Systemic delivery of adeno-associated virus vectors expressing G sarcoglycans and treatment of muscular dystrophy

Abstract

Described herein are methods for treating muscular dystrophy comprising administering a recombinant AAV (rAAV) scAAVrh74.MHCK7.hSGCG vector, methods for expressing the gamma-sarcoglycan gene in a patient, pharmaceutical compositions comprising rAAV, and methods for making rAAV.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 075,697, filed on 8 September 2020, 35 U.S.C. § 119(e), the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes sequence listings submitted electronically in ASCII format, which are incorporated herein by reference in their entirety. The ASCII copy, created on 24 August 2021, is named 8186WO00_Sequence_Listing_ST25.txt and is 23 kilobytes in size.

[0003] Therapeutic vectors, such as AAV vectors expressing γ-sarcoglycans, and methods for reducing and preventing fibrosis in subjects suffering from muscular dystrophy using these vectors are described herein. [Background technology]

[0004] Limb-girdle muscular dystrophy (LGMD) type 2C (LGMD2C) is an autosomal recessive disorder resulting from mutations in the gene encoding gamma-sarcoglycan (SGCG), which causes loss of a functional protein. It manifests as a progressive muscular dystrophy that begins in the girdle muscles and subsequently expands to the muscles of the lower and upper limbs, and can also affect the diaphragm (DIA) and heart (HRT), potentially leading to respiratory and heart failure in certain patients. There are no approved disease-modifying therapies for LGMD2C. Therefore, there is a need for effective therapies for patients with LGMD2C. [Overview of the project]

[0005] Methods for delivering gene therapy vectors, such as AAV expressing the γ-sarcoglycan gene and γ-sarcoglycan, to muscles to reduce and / or prevent fibrosis; and / or increase muscle strength; and / or treat mammalian subjects suffering from muscular dystrophy are described herein.

[0006] In one embodiment, a method for treating muscular dystrophy in a subject requiring the use of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG is described herein, comprising the step of administering the rAAV to the subject requiring the use of the rAAV, which is administered via a systemic route, based on a supercoiled plasmid as a quantitative standard, for approximately 2 × 10⁶ 12 vg / kg ~ approx. 5.0×10 14 Based on a linear plasmid as a dose or quantitative standard of vg / kg, 3.0 × 10 12 vg / kg ~ approx. 1.0×10 14 Administered at a dose of vg / kg; serum creatine kinase (CK) levels in subjects decreased after rAAV administration compared to serum CK levels before rAAV administration.

[0007] In another embodiment, a method is provided for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, wherein the level of gamma-sarcoglycan gene expression in the cells of the subject is increased after administration of rAAV compared to the level of gamma-sarcoglycan gene expression before administration of rAAV; the number of gamma-sarcoglycan-positive fibers in the muscle tissue of the subject is increased after administration of rAAV compared to the number of gamma-sarcoglycan-positive fibers before administration of rAAV; or motor function is improved in the subject compared to the motor function of the subject before administration of rAAV, and motor function is determined by a 100-meter walk time test and / or NSAD. In the embodiment, NSAD is increased in the subject compared to the NSAD of the subject before administration of rAAV.

[0008] In another aspect, the disclosure uses a linearized plasmid as a quantitative standard, yielding approximately 4.63 × 10⁻⁶ 12 vg / kg, approximately 1.85×10 13 vg / kg or 7.41 × 10 13 The present disclosure provides a method for treating limb-girdle muscular dystrophy in a subject of need, comprising administering rAAV intravenously at a volume of vg / kg to the subject, wherein rAAV comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10. In one embodiment, rAAV comprises the nucleotide sequence of SEQ ID NO: 10. In another embodiment, the disclosure describes a method for expressing a gamma-sarcoglycan gene in cells of a subject, comprising administering the subject a scAAVrh74.MHCK7.hSGCG construct comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 7 or 10. In one embodiment, the disclosure provides a method for increasing gamma-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, comprising administering the subject a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 7.

[0009] In one embodiment, a method for increasing the expression of alpha-sarcoglycans and / or beta-sarcoglycans in a subject requiring such effect is described herein, comprising administering to the subject an rAAV containing a scAAVrh74.MHCK7.hSGCG construct having a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 10. In another embodiment, a method for increasing the localization of alpha-sarcoglycans and / or beta-sarcoglycans to the cell membrane in a subject requiring such effect is provided herein, comprising administering to the subject a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 10. In yet another embodiment, a method for increasing sarcoglycan expression in muscle tissue or improving muscle function in a subject is provided, comprising administering to the subject an rAAV containing a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 7 or SEQ ID NO: 10. In another aspect, the disclosure provides a method for increasing sarcoglycan expression in muscle tissue of a subject, comprising administering the subject a construct comprising a nucleotide sequence encoding a first sarcoglycan and detecting an increase in the expression of at least a second sarcoglycan in the cell membrane of cells expressing the first sarcoglycan.

[0010] In another embodiment, a composition comprising an rAAV scAAVrh74.MHCK7.hSGCG vector, a buffer, an ionic strengthener, and a surfactant is described. In another embodiment, a pharmaceutical composition comprising recombinant AAV(rAAV)scAAVrh74.MHCK7.hSGCG is described herein, wherein scAAVrh74.MHCK7.hSGCG comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 7 or SEQ ID NO: 10. In another embodiment, scAAVrh74.MHCK7.hSGCG comprises a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 10. In another embodiment, scAAVrh74.MHCK7.hSGCG comprises the nucleotide sequence of SEQ ID NO: 10.

[0011] In another embodiment, a method is provided for generating recombinant AAV scAAVrh74.MHCK7.hSGCG, comprising introducing a plasmid into cells, wherein the plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8. In particular, the plasmid contains the nucleotide sequence of SEQ ID NO: 8. In another embodiment, the plasmid contains the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 10.

[0012] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding γ-sarcoglycan are described herein. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence that is identical to the nucleotide sequence described in SEQ ID NO: 1, and encodes a protein that retains γ-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan comprises the nucleotide sequence described in SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan consists of the nucleotide sequence described in SEQ ID NO: 1.

[0013] In another embodiment, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding a γ-sarcoglycan that is sequence-identical to the amino acid sequence of SEQ ID NO: 9 by 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%, and the protein retains γ-sarcoglycan activity.

[0014] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding a functional γ-sarcoglycan are described herein. In one embodiment, the polynucleotide sequence comprises a nucleotide sequence that, under stringent conditions, hybridizes to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, or its complementary strand.

[0015] The term "stringent" refers to conditions that are generally understood as stringent in the art. 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 0.015 M sodium chloride, 0.0015 M sodium citrate (65°C–68°C) or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide (42°C). See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (higher temperature, lower ionic strength, higher formamide, or other denaturing agents, etc.) may be used, but the rate of hybridization will be affected. In the case of hybridization of deoxyoligonucleotides, additional typical 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).

[0016] When ranges for physical properties such as molecular weight, concentration, or dosage are used herein, it is intended that these ranges include all combinations and subcombinations of those ranges and particular embodiments. The term “approximately” when referring to a number or numerical range means that the number or numerical range mentioned is an approximation within the variability of the experiment (or within the statistical error of the experiment), and therefore the number or numerical range may vary, for example, from 1% to 15% of the stated number or numerical range.

[0017] Other agents may be included in the hybridization and washing buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4(SDS), Ficol, Denhart's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, but other suitable agents may also be used. The concentrations and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically 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 Hybridization: A Practical Approach, Ch.4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to modulate these variables and allow DNAs of different sequence relevances to form hybrids.

[0018] In another embodiment, the recombinant AAV vectors described herein may be operably linked to muscle-specific regulatory elements. For example, muscle-specific regulatory elements include human skeletal actin gene elements, cardiac actin gene elements, muscle cell-specific enhancer-binding factors (MEFs), muscle creatine kinase (MCK), tMCK (tectonic MCK), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK), C5-12 (a synthetic promoter), mouse creatine kinase enhancer elements, skeletal fast-twitch muscle troponin C gene elements, slow-twitch muscle cardiac troponin c gene elements, slow-twitch muscle troponin I gene elements, hypoxia-inducible nuclear factor, steroid-inducible elements, or glucocorticoid-responsive elements (GREs).

[0019] In some embodiments, rAAV pAAV.MHCK7.hSGCG 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% of the nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about The nucleotide sequences include nucleotide sequences encoding polypeptides that are 99% identical, or that are 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 a nucleotide sequence that is 99% identical to SEQ ID NO: 9.

[0020] In one embodiment, the polynucleotide sequence encodes a protein that retains sarcoglycan activity, including beta and / or alpha-sarcoglycan activity. In another embodiment, the polynucleotide sequence encodes a protein that retains gamma-sarcoglycan activity.

[0021] In some embodiments, the muscle-specific promoter is tMHCK7 (SEQ ID NO: 2). A typical rAAV described herein is pAAV.tMCK.hSGCG, which contains the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 10.

[0022] AAV may be any serotype, e.g., 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 AAVrh.74. The production of pseudotype rAAV is disclosed, for example, in International Publication No. 01 / 83692. Other types of rAAV variants with capsid mutations, e.g., rAAV, should also be considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0023] Compositions comprising any of the rAAV vectors described herein are also intended.

[0024] In some embodiments, the Disclosure relates to a nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10 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 described in SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10. The present disclosure provides a composition or pharmaceutical composition comprising the scAAVrh74.MHCK7.hSGCG rAAV vector containing a nucleotide sequence encoding a polypeptide that is at least 65%, at least 70%, at least 75%, at least 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 9.

[0025] A method for treating muscular dystrophy in a subject in need thereof is provided, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, wherein the rAAV uses a systemic administration route and is based on a linearized plasmid as a quantitative standard, and is administered at a dose of 2×10 12 vg / kg to about 5.0×10 14 vg / kg.

[0026] A composition for treating muscular dystrophy is also provided, the composition comprising recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGC at a dose of about 2.0×10 12 vg / kg to about 5.0×10 14 vg / kg, based on a linearized plasmid as a quantitative standard, and the composition is formulated for systemic administration.

[0027] Furthermore, the use of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGC for the manufacture of a medicament for treating muscular dystrophy is provided, the medicament comprising scAAVrh74.MHCK7.hSGC at a dose of about 1.0×10 12 vg / kg to about 5.0×10 14 vg / kg, based on a linearized plasmid as a quantitative standard, and the medicament is formulated for systemic administration.

[0028] In any of the methods, compositions and uses provided, the level of gamma-sarcoglycan gene expression in the cells of the subject is increased after administration of the rAAV compared to the level of gamma-sarcoglycan gene expression before administration of the rAAV; the serum creatine kinase (CK) level in the subject is decreased after administration of the rAAV compared to the serum CK level before administration of the rAAV; and / or the number of gamma-sarcoglycan positive fibers in the muscle tissue of the subject is increased after administration of the rAAV compared to the number of gamma-sarcoglycan positive fibers before administration of the rAAV.

