Adeno-associated virus vector delivery of alpha-sarcoglycan and treatment of muscular dystrophy

Abstract

Described herein are methods of treating muscular dystrophy in a subject comprising administering a systemic route of administration at about 1.0×10 12 vg / kg to about 5.0×10 15 The recombinant AAV vector AAVrh74.tMCK.hSCGA is administered at a dose of vg / kg. Also disclosed are methods for expressing the α-sarcoglycan gene in cells or subjects in need, reducing serum CK levels, and increasing α-sarcoglycan-positive fibers in the muscle tissue of the subject.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 889,749 filed on August 21, 2019, U.S. Provisional Application No. 63 / 014,934 filed on April 24, 2020, and U.S. Provisional Application No. 63 / 022,843 filed on May 11, 2020, all of which are incorporated herein by reference in their entirety. Technical Field

[0003] Described herein are therapeutic vectors, such as AAV vectors that express α-sarcoglycan, and methods of using these vectors to treat limb-girdle muscular dystrophy, such as LGMD2D.

[0004] Incorporation by Reference into the Sequence Listing

[0005] This application contains a Sequence Listing in computer-readable form as a separate part of the disclosure (file name: 54652_SeqListing.txt; 18,768 bytes - ASCII text file; created on August 17, 2020), which is incorporated herein by reference in its entirety. Background Art

[0006] Muscular dystrophy (MD) is a group of genetic diseases. The group is characterized by progressive weakness and degeneration of skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while other forms of MD may not appear until middle age or later. The conditions differ in the distribution and degree of muscle weakness (some forms of MD also affect the heart muscle), age of onset, rate of progression, and inheritance pattern.

[0007] One group of MDs is the limb-girdle muscular dystrophy (LGMD). LGMD is a rare condition and varies from person to person in terms of age of onset, areas of muscle weakness, heart and respiratory involvement, rate of progression, and severity. LGMD may begin in childhood, adolescence, young adulthood, or even later. Both sexes are affected equally. LGMD causes weakness in the shoulder and pelvic girdle, and sometimes nearby muscles in the thighs and arms, which weaken over time. Leg weakness usually develops before arm weakness. Facial muscles are usually not affected. As the condition progresses, people may have problems walking and may need to use a wheelchair over time. Involvement of the shoulder and arm muscles may make it difficult to raise the arms overhead and lift objects. In some types of LGMD, the heart and respiratory muscles may be affected.

[0008] Specific testing for LGMD is now available through the national diagnostic program, the National Commissioning Group (NCG).

[0009] Subtype 2D LGMD (LGMD2D), commonly referred to as α-sarcoglycanopathy, is an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene (SGCA; α-sarcoglycan), resulting in a complete or reduced loss of functional protein as well as loss of other structural components of the dystrophin-related protein complex. Notably, loss of the α-sarcoglycan protein results in a progressive muscular dystrophy with deteriorating muscle function, with an onset age of 3 to 8 years. Symptoms include: delayed walking, proximal muscle weakness caused by fat replacement and fibrosis, elevated creatine kinase, scoliosis, and joint contractures. This debilitating disease often leads to wheelchair dependence and death from respiratory failure. Therefore, there remains a need for treatments for LGMD2D. Summary of the invention

[0010] Described herein are methods for treating muscular dystrophy in a subject in need thereof, comprising the step of administering a recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSGCA, wherein the rAAV is administered using a systemic route and is expressed at about 1.0×10 based on supercoiled DNA or plasmid as a quantitative standard. 12 About 5.0×10 15 In one aspect, the present disclosure relates to rAAV expressing an α-sarcoglycan gene, and methods of delivering α-sarcoglycan to muscle to reduce and / or prevent fibrosis; and / or increase muscle strength, and / or treat α-sarcoglycanopathy in a subject with muscular dystrophy.

[0011] In one aspect, described herein are recombinant AAV (rAAV) comprising a polynucleotide sequence encoding an α-sarcoglycan protein. In some embodiments, the polynucleotide sequence comprises a sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence set forth in SEQ ID NO: 1 and encodes a protein that retains α-sarcoglycan activity. In some embodiments, the polynucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1. In another embodiment, the polynucleotide encodes a protein that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence set forth in SEQ ID NO: 2. In another embodiment, the polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0012] In some embodiments, the rAAV comprises a nucleotide sequence containing a tMCK promoter. For example, the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO:3. In addition, the rAAV comprises a 5' inverted terminal repeat sequence of SEQ ID NO:5 and / or a 3' inverted terminal repeat sequence of SEQ ID NO:6. In some embodiments, the rAAV comprises a poly A sequence of SEQ ID NO:7. In one embodiment, the disclosed rAAV is serotype AAVrh.74.

[0013] In one embodiment, the polynucleotide comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 4. In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0014] The present disclosure provides a method of treating muscular dystrophy in a subject in need thereof, comprising the step of administering any of the rAAVs disclosed herein, wherein the rAAV is administered by a systemic route. In particular, in any of the disclosed methods, the rAAV is AAVrh74.tMCK.hSGCA, wherein the rAAV is administered using a systemic route of administration.

[0015] In any of the disclosed methods, rAAV is measured at about 1.0×10 12 vg / kg to about 5.0×10 15 For example, rAAV is administered at a dose of approximately 1.0 × 10 vg / kg based on supercoiled DNA or plasmid as a quantitative standard. 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg or about 5×10 13 vg / kg to about 2×10 14 vg / kg, or rAAV at about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 vg / kg dose.

[0016] In another embodiment, in any of the disclosed methods, rAAV is measured at about 1.85×10 13 vg / kg or 7.41×10 13 For example, rAAV is administered at a dose of approximately 1.0 × 10 vg / kg based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg dose.

[0017] In addition, in any of the disclosed methods, the systemic administration route is an intravenous route. For example, in any of the disclosed methods, the rAAV is administered by injection, infusion or implantation. In some embodiments, the rAAV is administered by an intravenous route through a peripheral limb vein.

[0018] In any of the disclosed methods, the muscular dystrophy is limb-girdle muscular dystrophy. For example, the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

[0019] In an exemplary embodiment, the method of treating muscular dystrophy comprises administering rAAV to a subject suffering from limb-girdle muscular dystrophy, wherein the rAAV is administered by intravenous infusion at a concentration of about 5×10 based on supercoiled DNA or plasmid as a quantitative standard. 13 vg / kg to about 2×10 14 vg / kg, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4.

[0020] In an exemplary embodiment, the present disclosure provides a method of treating muscular dystrophy in a subject in need thereof, the method comprising the step of administering rAAV to the subject, wherein the subject suffers from limb-girdle muscular dystrophy, and the rAAV is administered by intravenous infusion at a concentration of about 5×1013 vg / kg to about 2×1013 vg / kg based on supercoiled DNA or plasmid as a quantitative standard. 14 vg / kg, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. For example, in these methods, the expression level of the α-sarcoglycan gene in the subject's cells is increased after administration of the rAAV compared to the expression level of the α-sarcoglycan gene before administration of the rAAV.

[0021] In any of the disclosed methods, the level of α-sarcoglycan gene expression in the subject's cells is increased after administration of rAAV compared to the level of α-sarcoglycan gene expression before administration of rAAV; and / or wherein the serum CK level in the subject is decreased after administration of rAAV compared to the serum CK level before administration of rAAV; and / or wherein voluntary activity and specific force production are increased; wherein fibrosis is decreased; wherein resistance to contraction-induced injury in the tibialis anterior muscle is increased; and / or wherein the number of α-sarcoglycan-positive fibers in the subject's muscle tissue is increased after administration of rAAV compared to the number of α-sarcoglycan-positive fibers before administration of rAAV; or wherein fibrosis is decreased in the subject after administration of rAAV compared to before administration of rAAV; and / or wherein fibrosis is decreased in the subject after administration of rAAV compared to before administration of rAAV; and / or wherein specific force, fiber diameter size and / or eccentric contraction in the subject's muscle is increased after administration of rAAV compared to before administration of rAAV.

[0022] In some embodiments, α-sarcoglycan gene expression is detected by measuring α-sarcoglycan protein levels by Western blot and / or immunohistochemistry.

[0023] On the other hand, the present disclosure provides a method for expressing an α-sarcoglycan gene in a cell, comprising administering any of the disclosed rAAVs to a subject. For example, the present disclosure provides a method for expressing an α-sarcoglycan gene in a cell, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4 to a subject. In addition, in any of the methods, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the methods, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in a subject by detecting the number of vector genomes per microgram of genomic DNA.

[0024] The present disclosure provides a method for reducing serum CK levels in a subject in need thereof, the method comprising administering to the subject any of the disclosed rAAVs. For example, the present disclosure provides a method for reducing serum CK levels in a subject in need thereof, the method comprising administering to the subject a scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0025] In another aspect, the present disclosure provides a method for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject, comprising administering to the subject any of the disclosed rAAVs. For example, the present disclosure provides a method for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject in need thereof, the method comprising administering to the subject the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0026] The present disclosure also provides a method for increasing the expression of α-sarcoglycan in a subject in need thereof, comprising administering any of the disclosed rAAVs to the subject. For example, the present disclosure provides a method for increasing the expression of α-sarcoglycan in a subject in need thereof, comprising administering to the subject a scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4. In addition, in any of the disclosed methods, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the methods, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0027] The present disclosure provides a composition for treating muscular dystrophy in a subject in need thereof, wherein the composition comprises any one of the rAAVs disclosed herein, wherein the composition is formulated for administration by a systemic route. In particular, in any one of the compositions, the rAAV is AAVrh74.tMCK.hSGCA.

[0028] Any of the disclosed compositions comprises a supercoiled DNA or plasmid dosage of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg of rAAV. For example, based on supercoiled DNA or plasmid as a quantitative standard, the dose of rAAV is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg or about 5×10 13vg / kg to about 2×10 14 vg / kg, or a dose of rAAV of approximately 5 × 10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 vg / kg.

[0029] In another embodiment, in any of the disclosed compositions, rAAV is present at about 1.85×10 13 vg / kg or about 7.41×10 13 For example, rAAV is administered at a dose of approximately 1.0 × 10 vg / kg based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg dose.

[0030] In addition, any of the disclosed compositions are formulated for administration by an intravenous route, such as a composition formulated for administration by injection, infusion, or implantation. In some embodiments, the disclosed compositions are formulated for administration by an intravenous route via a peripheral limb vein.

[0031] Any of the disclosed compositions is used to treat limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0032] In an exemplary embodiment, the present disclosure provides a composition for treating a subject suffering from limb-girdle muscular dystrophy, wherein the composition comprises a supercoiled DNA or plasmid dose of about 5×10 13 vg / kg to about 2×10 14 vg / kg of rAAV, and wherein the composition is formulated for administration by intravenous infusion, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4.

[0033] In addition, the present disclosure provides a composition for treating limb-girdle muscular dystrophy in a subject in need thereof, wherein the composition comprises a supercoiled DNA or plasmid dose of about 5×1013 vg / kg to about 2×10 14 vg / kg of rAAV, and the composition is formulated for administration by intravenous infusion, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. For example, administration of the composition increases the expression level of the α-sarcoglycan gene in the subject's cells compared to the expression level of the α-sarcoglycan gene before administration of the rAAV.

[0034] In addition, administration of any of the disclosed compositions increases the level of α-sarcoglycan gene expression in cells of the subject compared to the level of α-sarcoglycan gene expression prior to administration of the composition; and / or wherein administration of the disclosed compositions reduces the serum CK level of the subject compared to the serum CK level prior to administration of the composition; and / or wherein voluntary activity and specific force production are increased; wherein fibrosis is reduced; wherein resistance to contraction-induced injury in the tibialis anterior muscle is increased; and / or wherein administration of the composition increases the number of α-sarcoglycan-positive fibers in muscle tissue of the subject compared to the number of α-sarcoglycan-positive fibers prior to administration of the composition; and / or wherein administration of the composition reduces fibrosis in the subject compared to before administration of rAAV; and / or wherein the composition reduces fibrosis compared to before administration of the composition; or wherein administration of the composition increases specific force, fiber diameter size, and / or eccentric contraction in the subject's muscle compared to before administration of the composition. In some embodiments, α-sarcoglycan gene expression is detected by measuring α-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0035] In another aspect, the present disclosure provides a composition for expressing an α-sarcoglycan gene in a cell, wherein the composition comprises any one of the disclosed rAAVs. For example, the present disclosure provides a composition for expressing an α-sarcoglycan gene in a cell, comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4. In addition, in any of the compositions, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the methods, the expression of the α-sarcoglycan gene in the cell is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0036] The present disclosure provides a composition for reducing serum CK levels in a subject in need thereof, the composition comprising any of the disclosed rAAVs. For example, the present disclosure provides a composition for reducing serum CK levels in a subject in need thereof, the composition comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0037] In another aspect, the present disclosure provides a composition for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject, wherein the composition comprises any one of the disclosed rAAVs. For example, the present disclosure provides a composition for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject, wherein the composition comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0038] The present disclosure also provides a composition for increasing the expression of α-sarcoglycan in a subject in need, wherein the composition comprises any one of the disclosed rAAVs. For example, the present disclosure provides a composition for increasing the expression of α-sarcoglycan in a subject in need, wherein the composition comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4. In addition, after administering any of the disclosed compositions, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in muscle biopsy. Alternatively, after administering any of the disclosed compositions, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in muscle biopsy. In other embodiments, after administering any of the disclosed compositions, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0039] The present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein the medicament is formulated for administration by a systemic route. In particular, the present disclosure provides the use of AAVrh74.tMCK.hSGCA for the preparation of a medicament for treating muscular dystrophy, wherein the medicament is formulated for administration by a systemic route of administration.

[0040] In any of the disclosed uses, the medicament comprises a supercoiled DNA or plasmid dose of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg of rAAV. For example, based on supercoiled DNA or plasmid as a quantitative standard, the dose of rAAV is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13vg / kg to about 3.0×10 14 vg / kg or about 5×10 13 vg / kg to about 2×10 14 vg / kg, or a dose of rAAV of approximately 5 × 10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 vg / kg.

[0041] In another embodiment, in any of the disclosed uses, the medicament comprises a dose of about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg of rAAV. For example, based on linearized DNA or plasmid as a quantitative standard, the drug contains a dose of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg of rAAV.

[0042] In addition, in any of the disclosed uses, the drug is formulated for administration via an intravenous route. For example, in any of the disclosed uses, the drug is formulated for administration via injection, infusion, or implantation. In some embodiments, the drug is formulated for administration via an intravenous route through a peripheral limb vein.

[0043] In any of the disclosed uses, the medicament is for treating limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0044] In an exemplary embodiment, the present disclosure provides a use of rAAV for the preparation of a medicament for treating limb-girdle muscular dystrophy, wherein the medicament is formulated for administration by intravenous infusion, and the dose of the rAAV is about 5×1013 vg / kg to about 2×1013 vg / kg based on supercoiled DNA or plasmid as a quantitative standard. 14 vg / kg, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. For example, administration of the drug to a subject in need thereof results in increased expression of the α-sarcoglycan gene in cells of the subject compared to the level of α-sarcoglycan gene expression prior to administration of the rAAV.

[0045] In any of the disclosed uses, administration of the drug to a subject in need thereof results in an increase in the level of alpha-sarcoglycan gene expression in the subject's cells compared to the level of alpha-sarcoglycan gene expression before administration of the drug; and / or administration of the drug to the subject results in a decrease in the subject's serum CK level compared to the serum CK level before administration of the drug; and / or wherein voluntary activity and specific force production are increased; wherein fibrosis is reduced; wherein resistance to contraction-induced injury in the tibialis anterior muscle is increased; and / or administration of the drug to the subject results in an increase in the number of alpha-sarcoglycan-positive fibers in the subject's muscle tissue compared to the number of alpha-sarcoglycan-positive fibers before administration of the drug, and / or administration of the drug to a subject in need thereof results in a decrease in fibrosis in the subject compared to before administration of the drug; and / or administration of the drug results in an increase in specific force, fiber diameter size, and / or eccentric contraction in the subject's muscle compared to before administration of the drug. In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0046] On the other hand, the present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for expressing an α-sarcoglycan gene in a cell of a subject in need thereof. For example, the present disclosure provides the use of a scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for expressing an α-sarcoglycan gene in a cell of a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. In addition, in any of the uses, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the uses, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0047] The present disclosure provides the use of any of the disclosed rAAVs for preparing a medicament for reducing serum CK levels in a subject in need thereof. For example, the present disclosure provides the use of a scAAVrh74.tMCK.hSGCA construct for preparing a medicament for reducing serum CK levels in a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4.

