared to the subject's motor function before administration of the rAAV, wherein motor function is determined by a 100 meter timed walk test.

[0013] In another aspect, the 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 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 × 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 a 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 a 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 a 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. 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 that includes 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 that includes a recombinant AAV (rAAV) scAAVrh74.MHCK7.hSGCB, where the scAAVrh74.MHCK7.hSGCB includes a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:19.

[0016] In another aspect, a method of generating 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, a recombinant AAV vector comprising a polynucleotide sequence encoding β-sarcoglycan is described herein. 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, 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 which protein retains β-sarcoglycan activity.

[0019] In another aspect, a recombinant AAV vector is described herein 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" is used to refer to conditions that are generally understood in the art as stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68°C or 0.015M sodium chloride, 0.0015M 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, NY1989). 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 concerned, 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 the 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 an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary, for example, between 1% and 15% of the stated numerical value or numerical range.

[0022] Other agents can be included in the hybridization and washing buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 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, but other suitable agents can be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, but under typical ionic strength conditions, the rate of hybridization is largely independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one of skill in the art to take these variables into account, allowing DNAs of different sequence similarity to form hybrids.

[0023] In another embodiment, the recombinant AAV vector described herein can be operably linked to a muscle-specific regulatory element. For example, the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-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, or 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 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 includes a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to the nucleotide sequence set forth in, for example, 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] The 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 are also contemplated, such as rAAV with capsid mutations. 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 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. In addition, the present disclosure provides a composition or pharmaceutical composition comprising a scAAVrh74.MHCK7.hSCGB rAAV vector comprising a nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19, or comprising a nucleotide sequence encoding 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 a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein the rAAV is administered in an amount of about 1.0×10 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 a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGC for the preparation of a medicament for treating muscular dystrophy, wherein the medicament comprises at least about 1.0×10 12 vg / kg ~ approx. 5.0×10 14 The use is provided wherein the medicament comprises scAAVrh74.MHCK7.hSGC at a dose of 100 mg / kg, 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 is increased 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 is decreased 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 is increased 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 methods, compositions and uses provided, motor function is improved in the subject, compared with the motor function of the subject before administration of rAAV, and the motor function is determined by 100-meter timed walking test.For example, motor function is improved 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 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 1.85×10 based on a linearized plasmid as a quantification standard. 13 vg / kg or approximately 7.41 × 1013 The dose of rAAV administered is approximately 5 × 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 an intravenous route is about 1.0×10 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 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 1.0×10 12 vg / kg ~ approx. 5.0×10 14vg / kg, wherein 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 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 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. For example, in any of the methods provided, the systemic administration route is an intravenous route, and the dose of rAAV administered is about 5.0×10 based on a supercoiled plasmid as a quantification standard. 13 In another embodiment, the dose of rAAV administered is about 2.0×10 vg / kg, based on a supercoiled plasmid as a quantification standard. 14 In another embodiment, the dose of rAAV administered is about 7.41x10 vg / kg, based on the linearized plasmid as a quantification standard. 13 In another embodiment, the dose of rAAV administered is about 1.85×10 vg / kg, based on the linearized plasmid as a quantification standard. 13 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, the dose of rAAV is administered at a concentration of about 10 mL / kg. In any of the methods provided, 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 a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, wherein motor function is demonstrably improved in the subject compared to the subject's motor function prior to administration of the rAAV, the motor function being determined by a 100 meter timed 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%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, or about 50%.

[0041] Methods of increasing levels of alpha-sarcoglycan 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 are provided for increasing levels of alpha-sarcoglycan in a subject in need thereof, 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 levels of alpha-sarcoglycan in a subject in need thereof. In some embodiments, alpha-sarcoglycan is co-localized to the membrane of a cell 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 a cell 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 methods, uses and compositions for treating muscular dystrophy provided, the subject is 4-15 years old, has a confirmed beta-sarcoglycan (SGCB) mutation in both alleles, is negative for AAVrh74 antibodies, and / or has a 100 meter walk test greater than 40% or normal. In any of the methods, uses and compositions for treating muscular dystrophy provided, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject, e.g., a subject in the range of 1-10 years old. In some embodiments, the subject is 4-15 years old. The subject, in one embodiment, is an adolescent subject, e.g., a subject in the range of 10-19 years old. Additionally, the subject, in one embodiment, is a young adult subject, e.g., a subject in the range of late teens or early twenties, e.g., the subject may be in the range of 15-29 years old. In some embodiments, the subject is a middle-aged adult or elderly subject, such that a middle-aged adult may be in the range of 25-55 years old and an elderly subject may be in the range of over 50 years old.

[0045] In some embodiments, the rAAV is administered by injection, infusion, or implantation. For example, the rAAV is administered by infusion over approximately 1-2 hours. Additionally, the rAAV is administered by intravenous route 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 at a concentration of about 1.0×10 based on supercoiled plasmid as a quantification standard using a systemic route of administration. 12 vg / kg ~ approx. 5.0×10 14In some embodiments, the 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 serotype AAVrh.74. 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 at a dose of about 1.0×10 12 vg / kg ~ approx. 5.0×10 14 The rAAV was administered at a dose of approximately 5.0×10 vg / kg, the subject was suffering from limb-girdle muscular dystrophy, and the rAAV was administered at approximately 5.0×10 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 × 10 13The rAAV is administered at a dose of 1000 mg / kg by intravenous infusion over approximately 1-2 hours, and the rAAV comprises the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0049] Additionally, the 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 at a dose 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 × 10 13 The compositions are provided in a dosage form formulated to deliver a dose of 100 mg / kg.