[0029] In another embodiment, in any of the provided methods, compositions, and uses, motor function is improved in the subject compared to the subject's motor function before administration of rAAV, and motor function is determined by a 100-meter walking time test. For example, 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 embodiments, motor function improves by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50%.

[0030] For example, in any of the methods, compositions, and uses provided, the systemic route of administration is an intravenous route. For example, rAAV is administered using an intravenous route, and the dose of rAAV administered is approximately 4.63 × 10⁶ based on a linearized plasmid as a quantitative standard. 12 vg / kg, approximately 1.85×10 13 vg / kg or 7.41 × 10 13 It is vg / kg.

[0031] In some embodiments, the dose of rAAV administered via an intravenous route is approximately 2.0 × 10⁶ based on a linearized plasmid as a quantitative standard. 13 vg / kg ~ approx. 5×10 14 That is the case.

[0032] Furthermore, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 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 It is vg. Furthermore, in any of the methods, compositions, or uses, the dose of rAAV is administered at a concentration of approximately 10 mL / kg. In any of the methods, compositions, or uses provided, the muscular dystrophy is limb-girdle muscular dystrophy.

[0033] Furthermore, a method is provided for treating muscular dystrophy in subjects requiring it, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, wherein rAAV is administered via a systemic route and based on a linearized plasmid as a quantitative standard, resulting in approximately 2.0 × 10⁶ units. 12 vg / kg ~ approx. 5.0×10 14 The method involves administering a dose of vg / kg; increasing the level of gamma-sarcoglycan gene expression in target cells after rAAV administration compared to the level of gamma-sarcoglycan gene expression before rAAV administration; decreasing serum CK levels in the target after rAAV administration compared to the serum CK levels before rAAV administration; or increasing the number of gamma-sarcoglycan-positive fibers in the target muscle tissue after rAAV administration compared to the number of gamma-sarcoglycan-positive fibers before rAAV administration. For example, in any of the methods provided, the systemic route of administration is intravenous, and the dose of rAAV administered is approximately 4.63 × 10⁻¹⁵ based on a linearized plasmid as a quantitative standard. 12 The values ​​are vg / kgvg / kg. In another embodiment, the dose of rAAV administered is approximately 1.85 × 10⁻¹⁶ based on a linearized plasmid as a quantitative standard. 13 The value is vg / kg. In another embodiment, the dose of rAAV administered is approximately 7.41 × 10⁶ based on a linearized plasmid as a quantitative standard. 13 The dosage is vg / kg. Furthermore, the total dose of rAAV administered is approximately 1.5 × 10⁻⁶. 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 It is vg. Furthermore, in either of the methods, the dose of rAAV is administered at a concentration of approximately 10 mL / kg. In either of the methods provided, the muscular dystrophy is limb-girdle muscular dystrophy.

[0034] In some embodiments, the disclosure includes a method for treating muscular dystrophy in a subject requiring the administration of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, wherein motor function is demonstrably improved in the subject compared to the subject's motor function before administration of rAAV, and motor function is determined by a 100m walk time test and / or NSD. In some embodiments, 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 embodiments, 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%.

[0035] A method is provided for increasing the levels of alpha-sarcoglycans and / or beta-sarcoglycans in a subject requiring such increase, comprising administering the subject a scAAVrh74.MHCK7.hSGCG construct containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10. Furthermore, a composition for increasing the levels of alpha-sarcoglycans and / or beta-sarcoglycans in a subject requiring such increase is provided, the composition comprising the scAAVrh74.MHCK7.hSGCG construct containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10. The use of the scAAVrh74.MHCK7.hSGCG construct containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10 for the manufacture of a pharmacopoeia for increasing the levels of alpha-sarcoglycans and / or beta-sarcoglycans in a subject requiring such increase is also provided. In some embodiments, the alpha-sarcoglycans and / or beta-sarcoglycans are localized to the membrane of cells expressing gamma-sarcoglycans encoded by scAAVrh74.MHCK7.hSGCG. In some embodiments, beta-sarcoglycans are localized to the membranes of cells expressing gamma-sarcoglycans encoded by scAAVrh74.MHCK7.hSGCG.

[0036] In some embodiments, the scAAVrh74.MHCK7.hSGCG construct includes an intron sequence. In one embodiment, the intron sequence includes the nucleotide sequence of SEQ ID NO: 6. In another embodiment, the scAAVrh74.MHCK7.hSGCG construct includes a poly(A) sequence. In one embodiment, the poly(A) sequence includes the nucleotide sequence of SEQ ID NO: 5. In another embodiment, the scAAVrh74.MHCK7.hSGCG construct includes a 5' inversion sequence (ITR). In one embodiment, the 5'ITR sequence includes the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the scAAVrh74.MHCK7.hSGCG construct includes a 3' inversion sequence (ITR). In one embodiment, the 3'ITR sequence includes the nucleotide sequence of SEQ ID NO: 4.

[0037] A method is also provided for increasing sarcoglycan expression in muscle tissue of a subject, comprising administering the subject a construct containing a nucleotide sequence encoding a first sarcoglycan, and detecting an increase in the expression of at least a second sarcoglycan in the cell membrane of cells expressing the first sarcoglycan. In some embodiments, the first sarcoglycan is γ-sarcoglycan (SGCG), and the second sarcoglycan is α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD).

[0038] In any of the methods, uses, and compositions provided for treating muscular dystrophy, subjects are aged 4–15 years, have confirmed gamma-sarcoglycan (SGCG) mutations in both alleles, are negative for AAVrh74 antibody, and / or have a >40% or normal 100-meter walk test. In any of the methods, uses, and compositions provided for treating muscular dystrophy, subjects are pediatric subjects. In some embodiments, subjects are pediatric subjects, such as subjects in the age range of 1–10 years. In some embodiments, subjects are aged 4–15 years. In one embodiment, subjects are adolescent subjects, such as subjects in the age range of 10–19 years. Furthermore, in one embodiment, subjects are young adult subjects, such as subjects in the late teens or early twenties, for example, subjects may be in the age range of 15–29 years. In some embodiments, subjects are middle-aged adults or elderly subjects, for example, middle-aged adults may be in the age range of 25–55 years, and elderly subjects may be in the age range of over 50 years.

[0039] In some embodiments, rAAV is administered by injection, infusion, or implantation. For example, rAAV is administered by infusion over a period of approximately 1-2 hours. Furthermore, rAAV is administered via an intravenous route through peripheral limb veins.

[0040] A method for treating muscular dystrophy in a subject requiring the administration of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, wherein rAAV is administered via a systemic route and based on a linearized plasmid as a quantitative standard, at a dose of approximately 1.0 × 10⁶. 12 vg / kg ~ approx. 5.0×10 14 Administered at a dose of vg / kg, rAAV contains the human γ-sarcoglycan nucleotide sequence of SEQ ID NO: 1. Furthermore, rAAV contains the MHCK7 promoter sequence of SEQ ID NO: 2. In some embodiments, rAAV is of serotype AAVrh.74. Furthermore, rAAV contains the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0041] In a typical embodiment, a method for treating muscular dystrophy in a subject requiring it includes the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG, where rAAV is administered via a systemic route and based on a linearized plasmid as a quantitative standard, at a dose of approximately 1.0 × 10⁶. 12 vg / kg ~ approx. 5.0×10 14 The drug was administered at a dose of vg / kg, and the subjects suffered from limb-girdle muscular dystrophy. Based on supercoiled plasmids as a quantitative standard, rAAV was approximately 1.25 × 10⁶. 13 vg / kg, approximately 5.0×10 13 vg / kg or approximately 2.0 × 10 14 Based on vg / kg, or a linearized plasmid as a quantitative standard, approximately 1.85 × 10⁶ 13 vg / kg, 7.41 × 10 13 vg / kg or approximately 4.63 × 10 12 The dose is administered by intravenous infusion over approximately 1-2 hours at a dose of vg / kg, and rAAV contains the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0042] This disclosure also provides the use of a dose of recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG for the manufacture of a pharmaceutical product for the treatment of limb-girdle muscular dystrophy, the dose of rAAV being approximately 1.25 × 10⁶ based on a supercoiled plasmid as a quantitative standard. 13 vg / kg, approximately 5.0×10 13 vg / kg or approximately 2.0 × 10 14 Based on vg / kg, or a linearized plasmid as a quantitative standard, approximately 1.85 × 10⁶ 13 vg / kg, approximately 4.63×10 12 vg / kg, or approximately 7.41 × 10⁻⁶ 13 The dosage is vg / kg, and the drug is formulated to deliver the dose by intravenous infusion over approximately 1-2 hours.

[0043] The Disclosure further provides a method for improving muscle 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 to SEQ ID NO: 1 or 7. Furthermore, a composition for improving muscle function in a subject is provided, comprising a construct comprising a nucleotide sequence having at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1 or 7 or 10. The use of a construct comprising a nucleotide sequence having at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1 or 7 or 10 is also provided for the manufacture of a pharmacopoeia for improving muscle function in a subject.

[0044] In any of the methods, uses, or compositions provided, the subject is affected by a genetic mutation in a sarcoglycan or a gene encoding muscular dystrophy. In some embodiments, the sarcoglycan is γ-sarcoglycan (SGCG), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD). In some embodiments, the sarcoglycan is γ-sarcoglycan.

[0045] In any of the provided methods, uses, or compositions, the level of gamma-sarcoglycan gene expression in the target cells is increased after administration of rAAV compared to the level of gamma-sarcoglycan gene expression before administration of rAAV.

[0046] Furthermore, in any of the provided methods, uses, or compositions, the expression of the gamma-sarcoglycan gene in cells is detected by measuring gamma-sarcoglycan protein levels in muscle biopsies taken before and after rAAV administration by Western blotting or immunohistochemistry.

[0047] In any of the provided methods, uses, or compositions, the level of gamma-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%, after administration of rAAV. It is increased by 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%, 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, the level of gamma-sarcoglycan protein is increased by at least 33%, as detected by measuring gamma-sarcoglycan protein levels by Western blotting in muscle biopsies taken before and after rAAV administration, or the level of gamma-sarcoglycan protein is increased by at least 38% or at least 39%, as detected by measuring gamma-sarcoglycan protein levels by immunohistochemistry in muscle biopsies taken before and after rAAV administration.

[0048] In any of the methods, uses, or compositions provided herein, serum CK levels in the subject decrease after administration of rAAV compared to serum CK levels before administration of rAAV. For example, serum CK levels in subjects 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% compared to serum CK levels before rAAV administration, 60–90 days after rAAV administration or 60 or 90 days after rAAV administration.