[0048] In another aspect, the present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject. For example, the present disclosure provides the use of a scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for increasing α-sarcoglycan-positive fibers in muscle tissue of a subject, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0049] The present disclosure also provides the use of any of the disclosed rAAVs for the preparation of a drug for increasing the expression of α-sarcoglycan in a subject in need thereof. For example, the present disclosure provides the use of a scAAVrh74.tMCK.hSGCA construct for the preparation of a drug for increasing the expression of α-sarcoglycan in a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. In addition, in any of the disclosed uses, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the disclosed uses, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0050] In any of the disclosed methods, compositions, or uses, the subject is a human subject between 4 and 15 years of age, or a human subject between 25 and 55 years of age, or a human subject over 50 years of age.

[0051] In any of the disclosed methods, compositions or uses, the subject is a pediatric subject, an adolescent subject or a young adult subject. Alternatively, the subject is a middle-aged adult or an elderly subject.

[0052] For example, in any of the disclosed methods, compositions, or uses, the subject is a human subject 4-15 years old with confirmed alpha-sarcoglycan (SGCA) mutations in both alleles, who is negative for AAVrh74 antibodies and / or has a >40% or normal 100 meter walk test.

[0053] In another aspect, the present disclosure provides a method of producing a rAAV for administration in any of the disclosed methods, compositions, or uses, the method comprising transferring an AAV vector plasmid to a host cell, wherein the AAV vector plasmid comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. For example, the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the vector plasmid comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 4, or 8, or the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4, or 8.

[0054] In any of the disclosed methods for producing rAAV, the method further comprises transferring the packaging plasmid and / or the helper virus to the host cell. In addition, in any of the disclosed methods for producing rAAV, the packaging cell comprises a stably integrated AAV cap gene and / or the packaging cell comprises a stably integrated AAV rep gene.

[0055] In another aspect, the present disclosure provides a host cell comprising an AAV vector plasmid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 4 or 8. For example, the host cell comprises an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 1, 4 or 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The scAAVrh74.tMCK.hSGCA gene cassette is shown.

[0057] Figure 2 Transgene expression in a dose escalation study following systemic treatment with scAAVrh74.tMCK.hSGCA is shown. (A) Transgene expression in a dose escalation study following systemic treatment with 1×10 12 vg, 3×10 12 vg and 6×10 12 vg(or 5×10 13 vg / kg, 1×10 14 vg / kg and 2×10 14 vg / kg, based on 20-g mice) systemically (intravenously) treated with α-sarcoglycan in various muscles of mice (n=6 / group). Compared with untreated controls, muscle fibers expressing α-sarcoglycan ranged from 70% to 93% 12 weeks after treatment. (B) Muscles from treated sgca - / - Western blot of muscles of mice confirmed hSGCA protein expression.Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; TRI, triceps; GLUT, gluteal; PSO, psoas; DIA, diaphragm; HRT, heart; WT, wild type.

[0058] Figure 3 The results showed that scAAVrh74.tMCK.hSGCA improved muscle morphology in sgca- / - mice regardless of the dose. (A) 12 vg, 3×10 12 vg and 6×10 12 vg(or 5×10 13 vg / kg, 1×10 14 vg / kg and 2×10 14vg / kg, based on 20-g mice) Hematoxylin and eosin images of various muscles of sgca- / - mice treated with scAAVrh74.tMCK.hSGCA. Representative 20x images show a significant reduction in centralized nuclei and overall normalization of fiber size, regardless of treatment dose. (B) Quantification of normalization of myofiber diameters of various muscles in confirmed treatment groups compared to vehicle-treated mice and wild-type controls (n=6 / group). (C) Quantification of centrally located nuclei in muscles of treated mice compared to untreated mice and wild-type controls (n=6 / group). Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteal; PSO, psoas; TRI, triceps; DIA, diaphragm; WT, wild-type.

[0059] Figure 4 sgca treated with scAAVrh74.tMCK.hSGCA is shown - / - (A) Picrosirius red staining shows reduced fibrosis in scAAVrh74.tMCK.hSGCA-treated mice, which is different from vehicle-treated sgca. - / - (A) Representative 20x images of the mice compared to those in the control group indicated a decrease in collagen deposition in various muscles. (B) Quantification of collagen levels in various muscles demonstrated a decrease in collagen levels in all three treatment groups compared to untreated mice and wild-type controls (n=6 / group). Abbreviations: PSO, psoas major; DIA, diaphragm; TRI, triceps; GLUT, gluteal muscle.

[0060] Figure 5 Functional benefits of skeletal muscle following treatment with scAAVrh74.tMCK.hSGCA are shown. (A) After 3 months of treatment, tibialis anterior (TA) muscles (left and right) were harvested to measure specific force and resistance to contraction-induced injury (normalized to TA weight). Specific force and quantitative eccentric contraction were increased in all treatment groups (with minimal differences between doses) compared to untreated controls (n=6 / group). (B) Diaphragm muscle strips were harvested to measure specific force. After 12 weeks of treatment, sgca-treated controls showed significant differences in specific force compared to untreated controls. - / - (C) After 12 weeks of treatment, the force was significantly increased in treated mice compared to untreated sgca- / -treated mice showed improved walking and vertical activity as measured by open field assay compared to controls (n = 6 / group). (D) Compared to untreated sgca - / -Serum creatine kinase levels were decreased in all treatment groups compared to controls. Data were analyzed by one-way ANOVA followed by Tukey's post hoc analysis for multiple comparisons. Unless otherwise stated, sgca treated with vehicle - / - Compared with mice, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Abbreviations: TA, tibialis anterior; DIA, diaphragm; WT, wild type.

[0061] Figure 6 There was no evidence of toxicity by blood chemistry following treatment with scAAVrh74.tMCK.hSGCA. Liver enzymes (ALT, AST, and ALP / K) and blood glucose (GLU) levels were analyzed for toxicity (n=6 / group). As indicated by the dotted lines, all chemical values ​​for treated mice were within normal / healthy limits for mice.

[0062] Fig. 7A Biodistribution analysis of systemic scAAVrh74.tMCK.hSGCA delivery is shown—in a 3×10 12 vg and 6×10 12 vg(or 1×10 14 vg / kg and 2×10 14 vg / kg, based on 20-g mice) intravenous delivery of scAAVrh74.tMCK.hSGCA - / - Distribution histogram of the average VG copies of transcript per microgram of DNA added to quantitative PCR in various tissues from mice. Figure 7B Display medium (sgca - / - LR (Lactate Ringer) or 1.0×10 12 vg (left western blot) or 6×10 12 vg (right western blot) of scAArh74.tMCK.hSGCA treated WT and sgca - / - Western blot of α-sarcoglycan expression in mouse liver. Each lane represents an independent mouse (M1: mouse 1; M2: mouse 2; M3: mouse 3). The lower panel represents vinculin used as a loading control. Abbreviations: DIA, diaphragm; TA, tibialis anterior; TRI, triceps.

[0063] Figure 8 By immunofluorescence staining ( Figure 8 A) and Western blot ( Figure 8 B) shows the expression of scAAVrh74.tMCK.hSGCA in 12-month-old sgca- / - Expression in skeletal muscle of mice. Figure 8 The diagram in C provides scAAVrh74.tMCK.hSGCAsgca - / - Percentage of positive myofibers in mice. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteal; TRI, triceps; PSOAS, psoas; diaphragm; WT, wild type; LRS, lactated ringers solution.

[0064] Fig. 9 Shown in SGCA at 12 months of age - / - Histological results of scAAVrh74.tMCK.hSGCA administration in mice. Fig. 9 a shows improved muscle pathology. Fig. 9 b shows a decrease in central nucleation and an increase in mean fiber size in gastrocnemius (GAS) and triceps (TRI) muscles. Fig. 9 c shows the reduction in fibrosis levels compared to untreated controls. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteal; PSO, psoas; TRI, triceps; diaphragm; WT, wild type.

[0065] Fig.10 showed functional improvement in aged mice after administration of scAAVrh74.tMCK.hSGCA. DETAILED DESCRIPTION

[0066] The present disclosure is based on the discovery that administration of rAAV comprising a polynucleotide expressing α-sarcoglycan results in a reduction or complete reversal of muscle fibrosis in an animal model of limb-girdle muscular dystrophy. As demonstrated herein in the Examples, administration of the rAAV described herein results in reversal of dystrophic features, including reduced CK levels, increased muscle strength, improved walking and vertical activity, and other motor functions.

[0067] Unless otherwise indicated, the practice of the present invention will employ conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. These techniques are fully explained in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (current edition); DNA Cloning: A Practical Approach, Vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., current edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., current edition); Transcription and Translation (B. Hames & S. Higgins, eds., current edition); CRC Handbook of Parvoviruses, Vols. I & II (P. Tijssen, ed.); Fundamental Virology, 2nd ed., Vols. I & II (B. N. Fields and D. M. Knipe, eds.); Freshney Culture of Animal Cells, A Manual of Basic Techniques. (1991) Current Protocols in Molecular Biology (Wiley Interscience, NY).

[0068] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0069] definition

[0070] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "a culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. Reference to "recombinant AAV" includes mixtures of two or more rAAV virions, and the like. Unless clearly indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0071] Unless explicitly indicated, the term "or" is used in the claims to mean "and / or" to refer to alternatives only, or the alternatives are mutually exclusive, although the present disclosure supports definitions and "and / or" referring only to alternatives.

[0072] Throughout this application, the term "about" is used to indicate that a value includes the statistical experimental error (the standard deviation of error) for the device or method employed to determine the value.

[0073] The term "vector" or "expression vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when combined with appropriate control elements and which can transfer gene sequences between cells. In one embodiment, the vector is a viral vector. The expression vector can contain a variety of control sequences, structural genes (e.g., genes of interest), and nucleic acid sequences that also serve other functions.

[0074] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus generated only in cells, in which certain functions are provided by co-infection helper viruses. Thirteen AAV serotypes have been characterized. Basic information and reviews of AAV can be found in, for example, Carter, 1989, "Parvovirus Handbook", Vol. 1, pp. 169 to 228 and Berns, 1990, "Virology", pp. 1743 to 1764, Raven Press, (New York). However, it is entirely expected that these same principles will be applicable to additional AAV serotypes, because it is well known that various serotypes are closely related in structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, "Parvoviruses and Human Disease", pp. 165 to 174, JRPattison, ed.; and Rose, "Comprehensive Virology" 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further indicated by heteroduplex analysis, which shows extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends, which correspond to "inverted terminal repeats" (ITRs). Similar infectivity patterns also indicate that the replication function of each serotype is under similar regulatory control.

[0075] As used herein, "AAV vector" refers to a vector comprising one or more polynucleotides of interest (or transgenes), the flanks of which are AAV terminal repeats (ITRs). When present in a host cell transfected with a vector encoding and expressing rep and cap gene products, this AAV vector can be replicated and packaged into infectious viral particles. In one embodiment, the AAV vector is a vector derived from an adeno-associated virus serotype, including but not limited to 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 rh10 and AAV rh74. The AAV vector may have one or more AAV wild-type genes that are completely or partially deleted, preferably rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are essential for the rescue, replication and packaging of AAV virions. Therefore, AAV vectors are defined herein as including at least those sequences required for viral replication and packaging (e.g., functional ITRs) in cis. The ITRs need not be wild-type nucleotide sequences and may be altered, for example, by insertion, deletion or substitution of nucleotides, as long as the sequence provides functional rescue, replication and packaging.

[0076] The term "AAV helper function" refers to an AAV-derived coding sequence that can be expressed to provide an AAV gene product that is in turn used in trans for productive AAV replication. Thus, AAV helper functions include the major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to have many functions, including: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and transcriptional regulation from an AAV (or other heterologous) promoter. The Cap expression product provides the necessary packaging function. AAV helper functions are used herein to supplement trans AAV functions that are missing in AAV vectors.

[0077] A "recombinant virus" refers to a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid sequence into the viral particle.

[0078] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. In one embodiment, the AAV virus particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell). In some embodiments, the production of AAV vector particles includes the production of an AAV vector because this vector is contained within the AAV vector particle. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as a "rAAV vector" or simply "rAAV particle". Therefore, the production of AAV vector particles necessarily includes the production of rAAV because this rAAV genome is contained within the rAAV vector particle.

[0079] For example, a wild-type (wt) AAV virus particle comprises a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein coating. AAV virus particles can be single-stranded (ss) AAV or self-complementary (SC) AAV. In one embodiment, a single-stranded AAV nucleic acid molecule with complementary sense (e.g., a "sense" or "antisense" strand) can be packaged into an AAV virus particle and the two strands have equal infectivity.

[0080] The term "recombinant AAV" or "rAAV" is defined herein as an infectious replication-defective virus composed of an AAV protein shell, which is encapsidated with a heterologous nucleotide sequence of interest flanked by AAV ITRs on both sides. In one embodiment, rAAV is produced in a suitable host cell, into which an AAV vector, AAV helper functions, and helper functions are introduced. In this way, the host cell is able to encode the AAV polypeptides required to package the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0081] The term "transfection" refers to the uptake of foreign DNA by a cell, and when foreign DNA is introduced into the cell membrane, the cell is "transfected". Many transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign DNA portions such as nucleotide integration vectors and other nucleic acid molecules into suitable host cells.

[0082] The term "transduction" refers to the delivery of a DNA molecule to a recipient cell in vivo or in vitro by a replication-defective viral vector, such as by a recombinant AAV virion.

[0083] The term "host cell" refers to microorganisms, yeast cells, insect cells and mammalian cells that can or have been used as recipients of AAV helper constructs, AAV vector plasmids, helper function vectors or other transfer DNA, for example. The term includes the descendants of the original cells that have been transfected. Therefore, as used herein, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It should be understood that the descendants of a single parent cell are not necessarily identical to the original parent in morphology or genome or total DNA complement due to natural, accidental or deliberate mutations.

[0084] The term "heterologous" refers to sequences that are not usually joined together and / or are not usually associated with a particular cell when it relates to a nucleic acid sequence (such as a coding sequence and a control sequence). Therefore, the "heterologous" region of a nucleic acid construct or vector is a nucleic acid fragment within or attached to another nucleic acid molecule that is not found to be associated with other molecules in nature. For example, the heterologous region of a nucleic acid construct can include a coding sequence, and its two sides are sequences that are not found to be associated with the coding sequence in nature. Another example of a heterologous coding sequence is a construct (for example, a synthetic sequence with codons different from natural genes) in which the coding sequence itself is not found in nature. Similarly, for purposes of the present invention, cells transformed with constructs that are not usually present in cells are considered to be heterologous. As used herein, allelic variation or naturally occurring mutation events do not produce heterologous DNA.

[0085] A coding sequence or a sequence encoding a specific protein is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminal and the translation stop codon at the 3' (carboxyl) terminal. The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located 3' of the coding sequence.

[0086] "Nucleic acid" sequence refers to a DNA or RNA sequence. Nucleic acids include base analogs of DNA and RNA, including but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, uracil, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxythymidine (oxybutoxosine), pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine and 2,6-diaminopurine.

[0087] The term DNA "control sequence" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc., which together provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these control sequences need to be present at all times, as long as the selected coding sequence is capable of replication, transcription, and translation in appropriate host cells.

[0088] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters may include "inducible promoters" (wherein the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (wherein the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters". In one embodiment, the promoter is a muscle-specific promoter, which includes but is not limited to human skeletal actin gene elements, cardiac actin gene elements, desmin promoter, skeletal α-actin (ASKA) promoter, troponin I (TNNI2) promoter, myocyte-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid inducible element and glucocorticoid response element (gre). In another embodiment, the promoter is MCK promoter, tMCK promoter or MHCK7 promoter.