[0050] The present disclosure also relates to the 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 5.0×10 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 × 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, 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 with 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 with 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 with 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 sarcoglycan or 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 subject's cells 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 methods, uses or compositions provided, 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%. 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 decreased after administration of the rAAV compared to serum CK levels before administration of the rAAV. For example, serum CK levels in a subject are 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, 60 days, or 90 days after administration of the rAAV, compared to serum CK levels 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 the 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 administration of rAAV. For example, the number of beta-sarcoglycan positive fibers in the muscle tissue of the 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 alpha-sarcoglycan protein level by immunohistochemistry or Western blot in muscle biopsy 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 SEQ ID NO:3 or SEQ ID NO:19, or includes the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0061] The present disclosure also provides for the use of the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a pharmaceutical 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 SEQ ID NO:3 or SEQ ID NO:19, 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 beta-sarcoglycan gene in cells, the expression of beta-sarcoglycan gene in cells is detected by measuring beta-sarcoglycan protein level by Western blot or immunohistochemistry in muscle biopsy before and after administration of scAAVrh74.MHCK7.hSGCB construct. For example, the cell has an AAV virus copy number of more than 1. In addition, the beta-sarcoglycan gene is measured in the subject by detecting rAAV vector genome copy more than 1 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 for the use of a 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, the method 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, 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 a subject's muscle tissue, 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 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 muscle tissue of a subject, wherein the scAAVrh74.MHCK7.hSGCB construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:19, 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 of 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 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 SEQ ID NO:3 or SEQ ID NO:19, or comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:19.

[0073] Also provided is a method of increasing 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 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 for the use of a scAAVrh74.MHCK7.hSGCB construct nucleotide sequence for the preparation of a medicament for increasing localization of alpha-sarcoglycan 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 of 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 SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:19, 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 for the use of a 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 SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:19, 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 the 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 is a method of 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 of generating rAAV comprises transferring an AAV vector plasmid into a host cell. In another embodiment, the plasmid comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO:24. In another aspect, the 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, such as human primary cells or established cell lines). In one embodiment, the mammalian cells include 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 the host cell. The host cell stably carrying the AAV vector plasmid can be used to generate rAAV. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCB.KAN plasmid.

[0083] The method of producing a recombinant AAV vector particle provided herein may further include a step of transferring a packaging plasmid and / or a helper virus into a host cell. For example, the method further includes a step in which the packaging cell contains a stably integrated AAVcap gene, and / or a step in which the packaging cell contains a stably integrated AAVrep gene. The present invention also provides a cell, the cell comprising a plasmid comprising a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 24, or a plasmid comprising the nucleotide sequence of SEQ ID NO: 24. Also provided is a cell comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 19.

[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 a mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In some embodiments, administering an 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 disorders that cause gradual loss of strength and muscle mass.Non-limiting examples of muscular dystrophy include congenital muscular dystrophies such as Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanopathy, congenital muscular dystrophy with partial LAMA2 deficiency, merosin-deficient congenital muscular dystrophy, 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, and the like. 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 simple epidermolysis bullosa, 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 following 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 of 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 invention are formulated for intramuscular or intravenous injection. In addition, the compositions of the invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation.

[0092] In addition, any of the compositions are formulated for administration to a subject suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-related 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 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 subjects suffering from muscular dystrophy (e.g., 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 a formulation or composition comprising a rAAV virion comprising a capsid from AAVrh74, a buffer, an ionic strength agent, and a detergent. In the provided formulation or composition, the rAAV is at about 1.0×10 12 vg / ml ~ approx. 5.0x10 14 Concentration of 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 and at a pH of 8.0, or the buffer comprises Tris at about 20 mM and at a pH of 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 (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. For example, the ionic strength agent comprises MgCl2 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 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, or the ionic strength agent comprises MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM.

[0097] In any of the formulations or compositions provided, the surfactant comprises one or more of sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. For example, the poloxamers comprise 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 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, and additional aspects are described in other sections, such as the detailed description. It is understood that the entire document is intended to relate as a unified disclosure, and that all combinations of features described herein are considered, 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 specific 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 an aspect of the invention individually. 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 a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB; The rAAV was administered at a dose of approximately 1.0 x 10 based on supercoiled plasmid as a quantification standard using a systemic route of administration. 12 vg / kg ~ approx. 5.0×10 14 vg / kg, 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. (Item 2) 1. A method of treating muscular dystrophy in a subject in need thereof, comprising administering a recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCB, the level of beta-sarcoglycan gene expression in 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 prior to 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% 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. (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) 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 5.0×10 13 vg / kg or approximately 2.0 × 10 14 Based on the dose of 1.85 × 10 vg / kg or linearized plasmid as a quantification standard, 13 vg / kg or 7.41 × 10 13 6. The method according to any one of items 1 to 5, wherein the antibody is administered in a dose of 100 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) 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 according to any one of items 1 to 10, wherein the rAAV comprises a 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 an MHCK7 promoter sequence of SEQ ID NO:4. (Item 13) 13. The method according to any one of items 1 to 12, wherein the rAAV is an rAAV of 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 according to 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 to a subject approximately 5.0×10 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 × 10 13 administering to the subject an intravenous infusion of rAAV at a dose of 1000 ng / 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 a subject's cells, 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: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 claim 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) 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 rAAV administration. (Item 26) The method according to 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) The method of claim 22, wherein expression of the beta-sarcoglycan protein is increased by at least 39% after administration of rAAV. (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) 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) 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 expression of alpha-sarcoglycan in a subject in need thereof, comprising administering to the subject an rAAV comprising a 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 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) 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) 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 items 34 to 36, wherein the alpha-sarcoglycan is co-localized to the membrane of a cell 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 a muscular dystrophy. (Item 40) 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, 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. (Item 43) The method of claim 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 according to any one of items 1 to 43, wherein the subject is a human subject aged 4 to 15 years. (Item 45) 44. The method according to 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 greater than 40% or normal. (Item 47) 44. The method according to any one of items 1 to 43, wherein the subject is a middle-aged adult or an elderly subject. (Item 48) 44. The method according to any one of items 1 to 43, wherein the subject is a human subject aged 25 to 55 years. (Item 49) 44. The method according to 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: (Item 51) The rAAV is about 1.0×10 12 vg / ml ~ approx. 5.0x10 14 vg / ml, or approximately 5.0x10 12 vg / ml ~ approx. 1.0x10 14 51. The composition according to item 50, having a concentration of 1000 mg / ml. (Item 52) The rAAV is about 2.0×10 13 vg / ml, 4 × 10 13 vg / ml, 5 × 10 13 51. The composition according to item 50, having a concentration of 1000 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 according to item 50, wherein the ionic strength agent comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM. (Item 58) 51. The composition according to item 50, wherein the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM. (Item 59) 51. The composition according to item 50, wherein 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. (Item 60) 51. The composition according to item 50, wherein the ionic strength agents comprise 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 the 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, the plasmid comprising 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 according to 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 according to item 74 or 75, comprising the nucleotide sequence of SEQ ID NO: 19. (Item 77) 77. The cell according to any one of items 74 to 76, wherein the cell is an insect cell, a mosquito cell, or a mammalian cell. [Brief description of the drawings]