[0049] In any of the methods, uses, or compositions provided herein, the number of gamma-sarcoglycan-positive fibers in the muscle tissue of interest is increased after administration of rAAV compared to the number of gamma-sarcoglycan-positive fibers before administration of rAAV. For example, the number of gamma-sarcoglycan-positive fibers is detected by measuring gamma-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration. For example, the number of gamma-sarcoglycan-positive fibers in the target muscle tissue after administration of rAAV was 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%. Or it is increased by 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%.

[0050] In any of the methods, compositions, and uses provided herein, the levels of alpha-sarcoglycan and / or beta-sarcoglycan in a subject are increased after administration of rAAV compared to the levels of alpha-sarcoglycan and / or beta-sarcoglycan before administration of rAAV. In any of the methods, compositions, and uses provided herein, the levels of beta-sarcoglycan in a subject are increased after administration of rAAV compared to the levels of beta-sarcoglycan before administration of rAAV. The levels of alpha-sarcoglycan or beta-sarcoglycan are detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry or Western blotting in muscle biopsies before and after rAAV administration.

[0051] Another embodiment provides a method for expressing a gamma-sarcoglycan gene in cells, comprising administering to a subject a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or that contains the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0052] A composition for expressing a gamma-sarcoglycan gene in cells is also provided, the composition comprising a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or that includes the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0053] This disclosure also provides the use of the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence for the manufacture of a pharmaceutical product for expressing a gamma-sarcoglycan gene in cells, wherein the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0054] In any of the methods, uses, or compositions provided for expressing the gamma-sarcoglycan gene in cells, the expression of the gamma-sarcoglycan gene in cells is detected by measuring gamma-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.MHCK7.hSGCG construct. For example, the cells have more than 1 AAV virus copy number. Furthermore, the gamma-sarcoglycan gene is measured in the subject by detecting more than 1 rAAV vector genome per nucleus in at least one cell. In one embodiment, the mean rAAV copy number in the muscle cells of the treated subject is at least 0.01 copies per nucleus. In another embodiment, the mean rAAV copy number in the muscle cells of the treated subject is at least 0.1 copies per nucleus. In yet another embodiment, the mean rAAV copy number in the muscle cells of the treated subject is at least 1 copy per nucleus. In yet another embodiment, the mean rAAV copy number in the muscle cells of the treated subject is at least 10 copies per nucleus.

[0055] A composition for reducing serum CK levels in subjects requiring it is also provided, the composition comprising a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or that includes the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0056] The disclosure also provides the use of the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence for the manufacture of a pharmaceutical for reducing serum CK levels in subjects requiring it, wherein the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0057] In any of these methods, uses, and compositions, serum CK levels in the subject are reduced by at least 82% by 60 days after rAAV administration compared to serum CK levels before rAAV administration.

[0058] In any of these methods, uses, and compositions, the number of gamma-sarcoglycan-positive fibers is detected by measuring gamma-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration. Furthermore, in any of these methods, uses, and compositions, the number of gamma-sarcoglycan-positive fibers is measured by detecting one or more rAAV vector genome copies per nucleus.

[0059] Another embodiment provides a method for increasing alpha-sarcoglycan expression in a subject requiring it, comprising administering to the subject a scAAVrh74.MHCK7.hSGCG construct that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or a nucleotide sequence containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0060] A composition is also provided for increasing the expression of alpha-sarcoglycans in subjects requiring it, the composition comprising a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or that includes the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0061] This disclosure also provides the use of the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence in the manufacture of a pharmaceutical product for increasing the expression of alpha-sarcoglycans in a subject, wherein the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0062] Also provided is a method for increasing the localization of alpha-sarcoglycans to the cell membrane in subjects requiring such localization, comprising administering to the subject a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or that contains the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0063] A composition is also provided for increasing the localization of alpha-sarcoglycans to the cell membrane in subjects requiring it, the composition comprising a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0064] This disclosure also provides the use of the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence for the manufacture of pharmaceuticals for increasing the localization of alpha-sarcoglycans and / or beta-sarcoglycans to cell membranes in subjects requiring it, wherein the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0065] In any of these methods, uses, and compositions, the level of alpha-sarcoglycan is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration. Furthermore, in any of the provided methods, uses, and compositions, alpha-sarcoglycan is localized to the membrane of cells expressing gamma-sarcoglycan encoded by scAAVrh74.MHCK7.hSGCG.

[0066] Another embodiment provides a method for increasing sarcoglycan expression in the muscle tissue of a subject requiring such enhancement, comprising administering to the subject a scAAVrh74.MHCK7.hSGCG construct that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or a nucleotide sequence containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0067] A composition is also provided for increasing sarcoglycan expression in muscle tissue in subjects requiring it, the composition comprising a scAAVrh74.MHCK7.hSGCG construct nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0068] The disclosure also provides the use of the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence for the manufacture of a pharmaceutical for increasing sarcoglycan expression in target muscle tissue where it is needed, wherein the scAAVrh74.MHCK7.hSGCG construct nucleotide sequence is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10, or comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 10.

[0069] In any of these methods, uses, and compositions for increasing sarcoglycan expression in muscle tissue, the subject is suffering from a gene mutation in a gene encoding sarcoglycan or muscular dystrophy. For example, in any of these methods, uses, or compositions, the sarcoglycan is γ-sarcoglycan (SGCG), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD).

[0070] A method for producing recombinant AAV vector particles is also provided, comprising culturing cells into which the plasmid described herein has been introduced, and recovering recombinant AAV particles from the supernatant of the introduced cells. Viral particles comprising any of the recombinant AAV vectors described herein are also intended. In one embodiment, a method for producing rAAV comprises introducing 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 cells comprising an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 8. The cells described herein include insect cells, e.g., Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells); or mammalian cells (preferably human cells, e.g., human primary cells or established cell lines). In one embodiment, the mammalian cells include 293 cells, COS cells, HeLa cells, or KB cells.

[0071] In another embodiment, the plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1, 7, or 10. In some embodiments, the vector plasmid contains one of the nucleotide sequences of SEQ ID NO: 1, 7, or 10. In some embodiments, the AAV vector plasmid is stably expressed in host cells. rAAV can be produced using host cells that stably support the AAV vector plasmid. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCG.KAN plasmid.

[0072] A method for producing recombinant AAV vector particles provided herein may further include a step of introducing a packaging plasmid and / or a helper virus into a host cell. For example, the method includes a step in which the packaging cell contains a stably incorporated AAV cap gene and / or the packaging cell contains a stably incorporated AAV rep gene. The present invention also provides a plasmid containing a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8, or a cell containing a plasmid containing the nucleotide sequence of SEQ ID NO: 8. Cells containing the nucleotide sequence of SEQ ID NO: 1 or 7 are also provided.

[0073] A method for reducing fibrosis in mammalian subjects requiring it is also provided. In this regard, the method comprises administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to a mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In some embodiments, administration of the AAV vector described herein (or a composition comprising the AAV vector described herein) reduces fibrosis in the skeletal or cardiac muscle of the subject.

[0074] As used herein, the term “muscular dystrophy” refers to a disorder characterized by a gradual decrease in strength and muscle mass. Non-exclusive examples of muscular dystrophy include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycan disorders, congenital muscular dystrophy due to partial LAMA2 deficiency, merosin-deficient congenital muscular dystrophy, type 1D congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, limb-girdle type 1A muscular dystrophy, limb-girdle type 2A muscular dystrophy, limb-girdle type 2B muscular dystrophy, limb-girdle type 2C muscular dystrophy, and limb-girdle type Examples of congenital muscular dystrophy include type 2D muscular dystrophy, limb-girdle type 2E muscular dystrophy, limb-girdle type 2F muscular dystrophy, limb-girdle type 2G muscular dystrophy, limb-girdle type 2H muscular dystrophy, limb-girdle type 2I muscular dystrophy, limb-girdle type 2J muscular dystrophy, limb-girdle type 2K muscular dystrophy, limb-girdle type IC muscular dystrophy, vertebral ankylosing muscular dystrophy with simple epidermolysis bullosa, oculopharyngeal muscular dystrophy, Ulrich type congenital muscular dystrophy, and Ulrich type contracture hyperextension muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject suffers from limb-girdle type 2C muscular dystrophy (LGMD2C).

[0075] As used herein, the term “fibrosis” refers to the excessive or uncontrolled deposition and abnormal repair processes of extracellular matrix (ECM) components in tissues at the time of injury, including skeletal muscle, cardiac muscle, liver, lungs, kidneys, and pancreas. The deposited ECM components include collagen, for example, collagen 1, collagen 2, or collagen 3.

[0076] In another embodiment, a method for increasing muscle strength and / or muscle mass in a mammalian subject is described herein, comprising administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to the mammalian subject. In one embodiment, the subject is a human.

[0077] In any of the methods of the present invention, the subject may have a muscular dystrophy such as limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy.

[0078] A method for treating muscular dystrophy in a mammalian subject is also provided, comprising administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to the mammalian subject. In some embodiments, the muscular dystrophy is limb-girdle muscular dystrophy.

[0079] In any of the methods of the present invention, rAAV is administered by intramuscular injection or intravenous injection. Furthermore, in any of the methods of the present invention, rAAV is administered systemically, such as by parenteral administration by injection, infusion, or implantation.

[0080] The compositions of the present invention are formulated for intramuscular or intravenous injection. Furthermore, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or transplantation.

[0081] In any of the provided formulations or compositions, the buffering agent comprises one or more of Tris, Trisine, Bistrisine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. For example, the buffering agent comprises Tris at pH 8.0 at a concentration of about 5 mM to about 40 mM, or the buffering agent comprises Tris at pH 8.0 at about 20 mM.

[0082] In any of the provided formulations or compositions, the ionic strengthening agent comprises one or more of the following: 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 strengthening agent may contain MgCl2 at a concentration of about 0.2 mM to about 4 mM, or the ionic strengthening agent may contain NaCl at a concentration of about 50 mM to about 500 mM, or the ionic strengthening agent may contain 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 strengthening agent may contain MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM.

[0083] In any of the provided formulations or compositions, the surfactant comprises one or more of sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. For example, the poloxamer comprises one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. The poloxamer may be present in a concentration of about 0.00001% to about 1%. A typical surfactant is poloxamer 188 at a concentration of about 0.001%.