[0089] The term "operably linked" refers to an arrangement of elements in which the components so described are configured to perform their usual functions. Thus, a control sequence operably linked to a coding sequence is capable of affecting the expression of the coding sequence. A control sequence need not be adjacent to the coding sequence, as long as it serves to direct its expression. Thus, for example, there may be an intervening untranslated but transcribed sequence between a promoter sequence and a coding sequence, and the promoter sequence may still be considered to be "operably linked" to the coding sequence.

[0090] A promoter "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind to the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

[0091] An "expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence or gene of interest. As described above, an expression cassette includes control elements, such as a promoter operably linked to the sequence or gene of interest (so as to direct transcription), and typically also includes a polyadenylation sequence. In certain embodiments of the invention, the expression cassettes described herein may be contained in a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selection markers, a signal that allows the plasmid construct to exist as single-stranded DNA, at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

[0092] When referring to a nucleotide sequence, "isolated" means that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other nucleotide sequences, chromatin material, etc. Thus, an "isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition.

[0093] For purposes of describing the relative position of a nucleotide sequence in a particular nucleic acid molecule throughout this application, for example, when a particular nucleotide sequence is described as being located "upstream," "downstream," "3" or "5" relative to another sequence, it is understood that this is the position of the sequence in the "sense" or "coding" strand of the DNA molecule, as is known in the art.

[0094] In the context of nucleic acid sequences or amino acid sequences, the terms "sequence identity", "sequence identity percentage" or "identity percentage" refer to the residues in two sequences that are identical when aligned to obtain maximum correspondence. The length of sequence identity comparison can exceed the full length of the genome, expecting a gene coding sequence of at least about 500 to 5000 nucleotides or the full length of the fragment. However, it is also possible to expect the identity between smaller fragments, for example, at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. The identity percentage of a sequence can be determined by techniques known in the art. For example, homology can be determined by comparing sequence information and using readily available computer programs (such as ALIGN, ClustalW2 and BLAST) to directly compare the sequence information between two polypeptide molecules. In one embodiment, when BLAST is used as an alignment tool, the default parameters are as follows: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR.

[0095] The term "subject" refers to any member of the animal kingdom, including but not limited to humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, etc. In some embodiments, the subject is a human whose age ranges from birth to 2 years old, from 1 year old to 10 years old, or from 4 years old to 15 years old, or from 10 years old to 19 years old, or from 20 years old to 40 years old, or from 15 years old to 29 years old, or from 25 years old to 55 years old, or from 40 years old to 60 years old, or over 50 years old or over 60 years old, or over 65 years old or over 70 years old. For example, the subject is a human child (2 to 12 years old), a human adolescent (10 to 19 years old). In some embodiments, the subject is an adult (18 years old or older). In particular, the subject is a young adult (15 to 29 years old), a middle-aged person (25 to 55 years old) or an elderly person (over 50 years old) or an elderly subject (over 65 years old) or a geriatric subject (over 70 years old).

[0096] A "therapeutic effect" refers to any therapeutic benefit conferred by a treatment described herein. For example, such an effect may be sustained expression in an appropriate target tissue of a protein or enzyme that is deficient or absent in the muscular dystrophy of interest. In addition, a therapeutic effect may be any reduction or elimination of one or more clinical or subclinical manifestations of the disease or condition of interest. For example, a decrease in CK levels, a decrease in fibrosis, an increase in resistance to contraction-induced injury in the tibialis anterior muscle, and / or an increase in specific force in the muscle and improved motor function provide a therapeutic benefit to a subject receiving LGMD-2D treatment.

[0097] In another aspect, the recombinant AAV vectors described herein comprise a polynucleotide sequence encoding an α-sarcoglycan that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, and the protein retains α-sarcoglycan activity. In another embodiment, the α-sarcoglycan comprises the polypeptide sequence set forth in SEQ ID NO: 2.

[0098] In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding a functional α-sarcoglycan, the polynucleotide sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or its complement. In another embodiment, the rAAV comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4. In another embodiment, the rAAV comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

[0099] The term "stringent" is used to refer to conditions that are generally understood to be stringent in the art. The stringency of hybridization is primarily determined by the concentration of temperature, ionic strength, and denaturants such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, New York, 1989). More stringent conditions (e.g., higher temperatures, lower ionic strengths, higher formamides or other denaturants) may also be used, however, the hybridization rate will be affected. In the case of deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37°C (for 14 base oligomers), 48°C (for 17 base oligomers), 55°C (for 20 base oligomers), and 60°C (for 23 base oligomers).

[0100] When ranges are used herein for physical properties, such as molecular weight, concentration or dosage, it is intended to include all combinations and subcombinations of ranges and specific embodiments therein. When referring to a number or a numerical range, the term "about" means that the number or numerical range referred to is an approximation within experimental variability (or statistical experimental error), so the number or numerical range may vary from, for example, 1% to 15% of the number or numerical range.

[0101] To reduce nonspecific and / or background hybridization, other reagents may be included in the hybridization and wash buffers. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable reagents may be used. The concentration and type of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8-7.4, however, under typical ionic strength conditions, the rate of hybridization is almost independent of pH. See Anderson et al. Nucleic Acid Hybridization: A Review Nucleic Acid Hybridisation: A Practical Approach》 Chapter 4, IRL Press Limited (Oxford, UK). One skilled in the art can adjust hybridization conditions to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.

[0102] Limb-girdle muscular dystrophy type 2D (LGMD2D) is a progressive muscular dystrophy characterized by muscle weakness, respiratory abnormalities, and, in rare cases, cardiomyopathy. LGMD2D is caused by mutations in the alpha-sarcoglycan gene, resulting in loss of the protein and concomitant loss of sarcoglycan and the dystrophin-associated glycoprotein complex. - / - ) mice recapitulate the clinical phenotypes of patients with LGMD2D, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduced production of absolute muscle force and voluntary activity. - / - The mouse provides a relevant model to test the safety and efficacy of gene replacement. Therefore, the present disclosure provides a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter truncated muscle creatine kinase (tMCK). A dose-escalation design was used to test the efficacy of scAAVrh74.tMCK.hSGCA in sgca - / - The efficacy and safety of 1 × 10 12 , 3×10 12 and 6×10 12 A single systemic injection of vg. in sgca treated with scAAVrh74.tMCK.hSGCA - / - In mice, the protein expression of α-SG on the sarcolemma of skeletal muscle was stable at all tested doses. In addition, scAAVrh74.tMCK.hSGCA effectively improved sgca - / - Histopathology of limb and diaphragm muscles of mice, as indicated by reduced fibrosis and central nucleation and normalized myofiber size. These molecular changes were accompanied by a significant increase in specific force production of the diaphragm and tibialis anterior muscles, protection from centrifugal force loss, and a decrease in serum CK. - / - Locomotor activity was improved at all doses of vector-treated mice compared to controls. Finally, a lack of vector-related toxicity was detected in serum chemistry panels and gross necropsy. Overall, the studies provide support for systemic delivery of scAAVrh74.tMCK.hSGCA for the treatment of LGMD2D in a clinical setting.

[0103] In another aspect, the recombinant AAV vector described herein can be operably linked to a muscle-specific control element. For example, the muscle-specific promoter comprises one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal α-actin (ASKA) promoter, troponin I (TNNI2) promoter, myocyte-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch troponin C gene element, slow-twitch cardiac troponin C gene element, slow-twitch troponin I gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-inducible element, and glucocorticoid response element (GRE).

[0104] In one embodiment, the muscle-specific promoter is the tMCK promoter, which comprises the sequence of SEQ ID NO: 3. An exemplary rAAV described herein is AAVrh74.tMCK.hSGCA, which comprises the nucleotide sequence of SEQ ID NO:4. In some embodiments, the polynucleotide sequence encoding AAVrh74.tMCK.hSGCA comprises a sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:4, or a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:1.

[0105] In another embodiment, the polynucleotide sequence encoding AAVrh74.tMCK.hSGCA comprises a nucleotide sequence encoding a polynucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 1. In another embodiment, the polynucleotide sequence encodes a protein that retains alpha-sarcoglycan activity.

[0106] In one embodiment, the rAAV comprises the 5' inverted terminal repeat sequence of SEQ ID NO: 5. In another embodiment, the rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO: 6. In some embodiments, the rAAV comprises the poly A sequence of SEQ ID NO: 7.

[0107] AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74 or a variant or derivative thereof. In one embodiment, rAAV is serotype AAVrh.74. The production of pseudotyped rAAV is disclosed in, for example, WO 2001 / 083692, which is incorporated by reference in its entirety. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).

[0108] Also contemplated are compositions comprising any of the rAAV vectors described herein.

[0109] A method for treating muscular dystrophy in a subject in need thereof is provided, comprising the step of administering a recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV is administered systemically and at a concentration of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg. For example, in any of the provided methods, the dose of rAAV administered is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 In another embodiment, the dose is about 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14vg / kg. In one embodiment, the rAAV is administered by a systemic route, which includes an intravenous route. In another embodiment, the rAAV is administered at about 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg is administered intravenously. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy.

[0110] In addition, the dose of rAAV administered was approximately 1.5×10 13 vg is about 3.5×10 16 vg, or 3×10 13 vg to 1×10 16 vg, or about 1.5×10 13 vg is about 2×10 15 vg, or about 1.5×10 13 vg is about 1×10 15 vg. In addition, in any of the methods, the dose of rAAV is administered at a concentration of about 10 mL / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy. In one embodiment, the muscular dystrophy is 2D limb-girdle muscular dystrophy. The doses expressed in vg or vg / kg in the present disclosure are based on titration identification methods using quantitative PCR (qPCR). qPCR-based titration methods are known in the art.

[0111] In addition, a method of treating muscular dystrophy in a subject in need thereof is provided, comprising the step of administering a recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV is administered by a systemic route at a concentration of about 1.0×10 12 vg / kg to about 2.0×10 15vg / kg; wherein the α-sarcoglycan gene expression level in the subject's cells is increased after rAAV administration compared to the α-sarcoglycan gene expression level before rAAV administration; wherein the serum CK level in the subject is decreased after rAAV administration compared to the serum CK level before rAAV administration; and / or wherein voluntary activity and specific force production are increased; wherein fibrosis is reduced; wherein resistance to contraction-induced injury in the tibialis anterior muscle is increased; and / or wherein the number of α-sarcoglycan-positive fibers in the subject's muscle tissue is increased after rAAV administration compared to the number of α-sarcoglycan-positive fibers before rAAV administration; wherein the fiber diameter size in the subject's muscle tissue is increased after rAAV administration compared to the number of fiber diameters before rAAV administration; or wherein the central nucleation in the subject's muscle tissue is reduced after rAAV administration compared to the central nucleation before rAAV administration. Muscle tissues include, but are not limited to, triceps, tibialis anterior, soleus, gastrocnemius, biceps, trapezius, gluteal, psoas, deltoid, quadriceps, and diaphragm. In one embodiment, muscle tissue includes the tibialis anterior, gastrocnemius, gluteal, psoas and triceps. Expression of alpha-sarcoglycan is determined by methods known to those of ordinary skill in the art. In one embodiment, expression is determined by Western blot, immunochemistry in muscle biopsy, and / or by measuring the number of vector genomes per microgram of genomic DNA.

[0112] In some embodiments, the present disclosure includes a method of treating muscular dystrophy in a subject in need thereof, comprising the step of administering a recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the motor function of the human subject is significantly improved compared to the motor function of the human subject before administration of rAAV.

[0113] Provided is a method of increasing alpha-sarcoglycan in a patient in need thereof, comprising administering to the patient the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0114] In any of the methods, uses, and compositions provided for treating muscular dystrophy, the subject is 4-15 years old, has confirmed alpha-sarcoglycan (SGCA) mutations in both alleles, is negative for AAVrh74 antibodies and / or has a 100-meter walk test of >40% or normal. In any of the methods, uses, and compositions provided for treating muscular dystrophy, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject, such as a subject with an age range of 1 to 21 years old. In some embodiments, the subject is 1 to 10 years old, or 2 to 12 years old, 4 to 15 years old, or 10 to 19 years old. In one embodiment, the subject is an adolescent subject, such as a subject with an age range of 12 to 21 years old. In addition, in one embodiment, the subject is a young adult subject, such as a subject with an age range of 15 to 29 years old or 18 to 39 years old. In some embodiments, the subject is a middle-aged adult or an elderly subject, such that the age range of the middle-aged adult can be 25 to 55 years old, the age range of the elderly subject can be over 50 years old, and the age range of the elderly subject can be over 65 years old. In some embodiments, the rAAV is administered by injection, infusion, or implantation. For example, the rAAV is administered by infusion over about 1 to 2 hours. In addition, the rAAV is administered by an intravenous route through a peripheral limb vein.

[0115] In a method of treating muscular dystrophy in a human subject in need thereof, the method comprises administering a recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV is administered systemically and at a concentration of about 1.0×10 12 vg / kg to about 5.0×10 14vg / kg, and the rAAV comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In another embodiment, the rAAV comprises the nucleotide sequence shown in SEQ ID NO: 1. In one embodiment, the rAAV encodes a protein comprising a polypeptide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. In another embodiment, the rAAV comprises a nucleotide sequence encoding a protein comprising the polypeptide sequence shown in SEQ ID NO:2. In addition, any of the disclosed rAAVs comprises a promoter, such as the tMCK promoter sequence of SEQ ID NO:3. In some embodiments, the rAAV is serotype AAVrh.74. In addition, the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:3. In one embodiment, the rAAV comprises the 5' inverted terminal repeat sequence of SEQ ID NO:5. In another embodiment, the rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO: 6. In another embodiment, the rAAV comprises the poly A sequence of SEQ ID NO:7.

[0116] AAV dosage can be determined by a variety of methods, including but not limited to LISA, reverse transcriptase activity assessment, FACS, transduction assay northern blot (e.g., semi-quantitative northern), dot blot analysis or PCR (e.g., qPCR). It is well known that AAV dosage can be determined by measuring the AAV vector genome using quantitative real-time PCR (qPCR). This qPCR method overcomes the inconsistent or arbitrary results of traditional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. The form of the plasmid may affect the dosage results of the qPCR method. In one embodiment, circular or supercoiled DNA or plasmid is used as a quantitative standard. In another embodiment, linearized DNA or plasmid is used as a quantitative standard.

[0117] The term "supercoiled DNA" or "supercoiled plasmid" refers to a DNA or plasmid that does not contain free ends. The term "linearized DNA" or "linearized plasmid" refers to a DNA or plasmid that contains a free 5' end and a free 3' end that are not connected to each other. In one embodiment, the linearized DNA or plasmid is obtained by restriction digestion of circular DNA (e.g., plasmid DNA) or by restriction digestion of dbDNA. In another embodiment, the restriction digestion is performed using an enzyme that produces at least one blunt end.

[0118] In an exemplary embodiment, a method for treating muscular dystrophy in a subject in need thereof is provided, comprising the step of administering a recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK7.hSGCA, wherein the rAAV is administered systemically and at a concentration of about 1.0×10 12 vg / kg to about 5.0×10 14 vg / kg. In one embodiment, rAAV is administered by intravenous infusion over about 1 to 2 hours at a dose of about 5.0×10 based on supercoiled DNA or plasmid as a quantitative standard. 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg, wherein the rAAV comprises the scAAVrh74.tMCK7.hSGCA construct nucleotide sequence of SEQ ID NO: 3. In another embodiment, the dose is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0119] The present disclosure also provides a method of increasing sarcoglycan expression in muscle tissue of a subject, comprising administering to the subject a scAAVrh74.tMCK.hSGCA construct comprising a nucleotide sequence at least 90% identical, at least 95% identical, or 99% identical to SEQ ID NO: 1 and / or 4.

[0120] The present disclosure further provides a method of improving muscle function in a subject, comprising administering to the subject a construct comprising a nucleotide sequence at least 90% identical, at least 95% identical, or 99% identical to SEQ ID NO: 1 and / or 4.