[0099] [Figure 1]Provides a schematic diagram of the therapeutic β-sarcoglycan transgene cassette. Self-complementary AAV vector containing 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] Figure 1. Human β-sarcoglycan expression in skeletal muscle. A) Immunofluorescence imaging of skeletal muscle, diaphragm, and heart from SGCB- / - mice injected intravenously with 3e12vg total dose of scAAVrh.74.MHCK7.hSGCB. Representative images with all muscles showing >98% transduction. 20x images shown. B) Western blotting showing expression of the hSGCB transgene (43 kDa) in muscles treated with clinical dose (#716) and high doses (#785, #786). N=6 for treatment groups, 100 kDa corresponds to 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 muscle 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. [Diagram 5]Figure 2 shows that overall gait in the x and y planes was significantly reduced in KO mice and slightly improved in MHCK7-treated mice. Vertical rearing activity in the hind limbs was slightly improved in MCHK7-treated mice (n=6). [Figure 6] 13 provides biodistribution westerns for muscle and organs from SGCB− / − mice systemically injected with two high doses (2.0×1014 vg / kg) scAAVrh.74.MHCK7.hSGCB. 43 kDa corresponds to β-sarcoglycan protein. 125 kDa corresponds to vinculin loading control. [Figure 7] 13 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] 13 provides β-sarcoglycan protein expression in muscle biopsies of human subjects 90 days after systemic administration of 5.0×10 13 vg / kg scAAVrh.74.MHCK7.hSGCB as detected and quantified by Western blot. [Figure 9] FIG. 1 shows that β-sarcoglycan protein expression upregulated the expression of the sarcoglycan complex, as demonstrated by detection and quantification of alpha-sarcoglycan by immunohistochemistry. [Figure 10] FIG. 1 shows restoration of membrane expression of β-sarcoglycan, α-sarcoglycan, and colocalization of β-sarcoglycan and α-sarcoglycan in patient #3 during the study. [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 the natural growth control group (FIG. 12A) and study subjects following administration of 5.0×10 vg / kg scAAVrh.74.MHCK7.hSGCB (FIG. 12B) are provided. NSAD data for six individual natural growth control patients is 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 PREFERRED EMBODIMENTS

[0100] The present disclosure is based on the discovery that administration of AAV vectors containing polynucleotides expressing β-sarcoglycan results in the reduction or complete recovery of muscle fibrosis in animal models of limb-girdle muscular dystrophy.As shown in the examples, administration of the AAV vectors described herein results in the recovery of dystrophic features, including the reduction of degenerated fibers, the reduction of inflammation, and the improvement of functional recovery due to protection against eccentric contraction by increasing 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 where certain functions are provided by a co-infecting helper virus. Currently, there are 13 serotypes of AAV that have been characterized. 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, it is well known that various serotypes are very closely related, both structurally and functionally, even at the genetic level, so 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. Puttson, 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 have 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 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) that are flanked by AAV terminal repeats (ITRs) that can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector that encodes and expresses the rep and cap gene products.

[0103] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is 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 that is 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 the 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 generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants are also contemplated, such as rAAVs with capsid mutations. 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 comprises a rAAV genome. The DNA plasmid is introduced into a cell that is permissive for infection with AAV's helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for the incorporation of the rAAV genome into infectious viral particles. The technology of producing rAAV particles is standard in the art, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell. The production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and the 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 WO01 / 83692, which is incorporated herein by reference in its entirety.

[0106] The method of generating packaging cells is to create a cell line that stably expresses all the components required for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking AAVrep and cap genes, AAVrep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. 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), 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 advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus 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 a packaging cell that produces infectious rAAV.In one embodiment, the packaging cell can be a stably transformed cancer cell, such as HeLa cell, 293 cell, and PerC.6 cell (similar 293 line).In another embodiment, the packaging cell is a cell that is not a transformed cancer cell, such as low-passage 293 cell (human fetal kidney cell transformed with adenovirus E1), MRC-5 cell (human fetal fibroblast), WI-38 cell (human fetal fibroblast), Vero cell (monkey kidney cell), and FRhL-2 cell (rhesus fetal lung cell).

[0109] Recombinant AAV (i.e., infectious, encapsidated rAAV particles) of the invention comprise a rAAV genome. Embodiments include, but are not limited to, a rAAV designated pAAV.MHCK7.hSCGB, which comprises the polynucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:19, as well as a rAAV designated 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 by 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 rAAV in a pharma- ceutically acceptable carrier. The compositions may also include other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients, 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 dextrin; 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 mg to 1000 mg of DNase resistant particles (DRP) or more. The dosage may be expressed in units of viral genomes (vg). The titer of the rAAV may be determined by supercoiled or linearized plasmid quantification standards.

[0113] Methods of 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 the rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In an embodiment of the present invention, an effective dose is a dose 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, causes 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, such as limb-girdle muscular dystrophy. Thus, provided is a method of 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. Combination therapy as used herein includes simultaneous or sequential therapy. Combination of the method of the present invention with standard medical treatment (e.g., steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort) is specifically contemplated, as well as combination with novel therapy. In this respect, this combination includes administering to the subject one or more steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort, before administering to the subject the rAAV of the method of the present invention, simultaneously with administering to the subject the rAAV, or after administering to the subject the rAAV.

[0115] In related embodiments of the combination therapy 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 is an expected response 2-4 weeks after gene transfer. One possible outcome to such antigen-specific T cell responses is clearance of the transduced cells and loss of transgene expression. To dampen the host's immune response to rAAV-based therapy, prior to treatment, e.g., 24 hours before the treatment procedure, subjects can be started on prophylactic presonisone or equivalent glucocorticoids orally at approximately 1 mg / kg / day, with a maximum dose of 60 mg / day. If necessary, equivalent glucocorticoids can also be administered intravenously at a dose of approximately 1 mg / kg / day. Treatment lasts approximately one month. A tapering protocol of the amount of presonisone or equivalent glucocorticoid can be implemented based on the individual subject's immune response to gene transfer, and can be assessed by ELISpot assay and by 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 method or linear standard qPCR titration method. In one embodiment, the therapeutically effective amount of rAAV is a dose of 5e13vg / kg based on a supercoiled plasmid as the quantification standard, or a dose of 1.85e13vg / kg based on a linearized plasmid as the quantification standard. In another embodiment, the therapeutically effective amount of rAAV is a dose of 2e14vg / kg based on a supercoiled plasmid as the quantification standard, or a dose of 7.41e13vg / kg based on a linearized plasmid as the quantification standard. In another embodiment, a therapeutically effective amount of scAAVrh74.MHCK7.hSGCB 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 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 5e13vg / kg to about 1e14vg / kg, or 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 doses of rAAV vector.