[0084] The preceding paragraph is not intended to define all aspects of the invention, and additional aspects are described in other sections, such as the detailed description of the invention. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if combinations of features are not found together in the same sentence, paragraph, or section of this document. The invention includes as additional aspects all embodiments of the invention that are narrower in any respect than the variations defined in the particular paragraph above. For example, if a particular aspect of the invention is described as a genus, it should be understood that all members of the genus are, individually, aspects of the invention. [Brief explanation of the drawing]

[0085] [Figure 1] Figure 1 shows the biodistribution of vector genome copies in various parts of skeletal muscle after administration of low, medium, and high doses of rAAV.MHCK7.hSGCBSGCG to mice. [Figure 2] Figure 2 shows human γ-sarcoglycan expression in skeletal muscle. Figure 2A shows immunofluorescence images of skeletal muscle, diaphragm, and heart of SGCG- / - mice intravenously injected with low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG. Figure 2B shows the percentage of fibers with SGCG protein expression at low, medium, and high doses. [Figure 3] Figure 3 shows the recovery of DAPC protein in SGCBSGCG- / - mice intravenously injected with low, medium, and high doses of scAAVrh.74.MHCK7.hSGCBSGCG. Figure 3A shows immunofluorescence images of DAPC protein expression. Figure 3B shows the percentage of fibers with DAPC protein expression at low, medium, and high doses. [Figure 4]Figure 4 shows the effect of systemic treatment with scAAVrh74.MHCK7.hSGCG on muscle pathology. Figure 4A shows H&E staining of quadriceps and diaphragm muscles from mice treated with BL / 6 WT, SGCG- / -, and low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG; Figure 4B shows trichrome staining for fibrosis quantification. [Figure 5] Figure 5 shows quantitative muscle morphometry in mouse-derived skeletal muscle treated with BL / 6 WT, SGCG- / -, and low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG. [Figure 6] Figure 6 shows the protection of force output in mice after treatment with low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG. [Figure 7] Figure 7 shows the physical activity of mice after treatment with low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG. [Figure 8] Figure 8 provides CK and chemical analysis of mice treated with low, medium, and high doses of scAAVrh.74.MHCK7.hSGCG. [Figure 9] Figure 9 provides a schematic map of the pAAV.MHCK7.hSGCG.KAN AAV vector plasmid. [Figure 10] Figure 10A shows a Western blot assay confirming γ-sarcoglycan protein expression across muscle tissue in treated mice. Figure 10B shows the relative expression of SGCG protein in treated mice compared to wild-type mice. [Modes for carrying out the invention]

[0086] This disclosure is based on the finding that administration of an AAV vector containing a polynucleotide expressing γ-sarcoglycan results in a reduction or complete reversal of myofibrosis or a restoration of sarcoglycan complex protein in an animal model of limb-girdle muscular dystrophy. As shown in the examples, administration of the AAV vector described herein resulted in a reversal of dystrophic features, including fewer degenerated fibers, increased walking ability, reduced CK levels, reduced inflammation, and improved functional recovery through protection against eccentric contractions due to increased force generation.

[0087] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells, with certain functions provided by co-infecting helper viruses. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related structurally, functionally, and even at the genetic level, it is quite expected that these same principles are also applicable to additional AAV serotypes. (See, for example, Blacklowe, 1988, 165-174, Parvoviruses and Human Disease, JRPattison (ed.); and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, and all give rise to three related capsid proteins, such as those expressed in AAV2. The degree of relevance is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along genome length, and the presence of similar self-annealing segments at the ends corresponding to “terminal inversion sequences” (ITRs). Similar infectivity patterns suggest that replication function in each serotype is under similar regulatory control.

[0088] As used herein, "AAV vector" refers to a vector containing one or more polynucleotides (or transgenes) of adjacent targets, with an AAV inverted terminal sequence (ITR). Such AAV vectors can be replicated and packaged into infectious viral particles when present in host cells transfected with a vector encoding and expressing rep and cap gene products.

[0089] An "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an inclusion polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically called an "AAV vector particle" or simply an "AAV vector." Thus, the production of an AAV vector particle necessarily involves the production of an AAV vector, and therefore the vector is contained within the AAV vector particle. AAV

[0090] The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA of the rAAV genome may be an AAV serotype that may result from the recombinant virus 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, AAV rh.10, and AAV rh.74. The production of pseudotype rAAV is disclosed, for example, in International Publication No. 01 / 83692. Other types of rAAV variants with capsid mutations, such as rAAV, should also be considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of various AAV serotype genomes are known in the art. AAV-1, AAV-5, AAV-6, AAVrh74, AAV-8, or AAV-9 may be used to promote skeletal muscle-specific expression.

[0091] The DNA plasmid of the present invention comprises an rAAV genome. The DNA plasmid is transferred to a cell tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus), which assembles the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function to be packaged are provided to the cell, are standard in the art. The production of rAAV requires the presence of the following components within a single cell (indicated herein as a packaging cell): the rAAV genome, the AAVrep and cap genes isolated from the rAAV genome (i.e., in the absence of the rAAV genome), and helper virus function. The AAV rep and cap genes may be derived from any AAV serotype, 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, from which the recombinant virus may be derived from and from an AAV serotype different from that of the rAAV genome ITR. The production of pseudotyped rAAV is disclosed, for example, in International Publication No. 01 / 83692, which is incorporated herein by reference in its entirety.

[0092] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a selective marker such as an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a neomycin resistance gene is incorporated into the cell genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077~2081), addition of a synthetic linker containing restriction enzyme endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65~73), or direct blunt endligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661~4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0093] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various approaches are described in Ratschin et al., Mol.Cell.Biol.4:2072(1984); Hermonat et al., Proc.Natl.Acad.Sci.USA,81:6466(1984); Tratschin et al., Mo1.Cell.Biol.5:3251(1985); McLaughlin et al., J.Virol.,62:1963(1988); and Lebkowski et al., 1988 Mol.Cell.Biol.,7:349(1988). Samulski et al. (1989, J. Virol., 63:3822-3828); US Publication No. 5,173,414; International Publication No. 95 / 13365 and corresponding US Publication No. 5,658,776; International Publication No. 95 / 13392; International Publication No. 96 / 17947; PCT / US98 / 18600; International Publication No. 97 / 09441 (PCT / US96 / 14423); International Publication No. 97 / 08298 (PCT / US96 / 13872); International Publication No. 97 / 21825 (PCT / US96 / 20777); International Publication No. 97 / 06243 (PCT / FR96 / 01064); International Publication No. 99 / 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. Patents 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on these sections of the documents relating to the production of rAAV.

[0094] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (congener 293 strain). In another embodiment, the packaging cells are non-transformed cancer cells such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus monkey lung cells).

[0095] The recombinant AAV of the present invention (i.e., infectious capsid-forming rAAV particles) comprises an rAAV genome. Examples of embodiments include, but are not limited to, an rAAV named pAAV.MHCK7.hSGCG containing the polynucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 7.

[0096] rAAV may be purified by standard methods in the art, such as column chromatography or a cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Patent No. 6,566,118 and International Publication No. 98 / 09657.

[0097] In another embodiment, the present invention envisions a composition comprising rAAV. The compositions described herein contain rAAV in a pharmaceutically acceptable carrier. The compositions may also include other components such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are preferably nontoxic to the recipient and inactive at the dose and concentration used, and include phosphates, citrates, 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 nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0098] The titer of rAAV to be administered by the method of the present invention may vary depending, for example, on the specific rAAV, the mode of administration, the therapeutic target, the individual, and the cell type(s) targeted, and may be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 Or it may be in the range of DNase-resistant particles (DRPs) or higher. The dosage may also be expressed in units of viral genome (vg). The titer of rAAV may be determined by a supercoiled plasmid quantification standard or a linearized plasmid quantification standard.

[0099] In one embodiment, the disclosure provides a method for determining the titer of an AAV vector, comprising determining the titer of the AAV vector by PCR using a first primer of SEQ ID NO: 13 and a second primer of SEQ ID NO: 14. In another embodiment, the method for determining the titer of an AAV vector comprises determining the titer of an AAV vector by using a probe comprising the sequence of SEQ ID NO: 15. In one embodiment, the probe comprises 5'-FAM-TGG ATC CCC-Zen-TGC ATG CGA AGA TC-3IABKFQ. In another embodiment, the AAV vector is scAAVrh74.MHCK7.hSGCG.

[0100] Methods for transducing target cells with rAAV in vivo or in vitro are considered in the present invention. An in vivo method involves administering an effective dose, or effective repeated dose, of a composition containing rAAV of the present invention to an animal (including humans) in need. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease being treated, delays or prevents progression to a disorder / disease state, delays or prevents progression of the disorder / disease, reduces the severity of the disease, results in disease remission (partial or complete), and / or prolongs survival. Examples of diseases intended for prevention or treatment using the methods of the present invention include muscular dystrophy, such as limb-girdle muscular dystrophy. Therefore, a method for transducing target cells with rAAV scAAVrh74.MHCK7.hSGCG, comprising the nucleotide sequence of SEQ ID NO: 1 or 7, is provided. This disclosure also provides a primer for SEQ ID NO: 11. In another embodiment, this disclosure provides a primer for SEQ ID NO: 12. In one embodiment, this disclosure provides a method for measuring SGCG expression in cells or subjects, the method comprising PCR analysis using the primers for SEQ ID NO: 11 and SEQ ID NO: 12. In one embodiment, the subject is suffering from LGMD. In one embodiment, the method comprises measuring the expression of the scAAVrh74.MHCK7.hSGCG vector in cells or patients.

[0101] Combination therapies are also considered in the present invention. Combinations used herein include both concurrent and sequential therapies. As with combinations with novel therapies, combinations of the methods of the present invention with standard pharmacotherapy (e.g., steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort) are specifically considered. In this regard, the combinations include administering one or more steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort, to a subject before, concurrently with, or after administration of the methods of the present invention to a subject of rAAV.

[0102] In related embodiments of the combination therapy envisioned by the present invention, the glucocorticoids include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0103] It is recognized that antigen-specific T cell responses may be present in subjects administered with rAAV vectors. This is a response expected 2–4 weeks after gene transfer. One possible consequence of such an antigen-specific T cell response is the clearance of transduced cells and loss of transgene expression. Prior to therapy, for example 24 hours before the therapeutic procedure, to attenuate the host immune response to rAAV-based therapy, subjects may be initiated orally with prophylactic prednisone or an equivalent glucocorticoid of approximately 1 mg / kg / day, with a maximum dose of 60 mg / day. IV administration of an equivalent glucocorticoid at an approximate dose of 1 mg / kg / day is also acceptable if necessary. Treatment continues for approximately one month. Tapering protocols for prednisone or an equivalent glucocorticoid can be performed based on the individual subject's immune response to gene transfer, which is assessed by monitoring liver function using ELISpot assays and GGT.