[0121] In some aspects, the subject has a genetic mutation in a gene encoding sarcoglycan protein or muscular dystrophy. In some aspects, the subject has a genetic mutation in a gene encoding alpha-sarcoglycan protein.

[0122] In any of the provided methods, the α-sarcoglycan gene expression level in cells of the subject is increased after administration of the scAAVrh74.tMCK7.hSGCA construct compared to the α-sarcoglycan gene expression level before administration of the scAAVrh74.tMCK.hSGCA construct.

[0123] Additionally, in any of the provided methods, expression of the α-sarcoglycan gene in cells is detected by measuring α-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0124] In any of the provided methods, the level of alpha-sarcoglycan protein is increased after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of alpha-sarcoglycan protein is increased by at least 33%, as detected by measuring the level of alpha-sarcoglycan protein by Western blot in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct, or by measuring the level of alpha-sarcoglycan protein by immunohistochemistry in muscle biopsies and / or by detecting the number of vector genomes per microgram of genomic DNA before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0125] In any of the provided methods, the subject's serum CK level is reduced after administration of the scAAVrh74.tMCK.hSGCA construct compared to the serum CK level before administration of the scAAVrh74.tMCK.hSGCA construct.

[0126] In any of the provided methods, the number of α-sarcoglycan-positive fibers in the subject's muscle tissue is increased after administration of the scAAVrh74.tMCK.hSGCA construct, compared to the number of α-sarcoglycan-positive fibers before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of α-sarcoglycan-positive fibers is detected by measuring α-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. For example, after administration of the scAAVrh74.tMCK.hSGCA construct, the number of α-sarcoglycan-positive fibers is increased in the subject's muscle tissue.

[0127] In any of the provided methods, the level of α-sarcoglycan in the subject is increased after administration of rAAV compared to the level of α-sarcoglycan before administration of the scAAVrh74.tMCK.hSGCA construct. The level of α-sarcoglycan is detected by measuring α-sarcoglycan protein levels by immunohistochemistry or western blot on muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0128] Another embodiment provides a method of expressing an α-sarcoglycan gene in a patient's cells, comprising administering to the patient the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. In any of the provided methods for expressing an α-sarcoglycan gene in a patient's cells, the expression of the α-sarcoglycan gene in the patient's cells is detected by measuring the α-sarcoglycan protein level by Western blot or immunohistochemistry in a muscle biopsy before and after administration of the scAAVrh74.tMCK.hSGCA construct. In one embodiment, the α-sarcoglycan gene is measured in the patient by detecting greater than one rAAV vector genome copy per nucleus. In another embodiment, the expression of the α-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0129] Also provided is a method of reducing serum CK levels in a patient in need thereof, the method comprising administering to the subject the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0130] Methods of increasing α-sarcoglycan-positive fibers in muscle tissue of a patient are provided, comprising administering to the subject the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. In any of these methods, the number of α-sarcoglycan-positive fibers is detected by measuring α-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0131] Another embodiment provides a method of increasing the expression of alpha-sarcoglycan in a subject in need thereof, comprising administering to the subject the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. In any of these methods, the level of alpha-sarcoglycan is detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0132] Also provided is a method for producing recombinant AAV vector particles, which includes culturing host cells transferred with any recombinant AAV vector described herein and recovering recombinant AAV particles from the supernatant of transfected cells. It is also contemplated that any of the recombinant AAV vectors described herein are viral particles. In one embodiment, the method for producing rAAV includes transferring the AAV vector plasmid to the host cell. In another embodiment, the recombinant AAV vector particles and / or AAV vector plasmids include at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88% or about 89% with SEQ ID NO: 8, more generally about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more identical nucleotide sequences. On the other hand, the present disclosure provides a cell comprising an AAV vector plasmid, the AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in a host cell. Host cells stably carrying the AAV vector plasmid can be used to produce rAAV. In one embodiment, the AAV vector plasmid is a pAAV.tMCK.hSGCA.KAN plasmid (SEQ ID NO: 8).

[0133] Also provided is a method for reducing fibrosis in a mammalian subject in need. In this regard, the method comprises administering a therapeutically effective amount of an AAV vector as described herein (or a composition comprising an AAV vector as described herein) to a mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In one embodiment, muscular dystrophy is LGMD2D. In some embodiments, administering an AAV vector as described herein (or a composition comprising an AAV vector as described herein) reduces fibrosis in the muscle tissue of the subject. In one embodiment, the muscle tissue includes the psoas major, diaphragm, triceps and / or gluteal muscles.

[0134] As used herein, the term "muscular dystrophy" refers to a condition in which strength and muscle mass gradually decrease. Non-limiting examples of muscular dystrophy diseases can include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanosis, congenital muscular dystrophy (e.g., congenital muscular dystrophy due to partial LAMA2 deficiency), partition protein-deficient congenital muscular dystrophy, ID congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, and congenital muscular dystrophy. 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, limb-girdle 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 2I muscular dystrophy, limb-girdle type 2J muscular dystrophy, limb-girdle type 2K muscular dystrophy, limb-girdle type IC muscular dystrophy, myospinal muscular dystrophy with epidermolysis bullosa simplex, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy, and Ullrich scleroatonic muscular dystrophy. In some embodiments, the subject has limb-girdle muscular dystrophy. In some embodiments, the subject has limb-girdle muscular dystrophy type 2D (LGMD2D).

[0135] As used herein, the term "fibrosis" refers to the excessive or unregulated deposition and abnormal repair process of extracellular matrix (ECM) components in tissues (including skeletal muscle, myocardium, liver, lung, kidney and pancreas) after injury. The deposited ECM components include collagen, such as collagen 1, collagen 2 or collagen 3, and fibronectin.

[0136] In another aspect, described herein is a method of increasing α-sarcoglycan positive fibers, fiber diameter size, muscle eccentric contraction, muscle strength, and / or expression of α-sarcoglycan in muscle tissue of a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein). Also described herein is a method of reducing fibrosis, central nucleation, CK levels, and / or collagen deposition in a subject, comprising administering to the subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein).

[0137] In any of the methods of the invention, the subject may be suffering from a muscular dystrophy, such as limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy. In one embodiment, the muscular dystrophy is LGMD-2D.

[0138] Also provided is a method of treating muscular dystrophy in a mammalian subject, comprising administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein). In some embodiments, the muscular dystrophy is limb-girdle muscular dystrophy.

[0139] In any of the methods of the invention, the rAAV is administered by intramuscular injection or intravenous injection. In addition, in any of the methods of the invention, the rAAV is administered systemically, such as parenterally by injection, infusion or implantation.

[0140] The compositions of the present invention are formulated for intramuscular or intravenous injection. Additionally, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.

[0141] In addition, any of the compositions are formulated for administration to a subject with a muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy). In some embodiments, the composition may further comprise a second recombinant AAV vector expressing α-sarcoglycan or a second recombinant AAV vector comprising the polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4.

[0142] In any of the uses of the present invention, the drug is formulated for intramuscular injection or intravenous injection. In addition, in any of the uses of the present invention, the drug is formulated for systemic administration, such as parenteral administration by injection, infusion or implantation. In addition, any of the drugs can be prepared for administration to a subject suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy). In some embodiments, the drug can also express a second recombinant AAV vector of alpha-sarcoglycan or a second recombinant AAV vector comprising a polynucleotide sequence in SEQ ID NO:1 or SEQ ID NO:4.

[0143] The foregoing paragraphs are not intended to define every aspect of the present invention, and additional aspects are described in other parts, such as the specific embodiments. 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 considered even if the combination of features is not found together in the same sentence or paragraph or part of this document. As additional aspects, the present invention includes all embodiments in which the scope of the present invention is narrower in any respect than the variations defined by the above-mentioned specific paragraphs. For example, where certain aspects of the present invention are described as categories, it should be understood that each member of the category is individually an aspect of the present invention.

[0144] AAV

[0145] The recombinant AAV genome of the present invention comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The production of pseudotyped rAAVs is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated.

[0146] The DNA plasmid of the present invention comprises the rAAV genome. The DNA plasmid is transferred into a cell that allows infection with a helper virus of AAV (e.g., adenovirus, adenovirus or herpes virus deleted by E1) to assemble the rAAV genome into infectious viral particles. The technology for producing rAAV particles is standard in the art, wherein the AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells. The production of rAAV requires the following components to be present in a single cell (represented herein as packaging cells): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not therein), and the helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITRs, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh10, and AAVrh. 74. The generation of pseudotyped rAAV is disclosed, for example, in WO 2001 / 083692, which is incorporated herein by reference in its entirety.

[0147] The method of producing packaging cells is to create a cell line that stably expresses all the necessary components for the production of AAV particles. For example, a plasmid (or plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and a selectable marker (e.g., a neomycin resistance gene) are integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. This approach has the advantage that the cells are selectable and is suitable for large-scale production of rAAV.Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0148] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al., (1989, Journal of Virology, 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777) WO 97 / 06243 (PCT / FR96 / 01064); WO 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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the document relating to rAAV production.

[0149] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (homologous 293 lines). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus embryonic lung cells).

[0150] The recombinant AAV of the present invention (ie, the infectious encapsidated rAAV particle) comprises the rAAV genome. Embodiments include, but are not limited to, the rAAV named pAAV.tMCK.hSGCA, which comprises the polynucleotide sequence shown in SEQ ID NO:3.

[0151] rAAV can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and are included in, for example, Clark et al., Human Gene Therapy, 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0152] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition described herein comprises rAAV in a pharmaceutically acceptable carrier. The composition may also include other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to the receptor and are preferably inert at the dose and concentration employed, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronic, or polyethylene glycol (PEG).

[0153] The titer of the rAAV to be administered in the methods of the present invention will vary depending on, for example, the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods standard in the art. The titer of the rAAV can range from about 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 About 1×10 14 Or more DNase-resistant particles (DRP). Dosage can also be expressed in units of viral genomes (vg) as measured by qPCR.

[0154] The present invention contemplates methods for transducing target cells with rAAV in vivo or in vitro. The in vivo method comprises the step of administering an effective dose or effective multiple doses of a composition comprising the rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the development of the condition / disease, the administration is preventive. If the dose is administered after the development of the condition / disease, the administration is therapeutic. In an embodiment of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the condition / disease state being treated, slows or prevents progression to the condition / disease state, slows or prevents the progression of the condition / disease state, reduces the extent of the disease, causes remission of the disease (partial or complete) and / or prolongs survival. An example of a disease considered to be prevented or treated with the methods of the present invention is muscular dystrophy, such as limb-girdle muscular dystrophy. Therefore, a method for transducing target cells with rAAV scAAVrh74.tMCK.hSGCA is provided, wherein the rAAVscAAVrh74.tMCK.hSGCA comprises the nucleotide sequence of SEQ ID NO:4.

[0155] In another embodiment, the present disclosure provides a method for producing rAAV scAAVrh74.tMCK.hSGCA, which includes transferring an AAV vector plasmid to a host cell. The method of transferring DNA to a host cell is well known in the art, including but not limited to transfection, infection, transformation, electroporation and transduction. In one embodiment, the vector plasmid comprises at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88% or about 89% with SEQ ID NO: 8, more usually about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more identical nucleotide sequence. In another embodiment, the vector plasmid comprises a nucleotide sequence at least 90%, 95% or 99% identical to SEQ ID NO: 8. In another embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. On the other hand, the present disclosure provides a cell comprising an AAV vector plasmid, wherein the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in a host cell. Host cells stably carrying the AAV vector plasmid can be used to produce rAAV. In one embodiment, the AAV vector plasmid is a pAAV.tMCK.hSGCA.KAN plasmid.

[0156] In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more usually about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO: 1, 4, or 8.

[0157] In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least about 90%, 95% or 99% identical to SEQ ID NO: 1, 4 or 8. In one embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4 or 8. In one embodiment, the method of producing rAAV further comprises transferring the packaging plasmid and / or helper virus to a host cell. In some embodiments, the packaging plasmid comprises an AAV rep and / or cap gene operably linked to a promoter. In one embodiment, the promoter is an AAV transcriptional promoter. In one embodiment, the host cell is a packaging cell. In one embodiment, the packaging cell comprises a stably integrated AAV cap gene. In another embodiment, the packaging cell comprises a stably integrated AAV rep gene.

[0158] As used herein, the term "host cell" refers to a cell that can be used to express an exogenous DNA sequence. Non-limiting examples of host cells include microorganisms, yeast cells, insect cells and / or mammalian cells. Host cells can be used as receptors for AAV auxiliary constructs, packaging plasmids, AAV vector plasmids, auxiliary function vectors or other DNA. As used herein, the term includes the offspring of the original cell after the exogenous DNA sequence is expressed in the original host cell. Non-limiting examples of host cells for AAV production include Sf9 insect cells and HEK 293T cells. AAV vector plasmids can be introduced into host cells, such as Sf9 or 293T, by infection (virus or baculovirus), transient transfection using reagents (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation) or other means known in the art. In another embodiment, the host cell line is stably integrated into its genome with the rAAV plasmid. Such stable cell lines can be established by incorporating a selection marker into a vector plasmid.

[0159] In one embodiment, the host cell is a packaging cell for producing AAV viral particles. Therefore, in another aspect, the present disclosure provides a host cell comprising an AAV vector plasmid, the AAV vector plasmid comprising a nucleotide sequence 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, 4 or 8.

[0160] The present invention also contemplates combination therapy. Combinations as used herein include both simultaneous treatment and sequential treatment. It is particularly contemplated that the method of the present invention is combined with standard medical treatment (e.g., steroids, corticosteroids and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone and deflazacort), as a combination with a new therapy. In this regard, the combination is included in the application of one or more steroids, corticosteroids and / or glucocorticoids to the subject before the rAAV of the method of the present invention is applied to the subject, while the rAAV is applied to the subject, or after the rAAV is applied to the subject, one or more steroids, corticosteroids and / or glucocorticoids are applied to the subject, including but not limited to one or more of prednisone, prednisolone and deflazacort.

[0161] In related embodiments of the combination therapies contemplated by the invention, glucocorticoids include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0162] It is recognized that antigen-specific T cell responses may occur in subjects administered with rAAV vectors. This is an expected response between 2 and 4 weeks after gene transfer. One possible consequence of such antigen-specific T cell responses is the clearance of transduced cells and the loss of transgene expression. In order to suppress the host's immune response to rAAV-based treatments, subjects can start oral prophylactic prednisone or similar glucocorticoids at approximately 1 mg / kg / day before treatment, for example, twenty-four hours before the treatment procedure, with a maximum dose of 60 mg / day. If necessary, IV administration of similar glucocorticoids at a dose of approximately 1 mg / kg / day is also permitted. Treatment will last for approximately one month. A gradual reduction regimen of prednisone or similar glucocorticoids can be implemented based on the individual subject's immune response to gene transfer, assessed by ELISpot assays, and by monitoring liver function with GGT.

[0163] The therapeutically effective amount of rAAV vector is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg of rAAV. In another embodiment, the dose is about 5.0×10 13 vg / kg, about 1.0×10 14 vg / kg or about 2.0×10 14 In another embodiment, the dose is 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14The present invention also contemplates compositions comprising rAAV vectors within these ranges.

[0164] The dose can also be expressed in units of viral genomes (vg). The titer of rAAV can be determined by supercoiled DNA or plasmid quantitative standards or linearized DNA or plasmid quantitative standards. The titer or dose of the AAV vector can vary based on the physical form of the plasmid DNA used as the quantitative standard. For example, the value of the titer or dose may vary due to the supercoiled standard qPCR titration method or the linear standard qPCR titration method. In one embodiment, the dose in the present disclosure is based on supercoiled DNA or plasmid as a quantitative standard. In another embodiment, the dose in the present disclosure is based on linearized DNA or plasmid as a quantitative standard. Therefore, in one embodiment, based on supercoiled DNA or plasmid as a quantitative standard, the therapeutically effective amount of the rAAV vector is in the range of about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 In another embodiment, the dose is about 5.0×10 13 vg / kg, about 1.0×10 14 vg / kg or about 2.0×10 14 In another embodiment, the dose is 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg.