[0119] Administration of an effective dose of the composition may be by routes 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) may be selected and / or adapted by one skilled in the art taking into account the infection and / or disease state to be treated, and the target cells / tissues expressing β-sarcoglycan.

[0120] The present invention provides for local 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 whole 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 transports the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into muscle, 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 may be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual manner 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, WO02 / 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 muscle by 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 pharma- ceutically acceptable carrier for ease of 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 at about 1.0x10 12 vg / ml ~ approx. 5.0×10 14 In another embodiment, the rAAV is at a concentration of about 5.0x10 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 2.0×10 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 1×10 13 vg / ml ~ 2 × 10 14 In another embodiment, the concentration is 2×10 13 vg / ml, 4 × 10 13 vg / ml, or 5 × 10 13vg / 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 (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. In one embodiment, the ionic strength agent comprises MgCl2 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 an adjuvant such as sesame or peanut oil or in aqueous propylene glycol, as well as 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 salts 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, as well as 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, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating agent 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, and the like. 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, together with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by mixing sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other required components from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces powder of active ingredient plus any additional desired components from their 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 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 a suitable medium and screening for cells with the DNA of interest using conventional techniques such as Southern blot and / or PCR, or 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, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth and cardiac muscles, 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 rAAV expressing β-sarcoglycan to a mammalian subject, preferably a human. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAV of the present invention. Transduction can be performed with gene cassettes that contain tissue-specific regulatory elements. For example, one embodiment of the present invention includes, but is not limited to, those derived from the actin and myosin gene families, such as the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], the human skeletal actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], the cardiac actin gene, 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, 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)), steroid-inducible elements, and glucocorticoid response elements (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] Since muscle tissue is not a vital organ and is easily accessible, it is an attractive target for in vivo DNA delivery. The present invention contemplates sustained expression of miRNA from transduced myofibrils.

[0129] By "muscle cell" or "muscle tissue" is meant 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 a described replication-deficient rAAV, resulting in expression of β-sarcoglycan by the recipient cell.

[0131] Accordingly, also described herein is a method of administering an effective dose (or doses administered essentially simultaneously or given 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 herein 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 take precedence.

[0133] The 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 disclosure provides a method of generating rAAV pAAV.MHCK7.hSCGB, comprising introducing an AAV vector plasmid into a host cell. Methods of introducing DNA into a host cell are known in the art, including, but not limited to, transfection, infection, transformation, electroporation, and transduction. In one embodiment, the vector plasmid comprises a nucleotide sequence that is 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. The host cell stably carrying the AAV vector plasmid can be used to generate rAAV. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCB.KAN plasmid. The pAAV.MHCK7.hSGCB.KAN plasmid is shown in FIG. 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 a nucleotide sequence of SEQ ID NO: 1, 3, 5, or 19. The method of generating rAAV, in one embodiment, further comprises transfecting a packaging plasmid and / or a helper virus into the 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. The term as used herein encompasses the progeny of the original cell after expressing 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, such as human primary cells or established cell lines). In one embodiment, the mammalian cells include 293 cells, COS cells, HeLa cells, or KB cells. The AAV vector plasmid can be introduced into the host cell (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 in their genome. Such a stable cell line can be established by incorporating a selection marker into the vector plasmid.

[0137] In one embodiment, the host cell is a packaging cell for producing AAV viral particles.Thus, in another aspect, the present disclosure provides a host cell comprising an AAV vector plasmid comprising a nucleotide sequence 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. EXAMPLES

[0138] Preclinical studies using scAAVrh74.MHCK7.hSGCB are described in WO2017 / 180976, which is incorporated 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 hour dark:light cycle. SGCB - / - Mice were identified by genotyping using PCR. All animals were housed in standard mouse cages and had free access to food and water.

[0140] Beta-sarcoglycan gene construction. Full-length human beta-sarcoglycan cDNA (GenBank accession no. NM_0034994.3) was codon-optimized and cloned into GenScript The cDNA sequences were synthesized by GenScript, Inc., 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 (www.genscript.com).

[0141] For the pAAV.tMCK.hSGCB construct, the cDNA was then cloned into a plasmid containing the AAV2 ITRs, and 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 promoter27, which contains a modification in the enhancer upstream of the promoter region that contains the transcription factor binding sites. The enhancer is composed of two E-boxes (right and left). The tMCK promoter modification contains 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 MHCK7 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. Vectors were purified and encapsidated vg titers were determined as previously described (using a Prism 7500 Taqman detector system, PE Applied Biosystems, Carlsbad, CA, USA). 30 Primers and fluorescent probes 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 injection of the vector into the tail vein of the mouse. Using a 30 gauge ultra-fine insulin syringe, 3 x 10 12 vg scAAVrh.74.MHCK7.hSGCB (2.0 × 10 14 Mice were injected with 100 mg of 10 ...