[0104] A therapeutically effective dose of rAAV vector is approximately 1 e13 vg / kg to 5 e14 vg / kg, or approximately 1 e13 vg / kg to 2 e13 vg / kg, or approximately 1 e13 vg / kg to 3 e13 vg / kg, or approximately 1 e13 vg / kg to 4 e13 vg / kg, or approximately 1 e13 vg / kg to 5 e13 vg / kg, or approximately 1 e13 vg / kg to 6 e13 vg / kg, or approximately 1 e13 vg / kg to 7 e13 vg / kg, or approximately 1 e13 vg / kg to 8 e13 vg / kg, or approximately 1 e13 vg / kg to 9 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 1 e14 vg / kg, or approximately 1 e13 vg / kg to approximately 2 e14 vg / kg, or 1 e13 vg / kg to approximately 3 e14 vg / kg, or approximately 1 e13 to approximately 4 e14 vg / kg, or approximately 3 e13 vg / kg to approximately 4 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 5 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 6 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 7 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 8 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 9 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 1 e 14vg / kg, or approximately 3e13vg / kg to approximately 2e14vg / kg, or 3e13vg / kg to approximately 3e14vg / kg, or approximately 3e13vg / kg to approximately 4e14vg / kg, or approximately 3e13vg / kg to approximately 5e14vg / kg, or approximately 5e13vg / kg to approximately 6e13vg / kg, or approximately 5e13vg / kg to approximately 7e13vg / kg, or approximately 5e13vg / kg to approximately 8e13vg / kg, or approximately 5e13vg / kg to approximately 9e13vg / kg, or approximately 5e13vg / kg to approximately 1e14vg / kg, or approximately 5e13vg / kg to approximately 2e1 4vg / kg, or 5e13vg / kg to approximately 3e14vg / kg, or approximately 5e13 to approximately 4e14vg / kg, or approximately 5e13vg / kg to approximately 5e14vg / kg, or approximately 1e14vg / kg to approximately 2e14vg / kg, or 1e14vg / kg to approximately 3e14vg / kg, or approximately 1e14 to approximately 4e14vg / kg, or approximately 1e14vg / kg to approximately 5e14vg / kg, 6e14vg / kg, 7e14vg / kg, 8e14vg / kg, 9e14vg / kg, 1e15vg / kg, 2e15vg / kg, 3e15vg / kg, 4e15vg / kg,The rAAV is in the range of doses of 5 e15 v g / kg, 6 e15 v g / kg, 7 e15 v g / kg, 8 e15 v g / kg, 9 e15 v g / kg, or 1 e16 v g / kg. The present invention also includes compositions comprising rAAV vectors in these ranges. In one embodiment, the dose is based on a linearized plasmid as a quantitative standard. In one embodiment, the dose is based on a supercoiled plasmid as a quantitative standard.

[0105] For example, therapeutically effective doses of rAAV vector are approximately 1e13vg / kg, 2e13vg / kg, 3e13vg / kg, 4e13vg / kg, 5e13vg / kg, 6e13vg / kg, 7e13vg / kg, 7.4e13vg / kg, 8e13vg / kg, 9e13vg / kg, 1e14vg / kg, 2e14vg / kg, 3e14vg / kg, and 4e14vg / kg. The titer or dose of the AAV vector may vary based on the physical form of the plasmid DNA as a quantitative standard. For example, the titer or dose value may vary based on a standard qPCR titration method for supercoiled or linear standard qPCR titration methods. In one embodiment, a therapeutically effective dose of rAAV is 5 e13 vg / kg based on a supercoiled plasmid as a quantitative standard, or 1.85 e13 vg / kg based on a linearized plasmid as a quantitative standard. In another embodiment, a therapeutically effective dose of rAAV is 2 e14 vg / kg based on a supercoiled plasmid as a quantitative standard, or 7.41 e13 vg / kg based on a linearized plasmid as a quantitative standard. In yet another embodiment, a therapeutically effective dose of rAAV is approximately 4.63 × 10⁻¹⁶ based on a linearized plasmid as a quantitative standard. 12 vg / kg, or approximately 1.25 × 10 based on supercoiled plasmids as a quantitative standard.13In another embodiment, a therapeutically effective amount of scAAVrh74.MHCK7.hSGCG is approximately 1e13vg / kg to approximately 5e14vg / kg, or approximately 1e13vg / kg to approximately 2e13vg / kg, or approximately 1e13vg / kg to approximately 3e13vg / kg, or approximately 1e13vg / kg to approximately 4e13vg / kg, or approximately 1e13vg / kg to approximately 5e13vg / kg, or approximately 1e13vg / kg to approximately 6e13vg / kg, or approximately 1e13vg / kg to approximately 7e13vg / kg, or approximately 1e13vg / kg ~ approximately 8 e13 vg / kg, or approximately 1 e13 vg / kg ~ approximately 9 e13 vg / kg, or approximately 1 e13 vg / kg ~ approximately 1 e14 vg / kg, or approximately 1 e13 vg / kg ~ approximately 2 e14 vg / kg, or approximately 1 e13 vg / kg ~ approximately 3 e14 vg / kg, or approximately 1 e13 ~ approximately 4 e14 vg / kg, or approximately 3 e13 vg / kg ~ approximately 4 e13 vg / kg, or approximately 3 e13 vg / kg ~ approximately 5 e13 vg / kg, or approximately 3 e13 vg / kg ~ approximately 6 e13 vg / kg, or approximately 3 e13 vg / kg ~ approximately 7 e13 vg / kg, or approximately 3 e13 vg / kg ~ Approximately 8 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 9 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 1 e14 vg / kg, or approximately 3 e13 vg / kg to approximately 2 e14 vg / kg, or approximately 3 e13 vg / kg to approximately 3 e14 vg / kg, or approximately 3 e13 to approximately 4 e14 vg / kg, or approximately 3 e13 vg / kg to approximately 5 e14 vg / kg, or approximately 5 e13 vg / kg to approximately 6 e13 vg / kg, or approximately 5 e13 vg / kg to approximately 7 e13 vg / kg, or approximately 5 e13 vg / kg to approximately 8 e13 vg / kg, or approximately 5 e13 vg / kg to approximately 9 e13vg / kg, or approximately 5e13vg / kg to approximately 1e14vg / kg, or approximately 5e13vg / kg to approximately 2e14vg / kg, or approximately 5e13vg / kg to approximately 3e14vg / kg, or approximately 5e13vg / kg to approximately 4e14vg / kg, or approximately 5e13vg / kg to approximately 5e14vg / kg, or approximately 1e14vg / kg to approximately 2e14vg / kg, or approximately 1e14vg / kg to approximately 3e14vg / kg, or approximately 1e14 to approximately 4e14vg / kg, or approximately 1e14vg / kg to approximately 5e14vg / kg, 6e14vg / kg, 7e14vg / kg,The dosages are in the range of 8e14vg / kg, 9e14vg / kg, 1e15vg / kg, 2e15vg / kg, 3e15vg / kg, 4e15vg / kg, 5e15vg / kg, 6e15vg / kg, 7e15vg / kg, 8e15vg / kg, 9e15vg / kg, or 1e16vg / kg. The present invention also includes compositions containing rAAV vectors in these dosages.

[0106] The effective dose of the composition may be administered by standard routes in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components of the rAAV of the present invention (in particular, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease condition being treated, as well as the target cells / tissues(s) that will express γ-sarcoglycans.

[0107] The present invention provides topical and systemic administration of effective doses of rAAV and compositions of the present invention. For example, systemic administration is administration to the circulatory system that affects the whole body. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration via injection, infusion, or transplantation.

[0108] In particular, the practical administration of rAAV according to the present invention can be achieved using any physical method for transporting the recombinant rAAV vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into muscle, bloodstream, and / or directly into the liver. It has been shown that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (although DNA-degrading compositions should be avoided in conventional methods using rAAV). The capsid protein of rAAV may be modified so that rAAV targets a specific target tissue of the subject, such as muscle. See, for example, International Publication No. 02 / 053703, the disclosure of which is incorporated herein by reference.

[0109] The pharmaceutical composition may be prepared as an injectable formulation or a topical formulation to be delivered to the muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. Therefore, in another embodiment, this application relates to a formulation comprising rAAV comprising an AAVrh74-derived capsid, a buffer, an ionic strengthener, and a surfactant. In one embodiment, the rAAV is 1.0 × 10⁻⁶ 12 vg / ml ~ approx. 1.0×10 16 vg / ml or approximately 1.0 × 10 12 vg / ml ~ approx. 5.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 5.0 × 10 based on a supercoiled plasmid as a quantitative standard. 12 vg / ml ~ approx. 1.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 5.0 × 10 based on a linearized plasmid as a quantitative standard. 12 vg / ml ~ approx. 1.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 2.0 × 10 based on a supercoiled plasmid as a quantitative standard. 13 The concentration is vg / ml. In one embodiment, rAAV is the scAAVrh74.MHCK7.hSGCG vector. In one embodiment, the concentration of rAAV in the composition or formulation is 1 × 10⁶ based on a supercoiled plasmid as a quantitative standard. 13 vg / ml ~ 2 × 10 14 The concentration is vg / ml. In another embodiment, the concentration is 2 × 10 based on a supercoiled plasmid as a quantitative standard. 13 vg / ml, 4 x 10 13 vg / ml, or 5×10 13The concentration is vg / ml. In one embodiment, the buffer comprises one or more of Tris, Trisine, Bistrisine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In another embodiment, the buffer comprises Tris having a pH of 8.0 at a concentration of about 5 mM to about 40 mM. In one embodiment, the buffer comprises Tris having a pH of 8.0 at about 20 mM. In one embodiment, the ionic strengthener 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 strengthener comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM. In another embodiment, the ionic strengthener comprises NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strengthening 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 strengthening 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 sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. 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 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, solutions in an adjuvant such as sesame oil or peanut oil, or aqueous solutions of propylene glycol, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered if desired, and the liquid diluent can first be isotonic with physiological saline or glucose.rAAV solutions, as free acids (DNA contains acidic phosphate groups) or pharmaceutically acceptable salts, can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0110] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0111] Sterile injectable solutions are prepared by incorporating the required amount of rAAV in a suitable solvent along with various other components listed above, and then, if necessary, by filter sterilization. Generally, dispersions are prepared by incorporating a sterile active ingredient into a sterile medium containing a basic dispersion medium and other components required from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques to produce a powder of the active ingredient and any additional desired components from its already filter-sterilized solution.

[0112] Transduction using rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are isolated from the target, transduced with rAAV, and reintroduced into the target. Alternatively, syngeneic or heterogeneic muscle cells can be used if these cells do not produce an inappropriate immune response in the target.

[0113] Suitable methods for transduction and re-transduction of transduced cells into a target are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells in a suitable culture medium, for example, and screening those cells for the target DNA using conventional techniques such as Southern blotting and / or PCR, or using a selection marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into a target by various techniques such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or injection into smooth muscle and cardiac muscle using a catheter, for example.