[0165] In another embodiment, the therapeutically effective amount of the rAAV vector is in the range of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×1013 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg of rAAV dose. For example, based on linearized DNA or plasmid as a quantitative standard, the therapeutically effective amount of rAAV vector is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg dose.

[0166] In one embodiment, a dose of 5.0×1013 vg / kg based on supercoiled DNA or plasmid as a quantitative standard is equivalent to 1.85×1013 vg / kg based on linearized DNA or plasmid as a quantitative standard. 13 In another embodiment, based on supercoiled DNA or plasmid 2.0×10 14 The dose of vg / kg is equivalent to 7.41×10 based on the linearized DNA or plasmid used as the quantitative standard. 13 Thus, in another embodiment, based on linearized DNA or plasmid as a quantitative standard, about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0167] Administration of an effective dose of the composition can be by routes standard in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal. One skilled in the art can select and / or match one or more routes of administration and one or more serotypes of the AAV components (particularly AAV ITRs and capsid proteins) of the rAAV of the present invention, taking into account the infection and / or disease state being treated and the one or more target cells / tissues in which α-sarcoglycan is to be expressed.

[0168] The present invention provides local and systemic administration of effective doses of rAAV and compositions of the present invention. For example, systemic administration is administration to the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the intestine and parenteral administration by injection, infusion or implantation.

[0169] In particular, the actual administration of the rAAV of the present invention can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of the animal. According to the present invention, administration includes, but is not limited to, injection into muscle, bloodstream and / or direct injection into the liver. Simply resuspending the rAAV in a phosphate buffered solution has been shown to be sufficient to provide a vehicle for muscle tissue expression, and there are no known restrictions on vectors or other components that can be co-administered with rAAV (although compositions that degrade DNA should generally be avoided for use with rAAV). The capsid protein of rAAV can be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference.

[0170] The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation that can be delivered to the muscle via transdermal transport. Many formulations for both intramuscular injection and transdermal delivery 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 aspect, the present application is directed to a formulation comprising rAAV containing an AAVrh74-derived capsid, a buffer, an ionic strength agent, and a surfactant. In one embodiment, the concentration of rAAV is about 1.0×10 12 vg / kg to about 5.0×10 14 In one embodiment, the concentration of rAAV is about 5.0×10 12 vg / kg to about 1.0×10 14 In another embodiment, the concentration of rAAV is about 5×10 13 vg / kg, about 1×10 14 vg / kg and / or about 2×10 14vg / kg. In one embodiment, the dosage is based on supercoiled DNA or plasmid as a quantitative standard. In one embodiment, rAAV is a scAAVrh74.tMCK.hSGCA vector. In one embodiment, the buffer comprises one or more of tris, tricine, Bis-tricine, HEPES, MOPS, TES, TAPS, PIPES and CAPS. In another embodiment, the buffer comprises tris with a pH of about 8.0 at a concentration of about 5mM to about 40mM. In one embodiment, the buffer comprises tris with a pH of about 8.0 of about 20mM. In one embodiment, the ionic strength agent comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl) and lithium acetate. In one embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 0.2mM to about 4mM. In another embodiment, the ionic strength agent comprises NaCl at a concentration of about 50mM to about 500mM. In another embodiment, the ionic strength agent comprises MgCl at a concentration of about 0.2mM to about 4mM and NaCl at a concentration of about 50mM to about 500mM. In another embodiment, the ionic strength agent comprises MgCl at a concentration of about 1mM and NaCl at a concentration of about 200mM. In one embodiment, the surfactant comprises one or more of sulfonate, sulfate, phosphonate, phosphate, poloxamer and cationic surfactant. In one embodiment, poloxamer includes one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338 and poloxamer 407. In one embodiment, the surfactant comprises a poloxamer at a concentration of about 0.00001% to about 1%. In another embodiment, the surfactant comprises poloxamer 188 at a concentration of about 0.001%. For the purpose of intramuscular injection, adjuvants such as sesame oil or peanut oil or aqueous propylene glycol solutions, as well as sterile aqueous solutions can be used. If desired, such aqueous solutions can be buffered, and the liquid diluent is first made isotonic with saline or glucose. As a free acid rAAV solution (DNA contains an acidic phosphate group) or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous medium used can be easily obtained by standard techniques well known to those skilled in the art.

[0171] The pharmaceutical form suitable for injectable use includes sterile aqueous solution or dispersion and sterile powder for temporary preparation of sterile injectable solution or dispersion. In all cases, the form must be sterile and must be fluid, so that there is a degree of ease of injection. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), its suitable mixture and vegetable oil. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant, appropriate fluidity can be maintained. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferred to include isotonic agents, such as sugar or sodium chloride. By using agents that delay absorption, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.

[0172] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in an appropriate solvent with the various other ingredients listed above, as required, and then filtered sterile. Dispersions are generally prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0173] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, isogenic or allogenic muscle cells can be used, where these cells will not produce an inappropriate immune response in the subject.

[0174] Suitable methods for transducing and reintroducing transduced cells into subjects are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells (e.g., in an appropriate culture medium), and those cells with the DNA of interest can be screened using conventional techniques (e.g., Southern blots and / or PCR), or by using selection markers. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by various techniques, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by using, for example, a catheter to inject into smooth muscle and myocardium.

[0175] Transduction of cells with the rAAV of the present invention results in sustained expression of α-sarcoglycan. Therefore, the present invention provides methods of administering / delivering rAAV expressing α-sarcoglycan to mammalian subjects (preferably humans). These methods include transducing tissues (including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction can be performed with a gene cassette containing tissue-specific control elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissue directed by muscle-specific control elements, including, but not limited to, control elements derived from the actin and myosin gene families, such as control elements derived from 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)]; derived from the human skeletal actin gene [Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)]; cardiac actin control elements of genes; muscle creatine kinase sequence elements [see Johnson et al., Mol. Cell. Biol., 9:3393-3399 (1989)] and murine creatine kinase enhancer (mCK) elements; control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-induced nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)); steroid-inducible elements and promoters including the glucocorticoid response element (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)); and other control elements.

[0176] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention contemplates sustained expression of miRNA from transduced myofibers.

[0177] "Muscle cell" or "muscle tissue" refers to a cell or cell population derived from any type of muscle (e.g., skeletal muscle and smooth muscle, such as from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyocytes.

[0178] The term "transduction" is used to refer to the in vivo or in vitro administration / delivery of a polynucleotide of interest (eg, a polynucleotide sequence encoding α-sarcoglycan) to a recipient cell via the replication-defective rAAV, thereby resulting in the recipient cell expressing α-sarcoglycan.

[0179] Thus, also described herein are methods of administering to a mammalian subject in need thereof an effective dose (or multiple doses administered substantially simultaneously or multiple doses administered at intervals) of a rAAV encoding α-sarcoglycan.

[0180] All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was expressly and individually indicated to be incorporated by reference. In the event of a conflict, the present application (including any definitions herein) controls. The numerical ranges described include each integer value within each range, and include the lowest and highest stated integers.

[0181] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0182] Example

[0183] Preclinical studies using AAVrh74.tMCK.hSGCA are described in International Patent Publication No. WO 2013 / 078316 and U.S. Pat. Nos. 9,434,928 and 10,105,453, which are incorporated herein by reference in their entireties.

[0184] Example 1

[0185] Materials and Methods

[0186] Animal models

[0187] All procedures were approved by the Institutional Animal Care and Use Committee of Nationwide Children's Hospital. - / - ) mice were housed and maintained as homozygous animals. The mice were maintained on a 12:12 hour dark:light cycle in the Teklad Global Rodent Diet (3.8% fiber, 18.8% protein, 5% fat diet). All animals were housed in standard mouse cages with free access to food and water.

[0188] Genotyping

[0189] DNA genotyping for identification of sgca - / -Mouse. DNA from the tail was isolated and analyzed by polymerase chain reaction (PCR) using OneTaq DNA polymerase (New England Biolabs, Ipswich, MA). A series of primers were used in the PCR analysis to determine the α-SG gene knockout status. The following primers and conditions were used: intron 1 (CAGGGCTGGGAGCTGGGTTCTG; SEQ ID NO: 9); mutant primer-intron 3 (CCCAGGGCCTTGATGCCT; SEQ ID NO: 10); and NEOTR (GCTATCAGGACATAGCGTTGGCTA; SEQ ID NO: 11). Genomic DNA was reacted for 30 cycles under the following conditions: 94°C, 5 min; 94°C, 1 min; 64°C, 1 min; 72°C, 2.5 min; and 72°C, 7 min.

[0190] α-SG gene construction

[0191] The adeno-associated virus (AAV) vector DNA plasmid pAAV.tMCK.aSG-neo was used to insert the tMCK expression cassette driving the codon-optimized human α-SG cDNA sequence (human cDNA, GenBank Accession No. U08895) into the self-complementary vector backbone pHpa7 to make the scAAVrh74.tMCK.hSGCA transgenic cassette. The only viral sequence included in the vector is the inverted terminal repeat sequence of AAV2, which is required for viral DNA replication and packaging of the rAAV vector genome. One of the inverted terminal repeat sequences (ITR) has a targeted lack of a terminal resolution site (TRS) to limit R replication from this IT, thereby promoting the production of a dimeric replication form for self-complementary vector packaging. The AAVrh74 virus has been shown to be safe and efficient in transducing muscle across the vascular barrier in mice, non-human primates (NHPs), and humans.

[0192] Vector production

[0193] Recombinant AAV, (sc)rAAVrh74.tMCK.hSGCA, was prepared by triple transfection. Encapsulated vg titers were determined using a qPCR-based titration method using the Prism 7500 Fast Taqman detector system (PE Applied Biosystems). The construct contains a chimeric intron that promotes high-level expression. The chimeric intron consists of the 5' donor site of the first intron of the human β-globin gene and the branch point and 3' splice acceptor site of the intron between the leader sequence and the body of the heavy chain variable region of the immunoglobulin gene. rAAV also contains a synthetic SV40 polyadenylation signal for efficient transcription termination. A schematic diagram of the expression cassette is shown below Figure 1 The vector was generated using the human α-sarcoglycan (α-SG) gene flanked by AAV2 ITR sequences and encapsulated in AAVrh74 virions. The construct contains the tMCK immediate early promoter / enhancer (GenBank Accession No. M21390) and uses the β-globin intron for high-level expression.

[0194] Gene delivery

[0195] Systemic delivery in mice was achieved by injecting the vector into the tail vein of sgca- / - mice. Mice were injected with 1×10 12 vg, 3×10 12 vg or 6×10 12 Total dose of vg (range for mice: 13-20 g; 5×10 13 vg / kg, 1×10 14 vg / kg and 2×10 14 vg / kg - Dosage is based on scAAVrh74.tMCK.hSGCA diluted in 200 to 250 μL volume of lactated Ringer's solution (based on 20-g mice, respectively) as a quantitative standard. All treated mice were injected at 4 to 5 weeks of age and euthanized 12 weeks after injection. In another embodiment, the dose is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0196] Serum creatine kinase measurement

[0197] In wild-type C57BL / 6 mice (n=6), vehicle (lactated Ringer's solution)-treated sgca - / - Mice (n=6) and scAAVrh74.tMCK.hSGCA treated with sgca - / -The levels of creatine kinase were measured in the serum of mice (n=6 / dose) using the creatine kinase SL assay and the corresponding manufacturer's protocol (Sekisui Diagnostics; Charlottetown, Ontario, Canada) (Catalog No. 326-10). Briefly, 25 μL of serum was mixed with 1 mL of working reagent and added to a cuvette. The kinetic assay was set up on a spectrophotometer and the absorbance at 340 nm was measured every 30 seconds for 180 seconds. The creatine kinase level was calculated using the absorbance readings and the following formula:

[0198] U / L=[ΔAbs. / min)*1.025*1000] / [1*6.22*0.025]=(ΔAbs. / min)*6592.

[0199] Tetanic contraction of the diaphragm for functional assessment

[0200] The mice were euthanized, the diaphragm (DIA) was dissected, and the rib attachments and central tendon were kept intact and placed in KH buffer. 2-4mm wide DIA slices were isolated. The DIA strips were firmly tied with braided surgical silk (6 / 0; Surgical Specialties, Reading, Pennsylvania) at the central tendon, and sutured by a portion of the ribs fixed at the far end of the strips. Each muscle was transferred to a water bath filled with oxygenated KH solution, and the water bath temperature was kept at 37 ° C. The muscles were arranged horizontally and directly tied between fixed pins and dual-mode force sensors-servomotors (305C; Aurora Scientific, Aurora, Ontario, Canada). Two platinum plate electrodes were placed in the organ bath to flank the side of the muscle length. The muscle was stretched to the optimal length to measure the twitch contraction, and then allowed to rest for 10 minutes before starting the tonic protocol. Once the muscle was stable, it was set to an optimal length of 1 g and subjected to a warm-up consisting of 3 1-Hz twitches every 30 seconds, followed by 3 150-Hz twitches per minute. After a 3-min rest, the DIA was stimulated at 20, 50, 80, 120, 150, 180 Hz with a 2-min rest period between each stimulus and a duration of 250 ms to determine the maximum tetanic force. Muscle length and weight were measured. Force was normalized for muscle weight and length.

[0201] Tetanic contraction of the tibialis anterior (TA) muscle for functional assessment

[0202] The TA assessment procedure followed the protocol listed in Hakim et al., Methods Mol Biol; 709:75–89 (2011). Mice were anesthetized intraperitoneally using a ketamine / xylazine mixture (100 mg / kg and 10 mg / kg, respectively). Under a dissecting microscope, the skin of the hind limb was removed to expose the TA muscle and patella. A double square knot was tied around the patellar tendon with a 4-0 suture. The distal TA tendon was then dissected out and a double square knot was tied around the tendon with a 4-0 suture as close to the muscle as possible, and then the tendon was cut. The exposed muscle was continuously moistened with saline. The mice were then transferred to a thermally controlled platform and maintained at 37°C. The knee was fixed to a metal pin with a patellar tendon suture, and the distal TA tendon was sutured to the horizontal arm of a force transducer (Aurora Scientific, Aurora, Canada). An electrode was placed near the sciatic nerve to stimulate it. Once the muscle was stable, the resting tension was set to the length at which the twitch contraction was the largest (optimal length). After a 3-min rest, the TA was stimulated at frequencies of 50, 100, 150, and 200 Hz, with a 1-min rest between each stimulation. After a 5-min rest, the muscle was subjected to a series of 10 isometric contractions with a 10% stretch-relengthening procedure at 1-min intervals. After the eccentric contractions, the mice were then euthanized, and the TA muscles were dissected, weighed, and frozen for histology.

[0203] Immunofluorescence

[0204] Immunofluorescence staining for hSGCA transgene was performed on frozen sections (12-μm thick) from TA, gastrocnemius (GAS), quadriceps (QD), psoas (PSOAS), gluteal (GLUT), triceps (TRI), DIA, and heart (HRT) muscles. The sections were incubated with rabbit monoclonal α-SG primary antibody (Abcam; Cambridge, UK; catalog number ab189254) at a dilution of 1:100. Four random 20x images covering four different quadrants of the muscle section were taken using a Zeiss (Germany) AxioCam MRC5 camera. The percentage of fibers positive for α-SG staining compared to the control was determined for each image, and the average was taken for each muscle. Positive α-SG fiber expression was defined as having at least 30% fiber staining brighter than the vehicle-treated sgca- / - control.

[0205] Western blot analysis

[0206] Samples from wild-type C57BL / 6 mice, vehicle-treated sgca- / - mice, and vehicle-administered sgca- / - mice were used for each western blot. Rabbit monoclonal α-SG antibody (Abcam, catalog number ab189254) diluted 1:10,000 and mouse monoclonal α-actin antibody (Sigma-Aldrich, catalog number A7811) diluted 1:5,000 were used for hSGCA blots. Rabbit monoclonal mouse vinculin antibody (Invitrogen, catalog number 70062) diluted 1:1,000 was also used. Anti-mouse (Millipore, catalog number AP308P) and anti-rabbit (Life Technologies, catalog number 656120) secondary horseradish peroxidase antibodies were used for enhanced chemiluminescent immunodetection. Western blot quantification was performed by densitometry using ImageQuantTL 1D 8.1.0 (GE Healthcare Life Sciences).