[0145] Immunofluorescence. Cryostat sections (12 μm) were incubated with monoclonal human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK, Cat. No. NCL-Lb-SARC) at a dilution of 1:50 in blocking buffer (1×TBS, 10% goat serum, 0.1% Tween) for 1 h at room temperature in a humid chamber. Sections were then washed three times with TBS for 20 min each and reblocked for 30 min. AlexaFluor 594-conjugated goat anti-mouse secondary IgG1 antibody (Life Technologies, Grand Island, NY, USA, Cat. No. A21125) was applied at a dilution of 1:250 for 45 min. Sections were washed three times for 20 min with TBS 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 with 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 min at 4 °C. Protein was quantified with a NanoDrop (Thermo Scientific, Waltham, MA, USA). Protein samples (20 μg) were electrophoresed on 3–8% polyacrylamide Tris-acetate gels (NuPage, Invitrogen, Carlsbad, CA, USA) at 150 V for 1 h 5 min and then transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ, USA) for 1 h 15 min at 35 V. Membranes were blocked in 5% nonfat dry milk in TBST for 1 hour and then incubated with rabbit polyclonal human beta-sarcoglycan antibody (Novus Biologicals, Littleton, CO, USA, Catalog No. NBP-1-90300 1:100 or 1:250 dilution) and monoclonal mouse gamma-tubulin antibody (Sigma-Aldrich, St Louis, MO, USA, Catalog No. T6557) at 1:5000 or mouse monoclonal mouse α-actinin antibody (Sigma-Aldrich, St Louis, MO, USA, Catalog No. A7811) at 1:5000 dilution. Rabbit polyclonal mouse cardiac troponin I antibody (Abcam, Cambridge, MA, Catalog No. ab47003) at 1:500 dilution and rabbit monoclonal mouse vinculin antibody (Invitrogen, Frederick, MD, Catalog No. 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 targeted and nontargeted contralateral muscles as previously described. (18, 30) A vector-specific primer-probe set was used to amplify sequences of an intronic region immediately downstream from the tMCK promoter that 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 stained with an anti-rat Ig HRP detection kit (BD The tissue was incubated with rat anti-mouse monoclonal antibodies using Pharmagen, San Jose, CA, USA, Cat: 551013: CD3 (Cat: 555273), CD4 (Cat: 550280), CD8 (Cat: 550281), and Mac-3 for macrophages (Cat: 550292). 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 images were taken for each muscle and each corresponding stain. The number of mononuclear cells was counted and 1 mm 2 The results were expressed as the total number of hits.

[0148] Immunofluorescence: Cryostat sections (12 μm) from tibialis anterior (TA), gastrocnemius (GAS), quadriceps femoris (QUAD), psoas major (PSOAS), psoas (GLUT), triceps brachii (TRI), and diaphragm muscles along with the heart were subjected to immunofluorescence staining for the hSGCB transgene by 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, Cat. No. NCL-Lb-SARC) at a dilution of 1:100. 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 (>50% fascial 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 TA, GAS, QUAD, PSOAS, GLUT, TRI, and diaphragm muscles. In addition, myofiber diameter was measured in GAS, PSOAS, and TRI muscles. Four random 20x 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 - / -(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 to late evening, when mice are most active. All mice were tested in an isolation room, under dim light and by the same caretaker each time. To reduce anxiety and minimize variable behaviors that may affect the mice's normal activity and thus the results of the assay, the tested mice were not housed individually (Voikar et al., Genes Brain Behav. 4:240-52, 2005). Mouse behavior was monitored using the Photobeam Activity System (San Diego Instruments, San Diego, CA). The system monitors the position and movement of mice 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 4 in individual chambers. The testing equipment was cleaned after each use to reduce reactive variable behavior of the mice that could have altered 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 added to represent total ambulation, and light breaks in the Z plane were added to obtain vertical activity within the 1-hour interval.

[0151] Example 2 Construction of scAAVrh.74.MHCK7.hSGCB A transgene cassette was constructed containing the codon-optimized full-length human SCGB cDNA shown in Figure 1. The cassette contains a consensus Kozak sequence (CCACC), an SV40 chimeric intron, a synthetic polyadenylation site, and the muscle-specific MHCK7 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 scAAVrh.74.MHCK7.hSGCB at a dose of 3.0 × 10 12 vg total dose (2.0x10 14 vg / kg) in six SGCBs - / -The transgene expression and efficacy of the vector was evaluated when delivered by tail vein injection in mice and delivered systemically over an extended time point of 24 weeks. Mice were injected at 4-5 weeks of age, and full necropsies on all six mice were performed 24 weeks post-injection. The following muscles were extracted for analysis: TA, gastrocnemius, quadriceps, gluteus, large PSOAS, triceps, diaphragm, and heart. Organs were also harvested for toxicology and biodistribution studies. In summary, hSGCB transgene expression was as 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 of treated mice were again nearly completely transduced. This was accompanied by improved muscle histopathology and improved function.

[0153] β-Sarcoglycan Expression Immunofluorescence (IF) staining for human β-sarcoglycan was used to determine hSGCB transgene expression in six types of skeletal muscles 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 for 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 an average transduction rate for each muscle from each mouse, and these data are presented in Appendix C. The results shown in the panels of Figure 2A below are representative images from treated mice, again demonstrating greater than 98% transduction in all muscles analyzed, including the diaphragm and heart. Finally, Western blots, also shown in FIG. 2B, show expression of hSGCB in TA and TRI muscles that is similar to that achieved following delivery of the initial clinical dose.

[0154] Histopathology of treated muscles As previously discussed, the SGCB - / -Muscles from mice show extensive myopathy, with both skeletal and cardiac muscles including prominent muscle fiber atrophy and hypertrophy with 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. Hematoxylin & Eosin staining in Figure 3 below shows that SGCB mice have a myopathy when compared to normal wild type mice. - / - We show this dystrophic phenotype in mice and amelioration of muscle pathology after treatment. Quantification of histological parameters shows a reduction in central nucleation (CN) in a number of different skeletal muscles as a result of β-sarcoglycan gene transfer. A more detailed analysis of muscle histopathology reveals a normalization of fiber size distribution with an increase in the mean fiber diameter of diseased mice treated with the vector in all three muscles examined (gastrocnemius, psoas and triceps brachii) (Figure 3).

[0155] Functional assessment 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 mouse diaphragm and heart. 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 diseased 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 in the delivery of our clinical dose.- / - Provided overall functional benefits to mice, ultimately resulting in the development of SGCB - / - Laser monitoring of open field cage activity was performed on all groups of mice to determine if it improved the phenotype of the mice. The graphs in Figure 5 below show a significant 58.6% decrease in overall ambulation in the x and y planes along with a 48.9% decrease in hindlimb vertical rearing in the KO mice compared to WT. Mice treated with high dose scAAVrh.74.MHCK7.hSGCB were overall more active compared to the KO by qualitative observation, as shown by quantification of activity in MCHK7 treated mice (n=6 per group) with a 36.2% increase in overall ambulation and a 39.0% increase in hindlimb vertical rearing (Figure 5).