[0114] Transduction of cells using the rAAV of the present invention results in sustained expression of γ-sarcoglycan. Therefore, the present invention provides a method for administering / delivering rAAV expressing γ-sarcoglycan to a target, preferably a human. These methods involve transducing tissue (including, but not limited to, muscle-like tissues, organs such as the liver and brain, and glands such as salivary glands) using one or more rAAVs of the present invention. Transduction may be carried out using a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present invention is derived from 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)), regulatory elements derived from human skeletal actin genes (Muscat et al., Mol Cell Biol, 7:4089~4099 (1987)), cardiac actin genes, muscle creatine kinase sequence elements (Johnson et al., Mol Cell Biol, 9:3393~3399 (1989)), and mouse creatine kinase enhancer (mCK) elements, regulatory elements derived from the skeletal fast contractile troponin C gene, slow contractile cardiac troponin C gene, and slow contractile troponin I gene: hypoxia-induced nuclear factor (Semenza et al., Proc The present invention provides a method for transducing muscle cells and muscle tissue by muscle-specific regulatory elements, including, but not limited to, steroid-induced elements and promoters containing glucocorticoid response elements (GREs) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603~5607 (1993)), as well as other regulatory elements.

[0115] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. This invention considers the sustained expression of miRNAs from transduced muscle fibers.

[0116] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., gastrointestinal tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyocytes.

[0117] The term "transduction" is used to refer to the administration / delivery of a target polynucleotide (e.g., a polynucleotide sequence encoding γ-sarcoglycan) to recipient cells, either in vivo or in vitro, via the described replication-deficient rAAV, resulting in the expression of γ-sarcoglycan by the recipient cells.

[0118] Therefore, methods for administering rAAV encoding γ-sarcoglycans in an effective dose (or doses given in a manner or interval such that they are essentially administered simultaneously) to mammalian subjects in need thereof are also described herein.

[0119] All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually incorporated by reference. In the event of any conflict, this application, including any definitions, shall prevail.

[0120] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0121] In another embodiment, the disclosure provides a method for generating rAAV pAAV.MHCK7.hSGCG, which includes introducing an AAV vector plasmid into a host cell. Methods for introducing DNA into a host cell are known in the art and include, but are not limited to, transfection, infection, transformation, electroporation, and transduction. In one embodiment, the vector plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8. In another embodiment, the vector plasmid contains the nucleotide sequence of SEQ ID NO: 8. In another aspect, the disclosure provides a host cell containing an AAV vector plasmid containing the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. rAAV can be produced using a host cell that stably carries the AAV vector plasmid. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCG.KAN plasmid, which is illustrated in Figure 9. [Table 1-A] [Table 1-B] [Table 1-C] [Table 1-D] [Table 1-E] [Table 1-F] [Table 1-G] [Table 1-H] [Table 1-I] [Table 1-J] [Table 1-K] [Table 1-L]

[0122] In one embodiment, the vector plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 1 or 7. In another embodiment, the vector plasmid contains the nucleotide sequence of SEQ ID NO: 1 or 7. In one embodiment, the method for generating rAAV further includes introducing a packaging plasmid and / or a helper virus into a host cell. In some embodiments, the packaging plasmid contains an AAV rep 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 contains a stably incorporated AAV cap gene. In another embodiment, the packaging cell contains a stably incorporated rep gene.

[0123] As used herein, the term “host cell” refers to a cell that can be used to express a foreign 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 functional vectors, or other DNA. As used herein, the term encompasses the offspring of the original cell after the foreign DNA sequence has been expressed 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, e.g., Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells); or mammalian cells (preferably human cells, e.g., human primary cells or established cell lines). In one embodiment, mammalian cells include 293 cells, COS cells, HeLa cells, or KB cells. AAV vector plasmids can be introduced into host cells, such as Sf9 or 293T, by transient transfection using infection (virus or baculovirus), reagents (e.g., liposomes, calcium phosphate), physical means (e.g., electroporation), or other means known in the art. In another embodiment, host cell lines are stably incorporated with the rAAV plasmid into their genomes. Such stable cell lines can be established by incorporating a selection marker into the vector plasmid.

[0124] In one embodiment, the host cell is a packaging cell for the production of AAV virus particles. Therefore, in another embodiment, the present disclosure provides a host cell comprising an AAV vector plasmid comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8. In one embodiment, the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. In another embodiment, the host cell comprises the nucleotide sequence of SEQ ID NO: 1, 7, or 10. [Examples]

[0125] Preclinical studies using scAAVrh74.MHCK7.hSGCG are described in International Patent Publication WO2019 / 152474, which is incorporated herein by reference in its entirety. Example 1 material and method

[0126] Animal Model: WT (C57BL / 6J) mice with a BL6 genetic background and SGCG- / - mice were mated and maintained as homozygous animals under standardized conditions at the Animal Resources Core of the Sarepta Gene Therapy Center of Excellence. Mice were maintained on a Teklad Global Rodent Diet (3.8% fiber, 18.8% protein, 5% fat solid feed) in a 12:12 dark:light cycle. All animals were housed in standard mouse PH with free access to food and water. Both sexes of mice were used in all experiments: WT (n=6, 5 ​​males [M] / 1 female [F]); untreated SGCG- / - (n=6, 4M / 2F); low dose (n=6, 6M / 0F); medium dose (n=6, 4M / 2F); high dose (n=6, 0M / 6F).

[0127] Genotype analysis

[0128] SGCG- / - mice were identified using DNA genotyping resolution. DNA was isolated from tail clippings and analyzed by PCR using OneTaq DNA polymerase (New England Biolabs, Ipswich, MA). A series of primers were used in the PCR analysis to determine the SGCG- / - status. The following primers and conditions were used: GGA GGA AGC GCT GCC TAT ACC TAT T (SEQ ID NO: 11); CAA ATG CTT GCC TCA GGT ATT TC; GCC TGC TCT TTA CTG AAG GCT CTT T (SEQ ID NO: 12). The reaction was performed for 30 cycles in genomic DNA under the following conditions: 94°C, 30 seconds; 58°C, 30 seconds; 68°C, 25 seconds; followed by 5 minutes at 68°C.

[0129] hSGCG gene construct (scAAVrh74.MHCK7.hSGCG) and vector production

[0130] Full-length human SGCG cDNA (NC_000013.11) was codon-optimized and used in all experiments of this study. The cassette contains a consensus Scozak sequence (CCACC), an SV40 chimeric intron, and a synthetic polyadenylation site (53 bp). Expression is brought about using a muscle-specific MHCK7 promoter. This promoter is well-established for enhancing transgene expression in the heart and diaphragm. The SGCG expression cassette was cloned between AAV2 terminal inversion sequences (ITRs), and the cassette was packaged into an AAVrh74 vector using a triple transfection method at the Vector Manufacturing Facility of the Center for Gene Therapy at Nationwide Children's Hospital. The AAVrh74 virus has been shown to be safe and highly efficient in transducing muscle across the blood-vascular barrier in mice, non-human primates, and humans. One of the rate-limiting steps in AAV gene transduction and subsequent transgene expression is the conversion of the single-stranded vector genome to a double-stranded genome via the synthesis of cDNA strands. Because the SGCG transgene is small in size, the inventors can bypass this critical rate limiting step by using a self-complementary vector cassette that packages the double-stranded transgene via complementary base pairing and mutant hairpin ITRs.

[0131] The vector was titrated using Taqman qPCR with a primer-probe set located in the MHCK7 promoter region. No variability from standard AAV production and purification was observed using this method. The rAAV vector was generated using a modified cross-packaging method previously reported by Rodino-Klapac et al. (J.Trans.Med.5:45,2007). Here, triple transfection using CaPO4 precipitation in HEK293 cells allows for the packaging of AAV2 ITR into different AAV capsid serotypes (28, 29). The produced plasmids were (i) pAAV.MHCK7.hSGCG, (ii) rep2-caprh.74 modified AAV helper plasmid encoding cap serotype 8-like isolate rh.74, and (iii) adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I / II RNA genes. The vector was purified, and the vg titer was determined by capsidization (using a Prism 7500 Taqman detector system; PE Applied Biosystems, Carlsbad, CA, USA). The 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: 13); MHCK7 reverse primer, 5'-GTC CCC CAC AGC CTT GTT C-3' (SEQ ID NO: 14); and MHCK7 probe, 5'-FAM-TGG ATC CCC-Zen-TGC ATG CGA AGA TC-3IABKFQ-3'.

[0132] Treatment cohort

[0133] In dose-escalation studies, systemic delivery was administered via tail vein injection of either three different doses of vector or saline. Vector doses were calculated based on linear qPCR. A total dose of 8.94 × 10⁴ was administered to 4-week-old SGCG- / - mice corresponding to the low, medium, and high doses, respectively. 10vg(4.63×10 12 vg / kg; n=6, 6M / 0F), total dose 3.63 × 10 11 vg(1.85×10 13 vg / kg; n=6, 4M / 2F), or total dose 1.26 × 10 12 vg(7.41×10 13 SCAAVRH74.MHCK7.HSGCG was injected at a dose of vg / kg (n=6, 0M / 6F). Furthermore, WT mice (n=6, 5M / 1F) and SGCG- / - control mice (n=6, 4M / 2F) were injected with physiological saline. Mice were injected at 4-5 weeks of age and euthanized 12 weeks after gene delivery.

[0134] Low, medium, and high doses were based on linearized plasmids as quantitative standards. The AAV vector was diluted in saline using a 30-gauge ultrafine insulin syringe. Mice were warmed by restraining them in a retaining tube with the tail positioned posteriorly via a tail slot to dilate blood vessels and facilitate injection. After locating the artery from the midline of the tail, injection was performed in one of the purple / blue lateral veins running along the tail artery. All treated mice were injected at 4-5 weeks of age and euthanized for observation 12 weeks after injection. Endpoints included, but were not limited to, biomarker expression (e.g., immunofluorescence), transduction (e.g., qPCR vector genome), histology (e.g., centronucleus, diameter, fibrosis), function (e.g., activity cage, physiology), and safety (e.g., clinical chemistry).

[0135] Tissue processing

[0136] Skeletal muscle was extracted from each mouse, placed on gauze moistened with physiological saline, then placed in a wooden chuck fitted with a cryogel, and fresh-frozen in cooled methylbutane. The organ was divided in half; one half was placed in 10% neutral buffered formalin, followed by paraffin embedding for sectioning, and then stained with hematoxylin and eosin (H&E). The remaining half of the organ was fresh-frozen for subsequent molecular studies.