[0207] Morphological analysis

[0208] 12-μm thick frozen sections of muscle from 16- to 17-week-old wild-type C57BL / 6 mice (n=6), vehicle-treated sgca- / - mice (n=6), and scAAVrh74.tMCK.hSGCA 16- to 17-week-old treated sgca- / - mice (n=6 / dose) were stained with hematoxylin and eosin (H&E) for analysis. The percentage of myofibers with central nuclei was determined in TA, GAS, QD, GLUT, PSOAS, and TRI. In addition, myofiber diameters were measured in TA, GAS, QD, TRI, and PSOAS muscles. Four random 20x images of each muscle per animal were taken using a Zeiss AxioCam MRC5 camera. Centrally nucleated fibers were quantified using Health's ImageJ software from the National Institutes of Health, and fiber diameters were measured using Zeiss AxiovisionLE4 software.

[0209] Biodistribution quantitative polymerase chain reaction (PCR) analysis

[0210] As previously described, Taqman quantitative PCR was performed to quantify the vector genome copy number present in the targeted and non-targeted contralateral muscles and non-targeted organs. The vector-specific primer probe set was used to amplify sequences in the intronic region directly downstream of the tMCK promoter, which is unique and located within the scAAVrh.74.tMCK.hSGCA transgenic cassette. The following primers and probes were used in this study: tMCK intron forward primer 5′-ACC CGA GAT GCC TGG TTATAA TT-3′; tMCK intron reverse primer 5′-TCC ATG GTG TAC AGA GCC TAA GAC-3′; and tMCK intron probe 5′-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C-IABkFQ-3′ (Integrated DNA Technologies). Copy numbers are reported as vector genomes per microgram of genomic DNA.

[0211] Sirius red staining and collagen quantification

[0212] Sirius red staining was performed to determine the level of collagen deposition in muscle tissue. 12-μm cryosections of GLUT, PSOAS, TRI and DIA muscles from 16-17 week old wild-type C57BL / 6 (n=6), vehicle-treated sgca- / - (n=6) and scAAVrh74.tMCK.hSGCA 16-17 week old treated sgca- / - (n=6 / dose) were stained. Four 20x images were taken per muscle per mouse, and the amount of collagen deposition was determined using the ImageJ software program. The average collagen percentage per muscle of all groups was calculated.

[0213] Laser Monitoring of Open Field Cage Activity

[0214] Open field activity chambers were used to determine the overall activity of the experimental mice. - / - (n=6) Control mice aged 16 to 17 weeks and scAAVrh74.tMCK.hSGCA treated mice aged 16 to 17 weeks - / -Mice (n=6 / dose) were analyzed. All mice were tested at the same time of the day in the early morning near the end of the night cycle when mice were most active. All mice were tested in an isolated room under dim light, with the same treatment procedure used each time. In addition, in order to reduce anxiety and keep behavioral variables that may affect the normal activities of mice and thus the results of the assay to a minimum, we tested mice that were not raised alone. The Photobeam activity system (San Diego Instruments, San Diego, California) was used to monitor mouse activity. This system uses a grid of invisible infrared beams that pass through the animal room from front to back and from left to right to monitor the position and movement of mice in the xyz plane. Activity was recorded at 5-min intervals for a 1-hour period. A few days before starting data collection, mice were adapted to the initial 1-hour training in the activity test room. Mice were tested in a single room, four per group. The test equipment was cleaned between each use to reduce the reactionary behavioral variables of mice that may change the results. The data was converted into a Microsoft Excel worksheet, and all calculations were completed in the Excel program. Individual beam breaks for each mouse moving in the x- and y-planes were summed to represent total walking during the 1-h time interval, and beam breaks in the z-plane were summed to obtain vertical activity.

[0215] Safety research

[0216] hematology

[0217] Whole blood was taken from cardiac puncture for blood chemistry. Blood was collected in serum separator tubes and centrifuged at 15,000 rpm for 10 min. Serum was collected, frozen and sent to Charles River Laboratory for chemical testing. Liver enzymes and glucose chemistry were prioritized in hematology analysis.

[0218] Histopathology

[0219] At necropsy, muscles were fresh frozen in liquid nitrogen cooled methylbutane; all other organs were harvested and fixed in formalin and embedded in paraffin. After treatment, tissues were stained with H&E, and slides and all tissues were sent to GEMPath for formal review by a veterinary pathologist.

[0220] Statistical analysis

[0221] Unless otherwise stated, data are presented as mean ± SEM (error bars) and were analyzed using one-way ANOVA with multiple comparisons between groups assessed by Tukey's post hoc test using GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

[0222] Example 2

[0223] Efficiency of systemic delivery of scAAVrh74.tMCK.hSGCA

[0224] A small pilot study was initiated to observe the effect of 12 Total dose (5×10 13vg / kg , based on 20-g mice, n = 4) were injected intravenously into sgca - / - The efficacy of gene delivery was performed in the lateral tail vein of mice. Immunofluorescence analysis was performed on harvested muscles 4 weeks after gene transfer. The expression of the hSGCA transgene in seven different limb skeletal muscles: TA, GAS, GLUT, QD, PSOAS, and TRI and DIA. When analyzed by immunofluorescence, mice lacking α-SG showed a complete absence of protein ( Figure 2 A; Representative images of TA, GAS, TRI, and DIA). 1×10 12 The therapeutic dose of total vg dose resulted in an average of 54±23.81% vector transduction in all skeletal muscles (including DIA) 4 weeks after gene delivery.

[0225] Systemic delivery to SGCA - / - Dose escalation study of scAAVrh74.tMCK.hSGCA in mice

[0226] To determine the safest and most effective dose, delivery of three different doses of the vector was investigated in a dose-escalation study in which 4-week-old sgca - / - The lateral tail vein of mice was treated with 1×10 12 Total dose (5×10 13 vg / kg), 3×10 12 Total dose (1×10 14 vg / kg) or 6×10 12 Total dose (2×10 14 vg / kg) of scAAVrh74.tMCK.hSGCA. Mice were euthanized 12 weeks after gene delivery to evaluate hSGCA transgene expression in TA, GAS, QD, GLUT, PSOAS, TRI, DIA, and HRT muscles using immunofluorescence. 12 Total dose (5×1013 vg / The mean hSGCA expression in mice treated with the lowest dose of 1000 mg / kg was 70.07 ± 3.71% in skeletal muscle (including DIA). 12 Total dose (1×10 14 The mean hSGCA expression in all skeletal muscles of mice treated with the middle dose of 6×10 12 Total dose (2×10 14 The mean hSGCA expression in mice treated with the highest dose of 100 mg / kg (vg / kg) was 93.86 ± 2.02% in all skeletal muscles. For clarity, doses were calculated based on supercoiled DNA or plasmids as quantitative standards. hSGCA expression in HRT muscle remained at 75% independent of dose. Representative images of tissues are shown in Figure 2 In A. Stable hSGCA expression shows the effect of gene delivery at all three doses. Gene delivery targeted multiple muscles in the forelimbs and hindlimbs, showing abnormal α-SG expression in mice at all three doses. Most importantly, the important diaphragm muscle also showed α-SG gene expression after delivery. Figure 2 The Western blot shown in B confirms protein expression in all muscles of all three dosing groups of treated mice. Mouse myocardium also showed α-SG expression following treatment.

[0227] Histopathological features of humans and mice lacking α-SG protein include central nucleation, irregular fiber size distribution, necrosis, and fibrosis. H&E staining was used to observe muscle morphology, including fiber size and central nucleation ( Figure 3 ).like Figure 3 As shown in A and 3B, sgca treated with vehicle - / - Compared with the control, scAAVrh74.tMCK.hSGCA treated sgca - / - Normalization of fiber size distribution was observed in the TA, QD, and TRI of mice, similar to that observed in wild-type controls. Fiber mean diameter size was significantly increased in the TA, QD, and TRI muscles at all doses compared to vehicle-treated sgca- / - control mice (Table 1).

[0228] Table 1

[0229]

[0230] * = p < 0.05, **** = p < 0, 0. Abbreviations: TA, tibialis anterior, AD: quadriceps; and TRI, triceps. Minimum dose: 1.0 × 10 12 vg; intermediate dose: 3.0×10 12vg; Maximum dose: 6.0×10 12 vg.

[0231] In sgca treated with scAAVrh74.tMCK.hSGCA - / - In mice, a reduction in central nucleation was also observed. - / - The skeletal muscle of mice had 68.72±3.01% of fibers with centrally located nuclei. After treatment with scAAVrh74.tMCK.hSGCA, the overall value of central nucleation of all muscle tissues was reduced at the lowest dose of scAAVrh74.tMCK.hSGCA, resulting in 55.60±3.25% of skeletal muscle fibers showing centrally located nuclei (Table 2).

[0232] Table 2

[0233]

[0234] ****=p<0.0001. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteal muscle; PSO, psoas major; AVG, mean; and TRI, triceps.

[0235] Mice treated with the middle dose had 61.85 ± 4.00% of myofibers with centralized nuclei, whereas nucleation was reduced to 37.93 ± 12.46% in myofibers treated with the highest dose ( Figure 3 C).

[0236] Fibrosis, in which tissue is overcome by collagen, often occurs in the muscles of LGMD patients, leading to the formation of scar tissue. Fibrosis was assessed using Sirius red staining to detect collagen I and III content as a marker of fibrosis. Figure 4 As shown in Figure 2, a steady reduction in red staining was observed in sgca- / - mice after treatment with scAAVrh74.tMCK.hSGCA. Quantification showed that compared with vehicle-treated sgca - / - Compared with control mice, the collagen content of all muscles in scAAVrh74.tMCK.hSGCA-treated sgca- / - mice was significantly reduced ( Figure 4 B) In summary, these data demonstrate successful systemic delivery of the hSGCA transgene, as evidenced by stable expression in muscle tissue and sgca - / - This was indicated by the improvement of histopathological features associated with α-SG protein deficiency in mice.

[0237] Example 3

[0238] scAAVrh74.tMCK.hSGCA rAAV improves diaphragm and tibialis anterior muscle function and increases exercise capacity

[0239] Since proximal muscle weakness and loss of function are the main symptoms of LGMD2D, and respiratory failure is the main cause of death in LGMD2D, improving the function and strength of the TA and DIA is crucial to increase the lifespan and quality of life of LGMD2D subjects. Strips of the DIA and whole TA muscles were used to confirm the correlation between hSGCA expression and muscle strength. Figure 5 As shown in A and 5B, in untreated sgca mice, compared with wild-type mice, - / - Deficits in specific force and resistance to contraction-induced injury were identified in the TA and DIA muscles of mice.

[0240] Compared with wild-type mice, sgca - / - The TA muscles of mice showed a significant functional deficit of 44% in terms of reduced specific force output (161.6 ± 8.20 mN / mm 2 and 291.7±6.17mN / mm 2 ; p < 0.0001); and greater force loss after a strict eccentric contraction protocol (sgca - / - mice lost 44.0±6.0%; wild-type mice lost 18.0±1.0%; p<0.0001)( Figure 5 A). Twelve weeks after tail vein delivery, Applicants noted a significant increase in specific force output following treatment with low, medium, and high doses of scAAVrh74.tMCK.hSGCA, which increased to 218 ± 11.94 mN / mm, respectively. 2 、227±11.7mN / mm 2 and 255±11.7mN / mm 2 . sgca treated with vehicle - / - Resistance to injury following the eccentric contraction protocol was also improved compared to mice treated with the low, medium, and high doses, with mice losing only 22.0 ± 4.0%, 22.0 ± 3.0%, and 12.0 ± 1.0%, respectively (compared to vehicle-treated sgca - / - mice, p<0.0001)( Figure 5 A).

[0241] In vehicle-treated sgca - / - In DIA of mice, the specific force generated showed a 41% decrease in strength (131.5±12.07 mN / mm 2 and 223.8±15.85mN / mm 2Improvements in force were observed following treatment with scAAVrh74.tMCK.hSGCA at all three doses, with the specific force of the DIA increasing to 179.2 ± 21.03 mN / mm in low-dose mice. 2 , increased to 201.2±22.94 mN / mm in the medium-dose mice 2 And in high-dose mice, it increased to 261.46±9.73mN / mm 2 ( Figure 5 B) These data show that sgca - / - The TA and DIA muscles in WT mice were deficient in strength and depleted more rapidly than in wild-type mice. However, after delivery of scAAVrh74.tMCK.hSGCA, functional recovery was achieved.

[0242] Other symptoms of LGMD2D include exercise intolerance and decreased activity and walking, possibly due to muscle damage, leading to pain and muscle fatigue. To assess the level of physical activity, sgca - / - and wild-type C57BL / 6 mice were subjected to an open field activity protocol similar to that used in previous reports. The walking-related activity of the mice was monitored to determine whether sgca - / - Does the lack of α-SG in mice lead to reduced walking? Figure 5 The graph in C depicts the expression of sgca compared with wild-type controls. - / - Reduced walking and vertical rearing in a mouse model. - / - The mean average ambulation beam breaks recorded in sgca mice was 2000 ± 159 beam breaks / hour, a 77.5% reduction in ambulation compared to 8911 ± 1193 beam breaks / hour in wild-type controls. - / - The mean vertical upright fascicle breaks recorded in mice were 24.75 ± 11.47 fascicle breaks / hour, a 97% decrease in vertical upright compared to 803.3 ± 55.03 fascicle breaks / hour in wild-type mice. Following treatment with scAAVrh74.tMCK.hSGCA, walking and vertical upright activity in mice increased 12 weeks after gene delivery. Mean walking was 24.75 ± 11.47 fascicle breaks / hour in mice treated with 1 × 10 12 The total dose of Vg increased to 3595 ± 55.03 bundle breaks / hour in mice treated with 3 × 10 12 The total dose of Vg increased to 5238 ± 861.9 bundle breaks / hour in mice treated with 6 × 10 12 The total dose of VG increased to 6487 ± 467.9 fascicle breaks / hour in mice treated with 1×10 12 The total dose of Vg increased to 377 ± 146.1 bundle breaks / hour in mice treated with 3 × 10 12The total dose of Vg increased to 321 ± 126.1 bundle breaks / hour in mice treated with 6 × 10 12 The total dose of vg increased to 448.8 ± 53.43 bundle breaks / hour ( Figure 5 C) sgca treated with vehicle - / - Physical activity of treated mice showed 44%-69% improvement in walking and 92%-94% improvement in vertical standing compared to untreated mice. In addition, serum creatine kinase levels were significantly reduced in all treatment groups compared to untreated mice ( Figure 5 D) In ​​conclusion, these data show that delivery of α-SG restores physical activity and prevents sgca - / - Muscle breakdown in mice.

[0243] Safety and biodistribution analysis of scAAVrh74.tMCK.hSGCA

[0244] As a safety measure, sgca administered in vehicle - / - Blood chemistry and hematology studies were performed on wild-type mice. All values ​​were within the normal reference range for mice ( Figure 6 ). In addition, sgca from scAAVrh74.tMCK.hSGCA administration - / - All muscle and organ tissue sections stained with H&E from wild-type and WT mice were sent to a veterinary pathologist for formal review. - / - No adverse effects were found in any of the samples from either WT or wild-type mice. In addition to efficacy, these data demonstrate that systemic delivery of all three doses of scAAVrh74.tMCK.hSGCA has an effective - / - It was well tolerated, safe and non-toxic to wild-type mice.

[0245] To test potential toxicity or safety issues of delivering scAAVrh74.tMCK.hSGCA, vector biodistribution quantitative PCR was performed to quantify the presence of vector genomes ( Fig. 7A ). The vector-specific tMCK.hSGCA primer-probe set was used to detect sgca administered from scAAVrh74.tMCK / hSGCA - / - Vector genome in all muscles and organs tested in mice. As expected, the vector genome was present in all tissues tested, with the highest copy number in liver, followed by muscle. - / - Western blot of α-sarcoglycan protein in the liver of sgca- / - mice treated with LR) or scAArh74.tMCK.hSGCA is shown in Figure 7B middle.