[0157] A higher dose of 3.0 × 10 12 Total dose of vg (2.0 × 10 14 Intravenous injection of scAAVrh.74.MHCK7.hSGCB at 100 mg / kg resulted in nearly complete transduction and restoration of hSGCB expression (>98%) in limb skeletal muscle, diaphragm, and critical myocardium (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 a β-sarcoglycan-induced increase in muscle mass was achieved in all muscles throughout the body using 100 mg / kg of β-sarcoglycan (vg / kg) offers great promise for modifying 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 to the observed increase in specific force output at the diaphragm and increased open field cage activity (Figures 4, 5).

[0158] Example 4 Toxicology and vector biodistribution The objective of this study was to evaluate the efficacy and safety of SGCB in males and females 24 weeks after delivery of the test article, scAAVrh.74.MHCK7.hSGCB. - / -The objective of the 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 total dose of 3.0 × 10 vg (2.0 × 10 14 To evaluate the safety of our vector, we administered 100 mg / kg of 100 mg ... - / - Hematoxylin & 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 hepatic 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 for 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 scAAVrh.74.MHCK7.hSGCB at 100 vg / kg, various skeletal muscles, including the diaphragm, along with the heart and five other organs, were injected with the vector in SGCB mice. - / - H&E sections of each tissue taken from mice 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 of the nonmuscle tissues evaluated histologically showed no treatment-related pathology, with only the livers of mice no. 789 and 790 showing small focal liver lesions.

[0163] To further evaluate clinical liver function, we assessed the levels of liver enzymes in the serum of these mice. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed to determine whether they were elevated compared to normal levels in two untreated BL6 WT mice and two untreated SGCB mice, along with six scAAVrh.74.MHCK7.hSGCB-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 are not elevated in ALT and AST, exhibiting average ALT and AST levels within the normal range of 89.5 U / L and 330.75 U / L, respectively.

[0164] Taken together, these data indicate that the test material was well tolerated by the study subjects. Furthermore, two age-matched untreated SGCB - / -Compared with reference specimens from mice, independent histopathology review demonstrated that administration of scAAVrh.74.MHCK7.hSGCB reduced the risk of developing treatment-associated 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 extent 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 of (n=2) 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. - / - A positive signal was determined for tissue samples collected from the animals. A positive signal was any of 100 or more 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 of the high dose (3.0 × 10 12 The vector genome copies detected in each tissue sample from mice (no. 785, no. 786, no. 789, no. 790) injected with scAAVrh.74.MHCK7.hSGCB at a total dose of 1.0 × 10 vg was compared with the clinical dose (1.0 × 10 12 vg copy numbers from the same tissue samples in mice (#712, #713) treated with 1000 mg / kg bw (total vg 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 copy numbers of the vector genome were similar in each tissue when compared to the control (vg / kg) cohort. These data indicate that the test samples were efficiently delivered to all examined tissues of the vector-treated mice. [Table 3]

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

[0169] As the qPCR results above show, intravenous delivery of high-dose scAAVrh.74.MHCK7.hSGCB results in variable levels of vector transcript distribution in most tissues, with the highest levels occurring in muscle. Therefore, the goal of this part of the study was to determine protein expression of the human β-sarcoglycan transgene in these tissues and 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 various amounts in all skeletal muscle and heart samples and was detected in the liver of mice #785 and #787. (Table 4, Figure 6.) To further investigate the expression in the liver, Western blotting of 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 shown in Figure 6 indicate that β-sarcoglycan protein expression is seen in four of the six livers of vector-administered mice (#785, #786, #787, and #788). Table 5 below lists the detailed β-sarcoglycan protein expression results from all six mice (#785, #796, #787, #788, #789, and #790) for which a complete biodistribution Western was performed. [Table 4]

[0171] Table 4 shows the 2.0×10 14 FIG. 1 provides β-sarcoglycan protein expression in individual tissues from six SGCB− / − mice treated systemically with vg / 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 deficiency in LGMD2E patients, systemic delivery could provide significant clinical benefit to these patients. 12 vg total dose (2.0 × 10 14 SGCB receiving intravenous tail vein injection of scAAVrh.74.MHCK7.hSGCB at 100 mg / 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-administered mice (no. 785, no. 786, no. 787, and no. 788) showed expression of the β-sarcoglycan transgenic protein previously shown with systemic dosing (Salva et al., Mol Ther, 2007.15(2):p.320-9). Two of the six treated animals (no. 789 and no. 790) were reported to have minimal to mild focal liver lesions, while 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 no. 789 and no. 790, liver enzyme levels alanine aminotransferase and aspartate aminotransferase were measured in the serum of all six treated mice. The results of this experiment, shown in Table 4, show 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 showed lower vg copy numbers (Table 3) in addition to the absence of expression of the β-sarcoglycan transgenic protein. Taken together, 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 1.0×10 vg / kg, sustained β-sarcoglycan expression in the liver was observed, whereas at a lower dose (5.0×10 13 No 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. The 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 spinal muscular atrophy using AAV (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 once via systemic infusion via a peripheral vein. The vector was delivered in approximately 10 ml / kg lactate Ringer as needed, infused over approximately 1-2 hours. Patients had glucocorticosteroid adrenal suppression that was minimal at best after 30 days, but as a precaution, the maximum dose for each enrollee 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 greater than 40% normal 100-meter walk test scores. Each subject received 5 × 10 13 Subjects received a dose of 100 mg / kg of prednisone per day. 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 prednisone one day prior to gene transfer, and doses were recorded for 30 days.

[0176] If biopsies are read and 50% or more of muscle fibers express SGCB in the TA and biceps 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 be treated with 2×10 14 vg / kg. Three of the patients in Cohort 2 will receive placebo lactate Ringer. These placebo subjects will be treated approximately one year later with the same dose as the subjects treated in their cohort.