[0137] Biodistribution qPCR analysis

[0138] The presence of test substance-specific DNA sequences in muscle and organs was assessed using a real-time qPCR assay with a vector-specific primer-probe set designed to amplify the sequence of the intron region immediately downstream of the MHCK7 promoter (N=9; n=3 per low, medium, and high dose group). Frozen tissues were sectioned into pre-cooled microcentrifuge tubes using a cryostat (15 sections, 20 microns thick). Genomic DNA was isolated using the DNeasy Blood & Tissue Kit according to the manufacturer's protocol. The resulting DNA samples were stored at -80°C until analysis. Test DNA was prepared by diluting each sample in sterile ultrapure water to the maximum possible concentration of 5 ng / μL or 10 ng / μL. Using stock plasmids, 1 × 10⁶ DNA was prepared. 6 Standard samples were prepared by starting at a concentration of copies / μL and serially diluting to 10 copies / μL. All samples and standards were tri-analyzed. The initial denaturation step was performed at 95°C for 20 seconds, followed by 95°C for 1 second and 60°C for 30 seconds for 40 cycles. qPCR was performed using the QuantStudio system and software. The following primers and probes were used in this study: MHCK7 intron forward primer 5'- CCA ACA CCT GCT GCC TCT AAA-3' (SEQ ID NO: 13), MHCK7 intron reverse primer 5'- GTC CCC CAC AGC CTT GTT C-3' (SEQ ID NO: 14), and MHCK7 intron probe 5'- TGG ATC CCC TGC ATG CGA AGA TC-3' (SEQ ID NO: 15) [5'6-FAM, 3' Iowa Black® FQ, Internal ZEN® Quencher (Integrated DNA Technologies)]. The primers and probes were diluted to final concentrations of 100 nM, 100 nM, and 200 nM per reaction, respectively. The copy number in each reaction was calculated using a standard curve. The following equation was used to determine the vector genome copy number per nucleus:

[0139] The number of copies per nucleus = 10^((CT - standard curve Y-intercept) / slope of the standard curve) * (1000 / filling volume per well (ng)) * (5.98 × 10⁶)

[0140] Immunofluorescence

[0141] Transgene expression and DAPC repair across muscle tissue were evaluated using immunofluorescence. Frozen sections (12 μm thick) from the tibialis anterior, gastrocnemius, quadriceps, psoas major, gluteus, triceps, diaphragm, and myocardium were subjected to immunofluorescence staining for transgenes using protocols previously used by the inventors. For the detection of γ-sarcoglycan protein, sections were incubated with a 1:100 dilution of γ-sarcoglycan rabbit polyclonal primary antibody (Novus, catalog no. NBP1-59744). For the detection of α-, β-, and δ-sarcoglycan proteins, sections were incubated with a 1:100 dilution of α-sarcoglycan rabbit polyclonal antibody (Abcam, catalog no. ab189254), β-sarcoglycan mouse propylphosphone antibody (Leica, catalog no. B-SARC-L-CE), and δ-sarcoglycan primary antibody. Four random 20× images covering four different quadrants of muscle sections were acquired using a Zeiss (Germany) Axi℃am MRC5 camera. The percentage of fibers positive for α-, β-, δ-, and γ-sarcoglycan protein staining compared to the control was determined for each image and averaged for each muscle. Counted fibers were defined by the structural appearance of the fiber cross-section. To facilitate scoring, total fibers were counted using National Institutes of Health (NIH) ImageJ software with the Cell Counter plugin. Positive fiber expression was defined as having at least 50% fibers that stained brighter than the vehicle-treated SGCG- / - saline control, as previously described. Positive fibers were scored based on the original image exposure; no adjustments were made to image brightness or contrast during the positive image scoring process. The remaining fibers were scored as negative. The test substance was concealed at the time of injection. The operator who administered the injection did not perform any analysis other than the injection. Since no expression or remaining protein was present in the untreated group, it was clear which animals received treatment and which did not undergo microscopic observation, and this was irrelevant to immunofluorescence quantification. Images were taken with the same exposure to reduce intensity variability.Quantitative data of immunofluorescence-positive fibers expressing α-, δ-, and γ-sarcoglycan proteins in six mice per treatment group are reported as mean ± SEM values.

[0142] Western blot analysis

[0143] Tissue sections (20 μm thick, 15 sections) were collected in a microcentrifuge tube and homogenized with 150 μL of homogenization buffer (125 mM Tris-HCl, 4% SDS, 4M urea) in the presence of one protease inhibitor cocktail tablet. After homogenization, the samples were centrifuged at 10,000 rpm at 4°C for 10 minutes, and the supernatant was collected. Protein concentration was determined using NanoDrop. Protein samples (20 μg) were electrophoresed on a 3-8% polyacrylamide Tris-acetic acid gel at 150 V for 70 minutes, and then transferred to a PVDF membrane at 35 V for 90 minutes. The membranes were blocked for 1 hour in 5% nonfat-dried milk in TBST, and then incubated with either a 1:2000 dilution of monoclonal rabbit γ-sarcoglycan antibody (Abcam, catalog no. ab203113) and a 1:50000 dilution of mouse α-actinin antibody (Sigma, catalog no. A7811) or a 1:5000 dilution of monoclonal rabbit bincrine antibody (Fisher, catalog no. 700062). Anti-mouse (Sigma, catalog no. AP308P) and anti-rabbit secondary HRP antibodies (Invitrogen, catalog no. 65-6120) were used for ECL immunodetection. Western blot detection and quantification were performed using the Alliance Q9 Advanced chemiluminescence imaging system and software. Exposure times were set to vary depending on the sample intensity using the automated acquisition mode. The volume of the protein bands was quantified as the sum of all pixel intensities within a defined range using the software's analysis mode and normalized to the corresponding packed control band. Relative protein expression was determined by dividing it by the WT volume ratio.

[0144] Morphometric analysis

[0145] H&E staining was performed to visualize muscle morphology, including fiber size and centrinuation, on frozen sections of muscle (12 μm thick) from 16-week-old WT mice (n=6), SGCG- / - mice (n=6), and SGCG- / - mice treated with SCAAVRH74.MHCK7.HSGCG (n=6 per dose, 12 weeks after treatment). The percentage of muscle fibers with centrinuation was determined for the tibialis anterior, gastrocnemius, quadriceps, gluteus, triceps, and psoas major muscles. Furthermore, the diameter of muscle fibers was measured using ferret diameters for the tibialis anterior, triceps, and gastrocnemius muscles. Quantification was performed per muscle from each treatment group and control cohort, ranging from 1600 to 2000 fibers. Four random 20× images per muscle per animal were acquired using a Zeiss Axi℃am MRC5 camera. Central nucleus fibers were quantified using NIH ImageJ software, and fiber diameter was measured using Zeiss Axiovision LE4 software.

[0146] Histopathological examination

[0147] During the autopsy, muscle tissue was fresh-frozen in liquid nitrogen-cooled methylbutane, and the tissue was stained with H&E. All other organs were collected, fixed in formalin, and embedded in paraffin. Slides and all tissues were sent to GEMPath, Inc. for formal review by a veterinary pathologist.

[0148] Masson's trichrome staining for quantitative analysis of fibrosis

[0149] Frozen muscle tissue sections (12 μm) were placed on Fisherbrand Superfrost charged microscope slides. The slides were stained and fixed in Bouin fixative for 60 minutes, then washed with tap water, followed by washing with distilled water until clear. The slides were then incubated in Weigert Iron hematoxylin solution for 5 minutes and washed with running water for 5 minutes. The slides were rinsed with distilled water, then immersed in Biebrich Scarlet acid for 2 minutes, and washed again with distilled water. The slides were transferred to phosphotungstate-phosphomolybdic acid for 10 minutes, immersed in aniline blue for 2 minutes, and thoroughly washed with distilled water. After incubation in acetic acid (1% aqueous solution) for 7 minutes, the slides were dehydrated in gradient ethanol, cleared in xylene, and covered with coverslips using Cytoseal 60 medium from Thermo Fisher Scientific (Waltham, MA, USA; catalog no. 8310). Images were acquired using a Jenoptik Prokyon camera mounted on a Nikon Eclipse Ni-U microscope, with Gryphax software 2.0.0.68v. Four random 20× images covering four different quadrants of muscle sections were taken for analysis of Masson's trichrome staining and percentage collagen quantification. Thresholds for red (muscle) and blue (collagen area) contrast were individually set using BIOQUANT Life Sciences software (2019). Collagen and muscle areas were calculated using measurement functions. Total tissue area was determined by adding the muscle area and collagen area. The percentage of collagen was calculated by dividing the collagen area by the total tissue area. The average percentage for each individual was calculated.

[0150] Contraction of the tibialis anterior muscle for functional assessment

[0151] The anterior tibialis muscle evaluation method followed the protocol described by Hakim et al. Mice were anesthetized with an intraperitoneal injection of a ketamine / xylazine mixture (137.5 mg / kg and 10 mg / kg, respectively). The skin of the hindlimbs was removed to expose the anterior tibialis muscle and the patella. The muscle length was measured after dissection and before placing the mouse, and the length was entered into the software. Care was taken to limit drying of the exposed muscle by constantly hydrating the exposed muscle with Kimwipe covers soaked in saline. Next, the distal tendon of the anterior tibialis muscle was dissected (left and right per animal, mean of both limbs used for analysis [n = 12 per cohort]), and double loop sutures were placed around the tendon as close to the muscle as possible using 4-0 sutures before cutting the tendon. The mouse was then transferred to a temperature-controlled platform and maintained at 37 °C. To stabilize the limb, a metal pin was placed behind the patellar tendon, and the knee was fixed to the platform with the distal tendon of the anterior tibialis muscle sutured to the lever arm of a force transducer (Aurora Scientific, Aurora, Canada). Electrodes were placed near the sciatic nerve and stimulated. An Aurora Scientific-designed warm-up protocol was initiated, setting the resting tension to a force of 3 - 4 g and maintaining it for 5 minutes, followed by muscle stimulation at 1 Hz (3 times, 30-second intervals), and additional muscle stimulation at 150 Hz (3 times, 60-second intervals). When the muscle had stabilized, the resting tension was set to the length at which the contraction of spasm was maximal (optimal length). After a 3-minute rest period, the anterior tibialis muscle was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute rest between each stimulation. After a 5-minute rest, the muscle was then isometrically contracted 10 times at 1-minute intervals using a 10% stretch elongation method. The duration of tetanic contraction continued for 200 minutes. After the eccentric contraction, the mouse was euthanized and both anterior tibialis muscles were dissected and frozen for histological and molecular studies.