[0246] To increase the efficiency of α-SG expression, in one embodiment, the hSGCA cDNA sequence is packaged into a self-complementary vector. Self-complementary AAV vectors contain inverted repeat genomes that promote dsDNA formation, allowing replication and transcription to occur without the need for multiple vector genomes to facilitate these processes. Thus, the use of self-complementary vectors eliminates the rate-limiting step to allow for more rapid expression of the transgene. Applicants have shown that intravascular delivery of scAAVrh74.tMCK.hSGCA in LGMD2D patients is effective with 1×10 12 and 3×10 12 doses were associated with increased α-SG expression 180 days after gene transfer.

[0247] Intravenous delivery of scAAVrh74.tMCK.hSGCA provided muscles with enhanced strength and resistance to contraction-induced damage in the tibialis anterior and diaphragm muscles in all three vehicle-treated groups compared to vehicle-treated controls. In addition, treatment with scAAVrh74.tMCK.hSGCA resulted in a significant decrease in CK. In addition, after treatment with scAAVrh74.tMCK.hSGCA, mice were able to ambulate and lean on their hind limbs more frequently compared to vehicle-treated mice.

[0248] Significant histopathology, including centrally located nuclei, wide variability in fiber size, inflammation, necrosis, and fibrosis, is commonly observed by muscle biopsy of patients with LGMD2D. Following hSGCA delivery, mice had reduced CN, more uniform distribution of myofiber size, and reduced collagen content, as well as an overall healthier appearance of muscle compared to vehicle-treated mice. The overall reduction in histopathology and scar tissue was accompanied by improvements in overall normal function and physiology of the muscles of vehicle-treated mice.

[0249] Finally, safety studies performed by quantitative PCR, serum chemistry analysis, and histopathology did not review for signs of toxicity. The tMCK promoter was only detected in targeted tissues (all muscles) and was absent in other (non-muscle) organs except the liver. Detection of tMCK in the liver is not uncommon or concerning as it is a clearance organ. Histopathological review of all tissues (including liver) by a board-certified veterinary pathologist concluded that systemic delivery of scAAVrh74.tMCK.hSGCA was not only safe in all tissues, but gene delivery significantly reduced sgca observed in vehicle-treated patients. - / - The amount of dystrophic pathology found in the skeletal muscle of the mice. Chemical analysis of blood samples from vehicle-treated mice also supported the lack of toxicity.

[0250] As in this disclosure, dose escalation studies provide preclinical data to support the lowest dose systematically tested here, i.e., 1×10 12 Total amount of vg (5×10 13 vg / kg) was sufficient to reduce signs and symptoms associated with α-SG protein loss. At the lowest dose tested, functional improvements in all muscles were observed in vehicle-treated mice, as demonstrated by increases in strength and motor behavior (walking and standing). 12 Total amount of vg (2×10 14 Safety studies showed no signs of toxicity at the highest delivered dose of 100 mg / kg (vg / kg).

[0251] Example 4

[0252] Elderly patients and durability

[0253] rAAVrh74.tMCK.hSGCA-mediated gene replacement has shown positive results in the treatment of LGMD-2D and other related diseases. This study was designed to test the ability of rAAVrh74.tMCK.hSGCA to treat more severely affected muscles in the elderly and to determine the long-term durability of the AAV viral vector.

[0254] All procedures were performed in accordance with the approval of the Research Institute at the Nationwide Children's Hospital Institutional Animal Care and Use Committee. Mice were maintained under standard conditions with a 12:12 h light:dark cycle and food and water were provided ad libitum. First, rAAVrh74.tMCK.hSGCA was injected via tail vein at three doses (1.0 × 10 12 , 3.0×10 12 and 6.0×10 12 vg) systemically administered to 12-month-old sgca showing severe muscle histopathology - / - Mice (n=5). Controls included sgca injected with lactated Ringer's solution (LRS) - / -Mice (n = 5) and BL6 wild-type mice injected with LRS (n = 4). At the end of 6 months after treatment, SGCA protein expression, histological rescue and functional improvement of muscles from treated mice were evaluated. Compared with the control, all three doses showed stable protein expression of α-SG at the sarcolemma, increased histopathology, increased spontaneous activity and specific force generation, prevention of centrifugal force loss and reduced serum CK. No carrier toxicity was detected. In old mice, treatment resulted in widespread high-level protein expression in muscle, reduced fibrosis, and increased resistance to contraction-induced damage in the tibialis anterior muscle.

[0255] In particular, for 12-month-old sgca - / - IV injection of mice with rAAVrh74.tMCK.hSGCA resulted in widespread high-level protein expression in muscles throughout the lower limbs, upper limbs, and proximal trunk muscles, including the diaphragm and heart ( Figure 8 ).

[0256] An overall improvement in muscle pathology was observed after administration of scAAVrh74.tMCK.hSGCA ( Fig. 9 a) and a reduction in central nucleation. In addition, after administration, the average fiber size increased to a level similar to that of WT fibers in the gastrocnemius (GAS) and triceps (TRI) muscles ( Fig. 9 b).

[0257] The level of collagen deposition was quantified as a measure of fibrosis. Administration of scAAVrh74.tMCK.hSGCA resulted in a decrease in fibrosis levels compared to untreated controls ( Fig. 9 c). Functional improvements following administration of scAAVrh74.tMCK.hSGCA were demonstrated by improved force output (specific force) in the tibialis anterior (TA) and diaphragm (DIA) muscles and increased resistance to contraction-induced injury in the TA muscle ( Fig.10 ).

[0258] To further investigate the long-term durability of gene therapy, 4-week-old sgca - / - Mice were systemically administered with rAAVrh74.tMCK.hSGCA. More than 24 months after treatment, vector genome copy number was measured by qPCR in all transduced muscles tested (TA, TRI, DIA, GLUT, PSOAS, GAS, and QUAD). Protein expression and localization were studied by immunofluorescence staining of treated muscles.

[0259] Although the present disclosure has been described in terms of specific embodiments, it will be understood that changes and modifications will occur to those skilled in the art. Therefore, only the limitations appearing in the claims are intended to define the present disclosure.

[0260] All documents mentioned in this application are hereby incorporated by reference in their entirety.

[0261] SEQ ID NO:1

[0262] SGCA cDNA codon-optimized sequence:

[0263]

[0264] SEQ ID NO:2

[0265] Human SGCA protein sequence:

[0266] MAETLFWTPLLVVLLAGLGDTEAQQTTLHPLVGRVFVHTLDHETFLSLPEHVAVPPAVHITYHAHLQGHPDLPRWLRYTQRSPHHPGFLYGSATPEDRGLQVIEVTAYNRDSFDTTRQRLVLEIGDPEGPLLPYQAEFLVRSHDAEEVLPSTPASRFLSALGGLWEPGELQLLNVTSALDRGGRVPLPIEGRKEGVYIKVGSASPFSTCLKMVASPDSHARCAQGQPPLLSCYDTLAPHFRVDWCNVTLVDKSVPEPADEVPTPGDGILEHDPFFCPPTEAPDRDFLVDALVTLLVPLLVALLLTLLLAYVMCCRREGRLKRDLATSDIQMVHHCTIHGNTEELRQMAASREVPRPLSTLPMFNVHTGERLPPRVDSAQVPLILDQH*

[0267] SEQ ID NO:3

[0268] tMCK promoter sequence: CCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGATCCACTACGGGTCTATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGACCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCCCGGGTCAC

[0269] SEQ ID NO:4

[0270] AAVrh74-tMCK-SGCA:

[0271] CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGGGAGTGGGGTT AACCAATTGG CGGCCGCAAACTTGCATGCC CCACTACGGGTCTAGGCTGC CCATGTAAGG AGGCAAGGCC TGGGGACACCCGAGATGCCTGGTTATAATT AACCCCAACA CCTGCTGCCC CCCCCCCCCC AACACCTGCTGCCTGAGCCTGAGCGGTTAC CCCACCCCGG TGCCTGGGTC TTAGGCTCTGTACACCATGG AGGAGAAGCT CGCTCTAAAAATAACCCTGT CCCTGGTGGATCCACTACGG GTCTATGCTG CCCATGTAAG GAGGCAAGGCCTGGGGACACCCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCC CCCCCCCCCCCAACACCTGCTGCCTGAGCC TGAGCGGTTA CCCCACCCCG GTGCCTGGGTCTTAGGCTCT GTACACCATG GAGGAGAAGCTCGCTCTAAA AATAACCCTGTCCCTGGTGG ACCACTACGG GTCTAGGCTG CCCATGTAAGGAGGCAAGGCCTGGGGACAC CCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCCCCCCCCCCCCAACACCTGCT GCCTGAGCCT GAGCGGTTAC CCCACCCCGGTGCCTGGGTC TTAGGCTCTG TACACCATGGAGGAGAAGCT CGCTCTAAAAATAACCCTGT CCCTGGTCCT CCCTGGGGAC AGCCCCTCCTGGCTAGTCACACCCTGTAGG CTCCTCTATA TAACCCAGGG GCACAGGGGC TGCCCCCGGGTCACCTGCAGAAGTTGGTCG TGAGGCACTG GGCAGGTAAG TATCAAGGTTACAAGACAGG TTTAAGGAGACCAATAGAAACTGGGCTTGT CGAGACAGAGAAGACTCTTG CGTTTCTGAT AGGCACCTAT TGGTCTTACTGACATCCACTTTGCCTTTCT CTCCACAGGT GTCCACTCCC AGTTCAATTA CAGCGCGTGGTACCACCATGGCCGAGACAC TGTTCTGGAC TCCTCTGCTG GTGGTGCTGCTGGCTGGACT GGGAGATACC GAGGCTCAGCAGACCACACT GCACCCACTGGTGGGCCGGG TGTTCGTGCA CACCCTGGAC CATGAGACATTTCTGAGTCTGCCAGAACAC GTGGCTGTGC CACCTGCTGT GCATATCACT TACCACGCCCATCTGCAGGGCCATCCTGAT CTGCCACGGT GGCTGAGATA CACCCAGAGATCACCCCACC ATCCTGGATT CCTGTATGGAAGCGCTACCC CAGAGGACAGGGGACTGCAG GTGATCGAAG TGACAGCTTA CAACCGCGACAGTTTTGATACTACCAGGCA GCGCCTGGTG CTGGAGATTG GGGATCCAGA AGGACCCCTGCTGCCTTATCAGGCCGAGTT CCTGGTGCGG TCACACGACG CTGAGGAAGTGCTGCCATCA ACACCCGCCA GCAGATTTCTGTCCGCTCTG GGAGGACTGTGGGAGCCAGG AGAACTGCAG CTGCTGAATG TGACTAGCGCTCTGGATAGGGGAGGAAGGG TGCCACTGCC AATCGAGGGA AGGAAGGAAG GGGTGTACATTAAAGTGGGAAGCGCTTCCC CATTCTCCAC CTGCCTGAAG ATGGTGGCTTCTCCTGATAG TCACGCTAGG TGCGCTCAGGGACAGCCACC ACTGCTGTCCTGTTATGACA CACTGGCCCC CCATTTTCGC GTGGACTGGTGCAACGTGACTCTGGTGGAT AAATCTGTGC CTGAGCCAGC TGACGAAGTGCCAACCCCTGGAGACGGAATCCTGGAGCAC GATCCTTTCT TTTGTCCTCC AACAGAAGCCCCAGACAGGG ATTTCCTGGT GGACGCTCTGGTGACTCTGC TGGTGCCTCTGCTGGTGGCT CTGCTGCTGA CCCTGCTGCT GGCTTATGTGATGTGCTGTCGGAGAGAGGG ACGGCTGAAG AGAGACCTGG CCACATCTGA TATCCAGATGGTGCACCATTGTACTATTCA CGGCAACACC GAGGAACTGC GCCAGATGGCTGCTTCTAGG GAGGTGCCAA GGCCACTGAGTACACTGCCT ATGTTTAATGTGCACACTGG CGAACGGCTG CCCCCTAGAG TGGATAGCGCCCAGGTGCCACTGATTCTGG ACCAGCATTG AGGCCGCAAT AAAAGATCTT TATTTTCATTAGATCTGTGTGTTGGTTTTT TGTGTGTCCT GCAGGGGCGC GCCTCTAGAGCATGGCTACG TAGATAAGTA GCATGGCGGGTTAATCATTA ACTACAAGGAACCCCTAGTG ATGGAGTTGG CCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGG GCGACCAAAG GTCGCCCGAC GCCCGGGCTT TGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAG

[0272] SEQ ID NO:5

[0273] 5’ITR

[0274] CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTTGGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGGGTT

[0275] SEQ ID NO:6

[0276] 3’ITR

[0277] CCACTCCCTC TCTGCGCGCT CGCTCGCTCA CTGAGGCCGG GCGACCAAAG GTCGCCCGACGCCCGGGCTT TGCCCGGGCG GCCTCAGTGA GCGAGCGAGC GCGC

[0278] SEQ ID NO:7

[0279] PolyA

[0280] GGCCGCAAT AAAAGATCTT TATTTTCATT AGATCTGTGT GTTGGTTTTT TGTG

[0281] SEQ ID NO:8

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] <110> Research Institute at Nationwide Children's Hospital <120> Adeno-associated viral vector delivery of α-sarcoglycan and Treatment of muscular dystrophy <130> 28335 / 54625 <140> PCT / US20 / 47339 <141> 2020-08-21 <150> US 62 / 889,749 <151> 2019-08-21 <150> US 63 / 014,934 <151> 2020-04-24 <150> US 63 / 022,843 <151> 2020-05-11 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 1164 <212> DNA <213> Homo sapiens <400> 1 atggccgaga cactgttctg gactcctctg ctggtggtgc tgctggctgg actgggagat 60 accgaggctc agcagaccac actgcaccca ctggtgggcc gggtgttcgt gcacaccctg 120 gaccatgaga catttctgag tctgccagaa cacgtggctg tgccacctgc tgtgcatatc 180 acttaccacg cccatctgca gggccatcct gatctgccac ggtggctgag atacacccag 240 agatcacccc accatcctgg attcctgtat ggaagcgcta ccccagagga caggggactg 300 caggtgatcg aagtgacagc ttacaaccgc gacagttttg atactaccag gcagcgcctg 360 gtgctggaga ttggggatcc agaaggaccc ctgctgcctt atcaggccga gttcctggtg 420 cggtcacacg acgctgagga agtgctgcca tcaacacccg ccagcagatt tctgtccgct 480 ctgggaggac tgtgggagcc aggagaactg cagctgctga atgtgactag cgctctggat 540 aggggaggaa gggtgccact gccaatcgag ggaaggaagg aaggggtgta cattaaagtg 600 ggaagcgctt ccccattctc cacctgcctg aagatggtgg cttctcctga tagtcacgct 660 aggtgcgctc agggacagcc accactgctg tcctgttatg acacactggc cccccatttt 720 cgcgtggact ggtgcaacgt gactctggtg gataaatctg tgcctgagcc agctgacgaa 780 gtgccaaccc ctggagacgg aatcctggag cacgatcctt tcttttgtcc tccaacagaa 840 gccccagaca gggatttcct ggtggacgct ctggtgactc tgctggtgcc tctgctggtg 900 gctctgctgc tgaccctgct gctggcttat gtgatgtgct gtcggagaga gggacggctg 960 aagagagacc tggccacatc tgatatccag atggtgcacc attgtactat tcacggcaac 1020 accgaggaac tgcgccagat ggctgcttct agggaggtgc caaggccact gagtacactg 1080 cctatgttta atgtgcacac tggcgaacgg ctgcccccta gagtggatag cgcccaggtg 1140 ccactgattc tggaccagca ttga 1164 <210> 2 <211> 387 <212> PRT <213> Homo sapiens <400> 2 Met Ala Glu Thr Leu Phe Trp Thr Pro Leu Leu Val Val Leu Leu Ala 1 5 10 15 Gly Leu Gly Asp Thr Glu Ala Gln Gln Thr Thr Leu His Pro Leu Val 20 25 30 Gly Arg Val Phe Val His Thr Leu Asp His Glu Thr Phe Leu Ser Leu 35 40 45 Pro Glu His Val Ala Val Pro Pro Ala Val His Ile Thr Tyr His Ala 50 55 60 His Leu Gln Gly His Pro Asp Leu Pro Arg Trp Leu Arg Tyr Thr Gln 65 70 75 80 Arg Ser Pro His His Pro Gly Phe Leu Tyr Gly Ser Ala Thr Pro Glu 85 90 95 Asp Arg Gly Leu Gln Val Ile Glu Val Thr Ala Tyr Asn Arg Asp Ser 100 105 110 Phe Asp Thr Thr Arg Gln Arg Leu Val Leu Glu Ile Gly Asp Pro Glu 115 120 125 Gly Pro Leu Leu Pro Tyr Gln Ala Glu Phe Leu Val Arg Ser His Asp 130 135 140 Ala Glu Glu Val Leu Pro Ser Thr Pro Ala Ser Arg Phe Leu Ser Ala 145 150 155 160 Leu Gly Gly Leu Trp Glu Pro Gly Glu Leu Gln Leu Leu Asn Val Thr 165 170 175 Ser Ala Leu Asp Arg Gly Gly Arg Val Pro Leu Pro Ile Glu Gly Arg 180 185 190 Lys Glu Gly Val Tyr Ile Lys Val Gly Ser Ala Ser Pro Phe Ser Thr 195 200 205 Cys Leu Lys Met Val Ala Ser Pro Asp Ser His Ala Arg Cys Ala Gln 210 215 220 Gly Gln Pro Pro Leu Leu Ser Cys Tyr Asp Thr Leu Ala Pro His Phe 225 230 235 240 Arg Val Asp Trp Cys Asn Val Thr Leu Val Asp Lys Ser Val Pro Glu 245 250 255 Pro Ala Asp Glu Val Pro Thr Pro Gly Asp Gly Ile Leu Glu His Asp 260 265 270 Pro Phe Phe Cys Pro Pro Thr Glu Ala Pro Asp Arg Asp Phe Leu Val 275 280 285 Asp Ala Leu Val Thr Leu Leu Val Pro Leu Leu Val Ala Leu Leu Leu 290 295 300 Thr Leu Leu Leu Ala Tyr Val Met Cys Cys Arg Arg Glu Gly Arg Leu 305 310 315 320 Lys Arg Asp Leu Ala Thr Ser Asp Ile Gln Met Val His His Cys Thr 325 330 335 Ile His Gly Asn Thr Glu Glu Leu Arg Gln Met Ala Ala Ser Arg Glu 340 345 350 Val Pro Arg Pro Leu Ser Thr Leu Pro Met Phe Asn Val His Thr Gly 355 360 365 Glu Arg Leu Pro Pro Arg Val Asp Ser Ala Gln Val Pro Leu Ile Leu 370 375 380 Asp Gln His 385 <210> 3 <211> 714 <212> DNA <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 3 ccactacggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 60 ggttataatt aaccccaaca cctgctgccc cccccccccc aacacctgct gcctgagcct 120 gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct 180 cgctctaaaa ataaccctgt ccctggtgga tccactacgg gtctatgctg cccatgtaag 240 gaggcaaggc ctggggacac ccgagatgcc tggttataat taaccccaac acctgctgcc 300 cccccccccc caacacctgc tgcctgagcc tgagcggtta ccccaccccg gtgcctgggt 360 cttaggctct gtacaccatg gaggagaagc tcgctctaaa aataaccctg tccctggtgg 420 accactacgg gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc 480 tggttataat taaccccaac acctgctgcc cccccccccc aacacctgct gcctgagcct 540 gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct 600 cgctctaaaa ataaccctgt ccctggtcct ccctggggac agcccctcct ggctagtcac 660 accctgtagg ctcctctata taacccaggg gcacaggggc tgcccccggg tcac 714 <210> 4 <211> 2476 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggggtt aaccaattgg 120 cggccgcaaa cttgcatgcc ccactacggg tctaggctgc ccatgtaagg aggcaaggcc 180 tggggacacc cgagatgcct ggttataatt aaccccaaca cctgctgccc cccccccccc 240 aacacctgct gcctgagcct gagcggttac cccaccccgg tgcctgggtc ttaggctctg 300 tacaccatgg aggagaagct cgctctaaaa ataaccctgt ccctggtgga tccactacgg 360 gtctatgctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat 420 taaccccaac acctgctgcc cccccccccc caacacctgc tgcctgagcc tgagcggtta 480 ccccaccccg gtgcctgggt cttaggctct gtacaccatg gaggagaagc tcgctctaaa 540 aataaccctg tccctggtgg accactacgg gtctaggctg cccatgtaag gaggcaaggc 600 ctggggacac ccgagatgcc tggttataat taaccccaac acctgctgcc cccccccccc 660 aacacctgct gcctgagcct gagcggttac cccaccccgg tgcctgggtc ttaggctctg 720 tacaccatgg aggagaagct cgctctaaaa ataaccctgt ccctggtcct ccctggggac 780 agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc 840 tgcccccggg tcacctgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt 900 acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg 960 cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt 1020 gtccactccc agttcaatta cagcgcgtgg taccaccatg gccgagacac tgttctggac 1080 tcctctgctg gtggtgctgc tggctggact gggagatacc gaggctcagc agaccacact 1140 gcacccactg gtgggccggg tgttcgtgca caccctggac catgagacat ttctgagtct 1200 gccagaacac gtggctgtgc cacctgctgt gcatatcact taccacgccc atctgcaggg 1260 ccatcctgat ctgccacggt ggctgagata cacccagaga tcaccccacc atcctggatt 1320 cctgtatgga agcgctaccc cagaggacag gggactgcag gtgatcgaag tgacagctta 1380 caaccgcgac agttttgata ctaccaggca gcgcctggtg ctggagattg gggatccaga 1440 aggacccctg ctgccttatc aggccgagtt cctggtgcgg tcacacgacg ctgaggaagt 1500 gctgccatca acacccgcca gcagatttct gtccgctctg ggaggactgt gggagccagg 1560 agaactgcag ctgctgaatg tgactagcgc tctggatagg ggaggaaggg tgccactgcc 1620 aatcgaggga aggaaggaag gggtgtacat taaagtggga agcgcttccc cattctccac 1680 ctgcctgaag atggtggctt ctcctgatag tcacgctagg tgcgctcagg gacagccacc 1740 actgctgtcc tgttatgaca cactggcccc ccattttcgc gtggactggt gcaacgtgac 1800 tctggtggat aaatctgtgc ctgagccagc tgacgaagtg ccaacccctg gagacggaat 1860 cctggagcac gatcctttct tttgtcctcc aacagaagcc ccagacaggg atttcctggt 1920 ggacgctctg gtgactctgc tggtgcctct gctggtggct ctgctgctga ccctgctgct 1980 ggcttatgtg atgtgctgtc ggagagaggg acggctgaag agagacctgg ccacatctga 2040 tatccagatg gtgcaccatt gtactattca cggcaacacc gaggaactgc gccagatggc 2100 tgcttctagg gaggtgccaa ggccactgag tacactgcct atgtttaatg tgcacactgg 2160 cgaacggctg ccccctagag tggatagcgc ccaggtgcca ctgattctgg accagcattg 2220 aggccgcaat aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgtcct 2280 gcaggggcgc gcctctagag catggctacg tagataagta gcatggcggg ttaatcatta 2340 actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 2400 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 2460 gcgagcgagc gcgcag 2476 <210> 5 <211> 110 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggggtt 110 <210> 6 <211> 104 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 60 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgc 104 <210> 7 <211> 53 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 ggccgcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtg 53 <210> 8 <211> 5749 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 atgcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaacc 120 aattggcggc cgcaaacttg catgccccac tacgggtcta ggctgcccat gtaaggaggc 180 aaggcctggg gacacccgag atgcctggtt ataattaacc ccaacacctg ctgccccccc 240 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 300 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtggatcca 360 ctacgggtct atgctgccca tgtaaggagg caaggcctgg ggacacccga gatgcctggt 420 tataattaac cccaacacct gctgcccccc cccccccaac acctgctgcc tgagcctgag 480 cggttacccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 540 tctaaaaata accctgtccc tggtggacca ctacgggtct aggctgccca tgtaaggagg 600 caaggcctgg ggacacccga gatgcctggt tataattaac cccaacacct gctgcccccc 660 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 720 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtcctccct 780 ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac ccaggggcac 840 aggggctgcc cccgggtcac ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc 900 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga 960 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc 1020 acaggtgtcc actcccagtt caattacagc gcgtggtacc accatggccg agacactgtt 1080 ctggactcct ctgctggtgg tgctgctggc tggactggga gataccgagg ctcagcagac 1140 cacactgcac ccactggtgg gccgggtgtt cgtgcacacc ctggaccatg agacatttct 1200 gagtctgcca gaacacgtgg ctgtgccacc tgctgtgcat atcacttacc acgcccatct 1260 gcagggccat cctgatctgc cacggtggct gagatacacc cagagatcac cccaccatcc 1320 tggattcctg tatggaagcg ctaccccaga ggacagggga ctgcaggtga tcgaagtgac 1380 agcttacaac cgcgacagtt ttgatactac caggcagcgc ctggtgctgg agattgggga 1440 tccagaagga cccctgctgc cttatcaggc cgagttcctg gtgcggtcac acgacgctga 1500 ggaagtgctg ccatcaacac ccgccagcag atttctgtcc gctctgggag gactgtggga 1560 gccaggagaa ctgcagctgc tgaatgtgac tagcgctctg gataggggag gaagggtgcc 1620 actgccaatc gagggaagga aggaaggggt gtacattaaa gtgggaagcg cttccccatt 1680 ctccacctgc ctgaagatgg tggcttctcc tgatagtcac gctaggtgcg ctcagggaca 1740 gccaccactg ctgtcctgtt atgacacact ggccccccat tttcgcgtgg actggtgcaa 1800 cgtgactctg gtggataaat ctgtgcctga gccagctgac gaagtgccaa cccctggaga 1860 cggaatcctg gagcacgatc ctttcttttg tcctccaaca gaagccccag acagggattt 1920 cctggtggac gctctggtga ctctgctggt gcctctgctg gtggctctgc tgctgaccct 1980 gctgctggct tatgtgatgt gctgtcggag agagggacgg ctgaagagag acctggccac 2040 atctgatatc cagatggtgc accattgtac tattcacggc aacaccgagg aactgcgcca 2100 gatggctgct tctagggagg tgccaaggcc actgagtaca ctgcctatgt ttaatgtgca 2160 cactggcgaa cggctgcccc ctagagtgga tagcgcccag gtgccactga ttctggacca 2220 gcattgaggc cgcaataaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg 2280 tgtcctgcag gggcgcgcct ctagagcatg gctacgtaga tagtagcat ggcgggtaa 2340 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 2400 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 2460 cagtgagcga gcgagcgcgc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 2520 cccaacagtt gcgcagcctg aatggcgaat ggcgattccg ttgcaatggc tggcggtaat 2580 attgttctgg atattaccag caaggccgat agtttgagtt cttctactca ggcaagtgat 2640 gttatacta atcaaagaag tattgcgaca acggttaatt tgcgtgatgg acagactctt 2700 ttatcggtg gcctcactga ttataaaaaac acttctcagg attctggcgt accgttcctg 2760 tctaaaatcc ctttaatcgg cctcctgttt agctcccgct ctgattctaa cgaggaaagc 2820 acgttatacg tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc 2880 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 2940 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc atcttcaaat atgtatccgc 3000 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg 3060 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 3120 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 3180 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 3240 agtcccgccc ctaactccgc cccatggctg actaattttt tttatttatg cagaggccga 3300 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 3360 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 3420 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 3480 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgccccgg 3540 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 3600 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 3660 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 3720 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 3780 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 3840 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 3900 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 3960 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 4020 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 4080 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 4140 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 4200 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt 4260 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg 4320 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag 4380 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa 4440 aagacagaat aaaaacgttg cgcaaactat taactggcga actacttact ctagcttccc 4500 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 4560 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 4620 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 4680 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 4740 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 4800 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 4860 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 4920 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 4980 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 5040 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 5100 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 5160 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 5220 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 5280 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 5340 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 5400 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 5460 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 5520 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 5580 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 5640 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 5700 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 5749 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 9 cagggctggg agctgggttc tg 22 <210> 10 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 10 cccagggcct tgatgcct 18 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 11 gctatcagga catagcgttg gcta 24 <210> 12 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 12 acccgagatg cctggttata att 23 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 13 tccatggtgt acagagccta agac 24 <210> 14 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <220> <221> misc_feature <222> (1) <223> FAM <220> <221> misc_feature <222> (25)..(25) <223> IABQ <400> 14 ctgctgcctg agcctgagcg gttac 25

Claims

1. Use of recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSGCA for the preparation of a medicament for treating limb-girdle muscular dystrophy in a subject in need thereof, wherein the dosage of the rAAV in the form of supercoiled DNA or plasmid as a quantitative standard is 5×10 13 vg / kg to 2×10 14 vg / kg, wherein the medicament is formulated for systemic administration, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:

4.

2. The use according to claim 1, wherein the dosage of the rAAV in the form of supercoiled DNA or plasmid as a quantitative standard is 5×10 13 vg / kg, 1×10 14 vg / kg or 2×10 14 vg / kg.

3. Use of recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSGCA for the preparation of a medicament for treating limb-girdle muscular dystrophy in a subject in need thereof, wherein the dosage of the rAAV with linearized DNA or plasmid as a quantitative standard is 1.0×10 13 vg / kg to 8.0×10 13 vg / kg, and wherein the medicament is formulated for a systemic route of administration, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:

4.

4. The use according to claim 3, wherein the dosage of AAVrh74.tMCK.hSGCA with linearized DNA or plasmid as the quantitative standard is 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

5. The use of any one of claims 1 to 4, wherein the α-sarcoglycan gene expression level in the subject's cells increases after administration of the drug compared to the α-sarcoglycan gene expression level before administration of the drug; or wherein the subject's serum creatine kinase (CK) level decreases after administration of the drug compared to the serum CK level before administration of the drug; or wherein the number of α-sarcoglycan-positive fibers in the subject's muscle tissue increases after administration of the drug compared to the number of α-sarcoglycan-positive fibers before administration of the drug.

6. The use according to any one of claims 1 to 4, wherein the systemic administration route is an intravenous route.

7. The use according to any one of claims 1 to 4, wherein the medicament is formulated for administration by injection, infusion or implantation.

8. The use according to any one of claims 1 to 4, wherein the medicament is formulated for administration by infusion.

9. The use according to any one of claims 1 to 4, wherein the medicament is formulated for administration by the intravenous route via a peripheral limb vein.

10. The use according to any one of claims 1 to 4, wherein the limb-girdle muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

11. The use according to any one of claims 1 to 4, wherein the medicament is formulated for intravenous infusion.

12. A polynucleotide sequence, wherein the polynucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

4.

13. A polynucleotide sequence, wherein the polynucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

8.

14. A plasmid comprising the polynucleotide sequence according to claim 12 or 13.

15. A method for producing rAAV, comprising transferring the plasmid of claim 14 or the polynucleotide sequence of claim 12 or 13 into a cell.

16. A cell comprising the polynucleotide sequence of claim 12 or 13.

17. The cell of claim 16, wherein the cell is a yeast cell, an insect cell, a mammalian cell or a bacterial cell.

18. The cell of claim 17, wherein the cell is a HeLa cell, a 293 cell, a PerC.6 cell or a Sf9 cell.

19. A recombinant AAV vector comprising the nucleotide sequence shown in SEQ ID NO:

4.

20. The recombinant AAV vector of claim 19, wherein the vector is of serotype AAV rh.

74.

21. A composition comprising the recombinant AAV of claim 19 or 20.

Images