[0177] Baseline measurements before injection (from day -60 to day -2) After obtaining informed consent and completing the enrollment procedures, a baseline patient history was collected, including a record of all medications and supplements the patient was taking. Baseline functional tests to establish a stable baseline were compared to functional test results collected in a previous natural growth history study for consistency of baseline tests. At the screening visit, the 100m time 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, the subject may continue to participate in the LGMD natural growth history study. The following assessments are performed to confirm the subject's eligibility for this study. Baseline tests that must be completed prior to treatment administration include: Baseline From day -60 to day -2 before gene transfer Informed consent Medical History Physical exam / vitals EKG Cardiac MRI (performed without anesthesia, but if the procedure is poorly tolerated and considering the importance of cardiac evaluation in this disease, we will discuss the option of performing anesthesia using a protocol acceptable to the Nationwide Children's protocol) 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 Serum total 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 may 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 (handheld dynamometer measurements) PROMIS Survey Set up your device for activity monitoring Pulmonary function tests (PFTs) including spirometry -Timed Functional Tests [100m timed test, climbing 4 steps, timed up and go] ·Workspace volume North Star Assessment for Limb-Girdle Muscular Dystrophy (NSAD) Baseline muscle biopsy of upper and lower extremity muscles may be performed using ultrasound guided. Selection will be dependent on clinical findings to target appropriate muscles for analysis with least risk to patient. Placebo delayed subjects will not receive a second baseline muscle biopsy. ·Chest X-ray Day -1 Physical examination and vital signs Start prednisone or a similar glucocorticoid Photos of possible injection sites ·Safety Laboratory: Complete blood count (CBC) with differential and including platelets Serum total 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 may 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 The expected antigen-specific T cell response to the AAV vector was expected between 2 and 4 weeks after gene transfer. One possible outcome for such an antigen-specific T cell response was clearance of the transduced cells and loss of transgene expression. To dampen the host's immune response to the AAV-based therapy, 24 hours before the procedure, subjects could be started on prophylactic presonisone or equivalent glucocorticoids orally at approximately 1 mg / kg / day, with a maximum dose of 60 mg / day. If necessary, equivalent glucocorticoids could also be administered intravenously at a dose of approximately 1 mg / kg / day. Treatment lasted approximately 1 month. A tapering protocol of the amount of presonisone or equivalent glucocorticoid was implemented based on the individual subject's immune response to gene transfer, assessed by ELISpot assay and by liver function monitoring with GGT.

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

[0180] Syringes containing the vectors 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 the Lung PICU the night before gene transfer and examined by either the PI or Co-I (day -1). Subjects were kept NPO after midnight the night before the gene transfer procedure. The procedure was performed in the patient room and under sterile conditions.

[0182] An intravenous catheter with a heparin lock was placed in a peripheral vein for vector delivery. A second intravenous catheter was placed for use if complications occurred at the first site. Photographs of these locations were taken on the day of gene transfer. Vectors were delivered intravenously while patients were awake. Patients received conscious sedation as per protocol if deemed necessary by the investigator. 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 infusion.

[0183] Post-transfer monitoring Patient vital signs were monitored every 15 minutes for 4 hours and hourly for the remaining 24 hours after infusion. Safety labs and urinalysis were checked the day after the procedure. Concomitant medications and all adverse / serious adverse events were also monitored and documented after injection. Subjects were discharged 1 day after gene transfer (if no side effects of safety concern were observed). Subjects returned for follow-up visits at days 7, 14, 30, 60, 90, 180 and 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 Serum total 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 may 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 Immunology testing Physical therapy assessments beginning on 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 testing Photos 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) - MRI of cardiac and skeletal muscles (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 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 evaluated 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 according to FDA guidelines.

[0185] CBER will be notified if there is any indication that the follow-up period should 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 are instructed to notify the investigator of any changes in 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 3 years after administration, incorporating the active phase of the protocol. If any newly identified risk is related to our product or if the subject suffers from any adverse events during this period, long-term follow-up will be initiated according to FDA guidelines.

[0187] Primary Outcome of Clinical Trial 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, improvement in 100-meter time of 10% or more compared to baseline Reduction of CK following 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 [Climb 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 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, with one subject showing elevated liver enzymes and bilirubin after cessation of steroids. This event resolved with an increase in steroids. Two of the subjects had elevated liver enzymes that resolved with an increase in 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 dosage escalation was performed. If SGCB expression was <50% in all treated subjects, the dose was increased to 2x10 for cohort 2 and placebo subjects. 14 vg / kg. If possible, biopsies 2 years after treatment will be 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. Quantification of expression was performed using direct immunofluorescence and Western blot testing of muscle biopsies. Bioquant® automated software will be used to quantify the number of muscle fibers expressing SGCB. Baseline patient demographics are shown in Table 5. [Table 5]

[0190] FIG. 7 provides representative images showing robust SGCB expression in muscle 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 across the cohort was 47%, and the mean percentage of SGCB-positive fibers was 51%. FIG. 8 provides Western blots showing detection of β-sarcoglycan expression in three subjects 90 days after vector administration. The Western blot data show 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 is 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 any 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 examined. 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 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. 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 the sarcoglycan complex to the membrane. Treatment with scAAVrh74.MHCK7.hSGCB restored sarcoglycan complexes to the membrane (FIG. 10). FIG. 10 shows restoration of β-sarcoglycan, α-sarcoglycan expression at the membrane of the patient under study, as well as colocalization of β-sarcoglycan and α-sarcoglycan. Colocalization of β-sarcoglycan and α-sarcoglycan indicates that scAAVrh74.MHCK7.hSGCB restored sarcoglycan complexes.

[0195] Example 7 LGMD2E patients treated with β-sarcoglycan gene transfer showed improvement in the 100-meter test 3 months after treatment. Treatment with scAAVrh74.MHCK7.hSGCB resulted in patients with clear improvements in the 100 meter timed test just three months after gene transfer (Figure 11). Timed walking tests, such as the 100 meter timed test, are used to measure function in subjects with muscular dystrophy. The tests in this study measured the baseline performance of the patients compared to their performance after treatment. Figure 11 shows the average percent change from baseline for three subjects over the first three months after gene transfer. The data show that there was an average increase of more than 15% above baseline after three months, indicating an 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 the patients 9 months after systemic administration of scAAVrh.74.MHCK7.hSGCB. Three patients participated in functional testing. For example, in the 100m time test, at baseline (before administration), one patient had limited hip extension and flexion when running 100m. However, 9 months after administration, the same patient showed improved hip extension and flexion when running, and a faster speed. In addition, in the trunk control test, another patient showed improvement in the time to get up test 9 months after administration. At baseline or before administration, this subject had poor trunk control, which also improved 9 months after administration. Also, in the sitting test, the patient was asked to stand up from a sitting position. For example, the remaining patient had a reduced time to get up 9 months after administration compared to before administration. These data are summarized in Table 10. [Table 10]

[0197] An age-matched natural growth course study compared the change from baseline in NSAD for untreated subjects (denoted natural growth course subjects, see Table 11) and subjects administered scAAVrh74.MHCK7.hSGCB, herein denoted the "North Star Assessment of Limb-Girdle Muscular Dystrophy." As shown in Figures 12A-C, natural growth course subjects showed a stable reduction in change in NSAD over 200 days, whereas treated subjects showed a stable improvement in change in NSAD over 270 days (Figures 12A and 12C), whereas treated subjects showed a stable improvement in change in NSAD 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 a temperature below −60° C. The frozen drug product must be thawed prior to clinical administration.