[0152] Formula:

[0153] Anterior tibialis muscle limb-specific muscle strength = absolute muscle strength / cross-sectional area

[0154] Absolute force = force at 150 Hz * 9.8 (9.8 mN = 1 gram)

[0155] Cross-sectional area = muscle weight (mg) / 1.06 (muscle density) * Length (mm) * 1 muscle weight (g) / [tibialis anterior limb muscle fiber length (cm) x 1.06 (g / cm3)]

[0156] Tetani contraction of the diaphragm for functional assessment

[0157] Mice were euthanized, and the diaphragm was excised intact, preserving the rib attachments and central tendon. The excised tissue was then placed in the previously described Kreb's-Henseleit (KH) buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.25 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM glucose). Diaphragm sections 2–4 mm wide were isolated from each animal per cohort (n=6). The diaphragm strips were securely tied at the central tendon with surgical silk sutures (6 / 0; Surgical Specialties, Reading, PA) and sutured through a fixed portion of the rib at the distal end of the strip. Each muscle was transferred to a water bath filled with oxidized KH solution maintained at 37°C. Muscles were aligned horizontally and directly connected between a fixed pin and a dual-mode force transducer servomotor (305C; Aurora Scientific, Ontario, Canada). Two platinum plate electrodes were positioned in the bathtub adjacent to the length of the muscle. The muscle was stretched to the optimal length for measuring single contractions and then rested for 10 minutes before starting the tetanic protocol. Once the muscle was stable, it was set to the optimal length of 1g and a warm-up consisting of three 1Hz single contractions every 30 seconds, followed by three 150Hz single contractions every minute. After a 3-minute rest period, the diaphragm was stimulated at 20, 50, 80, 120, 150, and 180Hz, with a 2-minute rest period between each stimulation, and the maximum tetanic muscle force was determined for each with a duration of 250ms. Muscle length and muscle weight were measured, and muscle force was normalized against muscle weight and length.

[0158] formula:

[0159] Diaphragm-specific muscle strength = absolute muscle strength at 150Hz / cross-sectional area

[0160] Absolute force = force at 150Hz * 9.8 (9.8 mN = 1 gram)

[0161] Cross-sectional area = muscle weight (g) / [diaphragm fiber length (cm) x 1.06 (g / cm3)]

[0162] Laser monitoring of open field cage activities

[0163] To assess the level of physical activity, SGCG- / - and WT mice were subjected to an open-field activity protocol similar to that used in previous reports. The overall activity of the experimental mice was determined using an open-field activity chamber. Four-week-old mice from the WT (n=6, 5M / 1F) and untreated SGCG- / - (n=6, 4M / 2F) control groups, as well as SGCG- / - mice treated with SCAAVRH74.MHCK7.HSGCG (low dose [n=6, 6M / 0F]; medium dose [n=6, 4M / 2F]; high dose [n=6, 0M / 6F]), were subjected to analysis according to previously described protocols with some modifications. Mice were treated at 4 weeks of age, and the endpoint was 12 weeks post-treatment. Cohorts were injected one week apart to eliminate age variability at the endpoint. Sessions were separated by cohort. All mice were tested at the same time between 6:10 AM and 8:30 AM, the period when mice are most active. All mice were tested in an isolated room by the same person each time, in the dark. To reduce anxiety and minimize behavioral variables that could affect the normal activity of the mice and the resulting assay results, the inventors tested mice that were not individually housed. Mouse activity was monitored using a Photobeam activity system (San Diego Instruments, San Diego, CA). This system monitors the position and movement of mice in the xyz plane using a grid of invisible infrared beams that traverse the animal chamber from front to back and left to right. Activity was recorded in 1-hour cycles at 5-minute intervals. Mice were acclimatized to the activity testing chamber for the first 1-hour session for 3 and 4 days prior to the start of data collection. Mice were tested in individual chambers. The testing equipment was cleaned between each use to reduce behavioral variables of mouse responses that could alter the results. Data was converted to a Microsoft Excel worksheet, and all calculations were performed within the Excel program. The individual beam breaks for movement in the x and y planes were added together for each mouse to represent the total walking motion, and the beam breaks in the z plane were added together to obtain the vertical activity within a one-hour interval.

[0164] Serum chemistry and hematology

[0165] As a measure of safety, blood chemistry and hematology tests were performed in SGCG− / − mice and WT mice administered the vector. Whole blood was removed by cardiac puncture from treated WT mice and SGCG− / − mice. Blood was collected into serum separator tubes and centrifuged at 3,500 rpm for 10 minutes. Serum was collected, frozen, and sent to Nationwide Children’s Hospital for processing and evaluation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) liver enzyme levels.

[0166] Serum creatine kinase measurement

[0167] Following the manufacturer's protocol (Sekisui Diagnostics; Charlottetown, PE, Canada; catalog number 326-10), the creatine kinase SL assay was used to measure creatine kinase levels in the serum of WT mice (n = 6), mice treated with vehicle SGCG− / − lactated Ringer's solution (LR) (n = 6), and SGCG− / − mice treated with medium and high doses of SCAAVRH74.MHCK7.HSGCG (n = 6) (data for the low dose were not collected due to insufficient sample volume). Briefly, 25 μL of serum was mixed with 1 mL of working reagent and added to a cuvette. A kinetic assay was set up on a spectrophotometer to measure absorbance at 340 nm every 30 seconds for 180 seconds. Creatine kinase levels were calculated using the measured absorbance values and the formula listed below:

[0168] U / L = [(Δ absorbance / min) * 1.025 * 1000] / [1 * 6.22 * 0.025] = (Δ absorbance / min) * 6592.

[0169] Statistical analysis

[0170] Statistical analysis was performed using GraphPad Prism 7.01 software. Data were presented as mean ± SEM (error bars). One-way ANOVA with Tukey's multiple comparison test was performed for the analysis of blood chemistry, serum creatine kinase, diaphragmatic and anterior tibialis muscle physiology, and cage activity. Two-way ANOVA with Tukey's multiple comparison test was performed for the analysis of central nucleation and eccentric contraction. Kruskal-Wallis test with Dunn's multiple comparison test was performed for the analysis of fiber diameter.

[0171] A single systemic injection of SCAAVRH74.MHCK7.hSGCG resulted in successful systemic delivery, as indicated by the biodistribution of the vector genome copy (Figure 1). Intravenous administration of the scAAVrh74.MHCK7.hSGCG AAV vector to SGCG- / - mice in the presence of significant histopathology in the muscle resulted in transgene expression across the tibialis anterior (TA), DIA, and HRT muscles throughout the entire dose (Figure 2).

[0172] Furthermore, administration of the scAAVrh74.MHCK7.hSGCG vector to SGCG- / - mice in the presence of significant histopathology in muscle tissue resulted in dose-dependent restoration of DAPC protein in the muscle cell membrane (Figure 3). In particular, prior to treatment, SGCG- / - mice showed no or reduced expression of α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), and δ-sarcoglycan (SGCD) in the muscle cell membrane (Figure 3). Treatment with the scAAVrh74.MHCK7.hSGCG vector increased the expression of SGCA, SGCB, and SGCD subunits in the muscle cell membrane of SGCG- / - mice, as measured by immunofluorescence percent-positive fibers (Figure 3).

[0173] SGCG- / - mice exhibited significant histopathology in the muscles, accompanied by high levels of central nucleation, compared to wild-type mice (WT). After treatment with scAAVrh74.MHCK7.hSGCG, overall muscle pathology improved, and a reduction in central nucleation was observed (Figure 4A). Overall fibrous tissue deposition improved, and the level of fibrosis decreased as the dose increased compared to the level in untreated SGCG- / - mice (Figure 4B).

[0174] All three doses showed an increase in fiber diameter, exhibiting normalized fiber sizes similar to WT fibers in the TA, gastrocnemius (GAS), and triceps (TRI) muscles (Figure 5). Functional improvements were observed, including significantly increased muscle strength (force production) and resistance to contraction-induced injury in the TA and DIA muscles (Figure 6). Deficiencies in intrinsic muscle strength and resistance to contraction-induced injury were identified in the intrinsic muscle strength of the tibialis anterior and diaphragmatic muscles of SGCG- / - mice compared to WT mice. Treatment with medium and high doses of scAAVrh74.MHCK7.hSGCG significantly improved intrinsic muscle strength in both muscles compared to untreated SGCG- / - mice (tibialis anterior: low dose p=0.571, medium dose p=0.008, high dose p=0.0001; diaphragm: low dose p=0.388, medium dose p=0.088, high dose p=0.001; Figure 6). Numerical improvements in eccentric contraction were also observed with high-dose treatment.

[0175] Compared to WT controls, reduced walking and vertical housing were observed in the SGCG- / - mouse model (Figure 7). Laser monitoring of open field cage activity showed increased walking and movement in SGCG- / - mice treated with SRP-9005.

[0176] The treatment was associated with a decrease in CK levels. Liver enzymes (ALT and AST) returned to normal ranges in treated mice (Figure 8). Quantitative muscle morphometry showed increased fiber diameter at all three doses, exhibiting normalized fiber sizes similar to WT fibers in the tibialis anterior, gastrocnemius, and triceps muscles (Table 2). [Table 2]

[0177] Western blot analysis. Gamma-sarcoglycan

[0178] Western blotting confirmed γ-sarcoglycan protein expression across muscle tissue in mice treated with the lowest dose (Figure 10A). γ-sarcoglycan expression was dose-dependent, maintaining at least 100% of wild-type (WT) expression in the heart at low, medium, and high doses (Figure 10B).

[0179] This disclosure describes specific embodiments, but it will be understood that modifications and alterations can be made to those skilled in the art. Therefore, only the limitations set forth in the claims should be made in this disclosure.

[0180] All documents referenced in this application are incorporated herein by reference in their entirety.

Claims

1. A composition comprising recombinant adeno-associated virus (rAAV) scAAVrh74.MHCK7.hSGCG for treating limb-girdle muscular dystrophy in subjects requiring it, The rAAV comprises the nucleotide sequence of sequence number 10, The aforementioned rAAV, using a systemic administration route, yielded 7.41 × 10⁶ based on a linearized plasmid as a quantitative standard. １３ A composition administered at a dose of vg / kg, wherein the serum creatine kinase (CK) level in the subject decreases after administration of the rAAV compared to the serum CK level before administration of the rAAV.

2. The composition according to claim 1, wherein the rAAV is administered via an intravenous route.

3. The composition according to claim 1, wherein the level of gamma-sarcoglycan protein expression is increased by at least 30% after administration of rAAV compared to the level of gamma-sarcoglycan protein before administration of rAAV.

4. The composition according to claim 1, wherein the number of gamma-sarcoglycan-positive fibers in the target muscle tissue is increased by at least 40% after administration of rAAV compared to the number of gamma-sarcoglycan-positive fibers before administration of rAAV.

5. The composition according to claim 1, wherein the serum CK level in the subject decreases by at least 82% by 90 days after administration of rAAV compared to the serum CK level before administration of rAAV.

6. The composition according to claim 1, wherein the levels of alpha-sarcoglycan and / or beta-sarcoglycan in the subject are increased after administration of rAAV compared to the level of alpha-sarcoglycan before administration of rAAV.

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