[0200] scAAVrh74.MHCK7.hSGCB is stored at temperatures below -60°C, at which temperature the material is stable under long-term storage conditions. scAAVrh74.MHCK7.hSGCB vials are thawed at room temperature (20°C to 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 titers for scAAVrh74.MHCK7.hSGCB lots 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 scAAVrh74.MHCK7.hSGCB-mediated gene replacement 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 treated systemically with scAAVrh74.MHCK7.hSGCB. High levels of vector genome copy number were detected by PCR in all transduced muscles >24 months after treatment. Furthermore, immunofluorescence staining of treated muscles showed no reduction in protein expression levels (>95%) in all muscles compared to previous time points, and hSGCB protein remained correctly localized in the spine.

[0204] Second, mouse models of LGMD2E (β-sarcoglycan) will be treated at older ages (e.g., 12 months) with systemic delivery of the scAAVrh74.MHCK7.hSGCB vector. At an endpoint 6 months after treatment, muscles from these mice will be evaluated for protein expression, histological rescue, and functional improvement. Gene expression in muscle will be observed throughout the lower limb, upper limb, and proximal trunk muscles, notably including the diaphragm and heart. Furthermore, levels of fibrosis will be compared to untreated controls. Additionally, functional studies will include evaluation of force output of 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, and therefore 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 mdx mice.J Appl Physiol 2011;110:1656-1663. 33 Wein N,Vulin A,Falzarano MS,Szigyarto CA,Maiti B,Findlay A et al.Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.Nat Med 2014;20:992-1000.

Claims

1. A polynucleotide comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO:

24.

2. The polynucleotide of claim 1 comprising the nucleotide sequence of SEQ ID NO:

24.

3. A plasmid comprising the polynucleotide of claim 1 or 2.

4. A method for producing a recombinant AAV, comprising transfecting a cell with a polynucleotide according to any one of claims 1 to 3.

5. The method of claim 4, further comprising transfecting the cells with a packaging plasmid and / or a helper virus.

6. The method of claim 4 or 5, wherein the cell contains a stably integrated AAV cap gene.

7. The method of any one of claims 4 to 6, wherein the cell contains a stably integrated AAV rep gene.

8. 8. The method of any of claims 4-7, wherein the rAAV comprises one or more of the capsid proteins of 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, or mutants thereof.

9. An isolated cell comprising the polynucleotide of claim 1 or 2.

10. An isolated cell comprising the plasmid of claim 3.

11. 11. The isolated cell of claim 9 or 10, wherein the cell is an insect cell, a silkworm cell, a mosquito cell, a bacterial cell or a mammalian cell.

12. 11. The isolated cell of claim 9 or 10, wherein the cell is an S2 cell, a Kc cell, a Bme21 cell, a C6 / 36 cell, a 293 cell, a COS cell, a HeLa cell or a KB cell.

13. An rAAV comprising a nucleotide sequence identical to the sequence of SEQ ID NO:

19.

14. A polynucleotide comprising a nucleotide sequence identical to the sequence of SEQ ID NO:

19.

15. An rAAV comprising the polynucleotide described in claim 14.

16. The rAAV of claim 15, wherein the rAAV is of an rAAV serotype selected from 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, AAV rh.74, or a mutant of each of these.

17. The rAAV of claim 16, wherein the rAAV is of serotype AAV rh.74 or a mutant thereof.

18. A composition comprising an rAAV described in any one of claims 15 to 17 and a pharma- ceutically acceptable carrier.

19. The composition of claim 18, further comprising a buffer, an ionic strength agent, and a surfactant.

20. The composition of claim 18 or 19, wherein the rAAV is at a concentration of about 1.0×10 12 vg / ml to about 5.0×10 14 vg / ml, or about 5.0×10 12 vg / ml to about 1.0×10 14 vg / ml.

21. The composition of claim 19 or 20, wherein the buffer comprises one or more of Tris, Tricine, Bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS.

22. The composition of claim 19 or 20, wherein the buffer comprises Tris at a concentration of about 5 mM to about 40 mM, pH 8.

0.

23. The composition of claim 19 or 20, wherein the buffer comprises Tris at about 20 mM and pH 8.

0.

24. The composition of any one of claims 19 to 23, 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.

25. The composition of any one of claims 19 to 23, wherein the ionic strength agent comprises MgCl 2 at a concentration of about 0.2 mM to about 4 mM.

26. The composition of any one of claims 19 to 23, wherein the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM.

27. The composition of any one of claims 19 to 23, wherein the ionic strength agent comprises MgCl 2 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.

28. The composition of any one of claims 19 to 23, wherein the ionic strength agent comprises MgCl 2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM.

29. The composition of any one of claims 19 to 28, wherein the surfactant comprises one or more of a sulfonate, sulfate, phosphonate, phosphate, poloxamer, and cationic surfactant.

30. The composition described in claim 29, wherein the poloxamer includes one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407.

31. The composition of any one of claims 19 to 29, wherein the surfactant comprises the poloxamer at a concentration of about 0.00001% to about 1%.

32. The composition of any one of claims 19 to 29, wherein the surfactant comprises poloxamer 188 at a concentration of about 0.001%.

33. The composition of claim 18, further comprising Tris (pH 8.0), magnesium chloride, sodium chloride, and poloxamer 188.

34. An isolated cell comprising the polynucleotide described in claim 14.

35. The isolated cell of claim 34, wherein the cell is an insect cell, a silkworm cell, a mosquito cell, a bacterial cell or a mammalian cell.

36. The isolated cell of claim 34, wherein the cell is an S2 cell, a Kc cell, a Bme21 cell, a C6 / 36 cell, a 293 cell, a COS cell, a HeLa cell or a KB cell.

Images