Adeno-associated virus vector delivery of alpha-sarcoglycans, and treatment of muscular dystrophy.

Abstract

To provide adeno-associated virus vector delivery of alpha-sarcoglycan and treatment of muscular dystrophy.SOLUTION: Described herein are methods of treating muscular dystrophy in a subject, comprising administration of a recombinant AAV vector AAVrh74.tMCK.hSCGA using a systemic route of administration and at a dose of about 1.0×1012 vg / kg to about 5.0×1015 vg / kg. Further disclosed are methods of expressing alpha-sarcoglycan gene in a cell or in a subject in need thereof, decreasing a serum CK level, and increasing alpha-sarcoglycan positive fibers in muscle tissue of a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 62 / 889,749 filed on 21 August 2019, U.S. Provisional Application No. 63 / 014,934 filed on 24 April 2020, and U.S. Patent Application No. 63 / 022,843 filed on 11 May 2020, all of which are incorporated herein by reference in their entirety.

[0002] This specification describes therapeutic vectors, such as AAV vectors expressing alpha-sarcoglycans, and methods for treating limb-girdle muscular dystrophy, such as LGMD2D, using these vectors.

[0003] Inclusion by referencing the sequence list This application, as a separate part of the disclosure, provides a computer-readable sequence listing. (September 24, 2024 Created in: File name: 140056-0691_SL.xml, 29,783 bytes, XML This includes a text file, which is incorporated herein by reference in its entirety. [Background technology]

[0004] Muscular dystrophy (MD) is a group of genetic disorders characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorder varies in terms of the distribution and extent of muscle weakness (some forms of MD also affect the myocardium), age of onset, rate of progression, and mode of inheritance.

[0005] One group of muscular dystrophy (MD) is limb-girdle muscular dystrophy (LGMD). LGMD is a rare condition, and symptoms vary from person to person in terms of age of onset, area of ​​muscle weakness, involvement of the heart and respiratory system, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or later. Both sexes are equally affected. LGMD causes weakness in the shoulder and pelvic girdle, and the muscles near the upper limbs and arms may also weaken over time. Leg weakness often appears before arm weakness. Facial muscles are usually unaffected. As the condition progresses, people may have difficulty walking and may need to use a wheelchair over time. When the shoulder and arm muscles are involved, it may become difficult to raise the arms overhead or lift objects. Depending on the type of LGMD, the heart and respiratory muscles may be involved.

[0006] Specialized testing for LGMD is now available through the National Commissioning Group (NCG), a nationwide program for diagnosis.

[0007] LGMD subtype 2D (LGMD2D), often called α-sarcoglycanopathy, is an autosomal recessive disorder caused by mutations in the alpha-sarcoglycan gene (SGCA; α-sarcoglycan), resulting in the complete or reduced loss of a functional protein, along with the loss of other structural components of the dystrophin-associated protein complex. In particular, the loss of the alpha-sarcoglycan protein leads to a progressive muscular dystrophy with declining muscle function, which develops between the ages of 3 and 8. Symptoms include delayed gait, proximal muscle weakness due to fat replacement and fibrosis, elevated creatine kinase levels, scoliosis, and joint contractures. The debilitating disease often leads to wheelchair dependence and death due to respiratory failure. Therefore, there remains a need for treatment of LGMD2D. [Overview of the Initiative] [Means for solving the problem]

[0008] Described herein is a method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, wherein rAAV is administered via a systemic route, based on superhelical DNA or plasmid as a quantitative standard, at a dose of approximately 1.0 × 10⁶. 12 vg / kg ~ approx. 5.0×10 15 It is administered in a dose of vg / kg. In one embodiment, the disclosure relates to a method for delivering alpha-sarcoglycan to muscles by rAAV expressing the alpha-sarcoglycan gene, thereby reducing and / or preventing fibrosis, and / or increasing muscle strength, and / or treating alpha-sarcoglycanopathy in subjects suffering from muscular dystrophy.

[0009] In one embodiment, what is described herein is a recombinant AAV (rAAV) comprising a polynucleotide sequence encoding an alpha-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 shown in SEQ ID NO: 1, and encodes a protein that retains alpha-sarcoglycan activity. In some embodiments, the polynucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, 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 shown in SEQ ID NO: 2. In another embodiment, the polynucleotide encodes a protein containing the amino acid sequence shown in SEQ ID NO: 2.

[0010] In some embodiments, the rAAV comprises a nucleotide sequence comprising a tMCK promoter. For example, the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO: 3. Additionally, 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 polyA sequence of SEQ ID NO: 7. In one embodiment, the disclosed rAAV is an rAAV of serotype AAVrh.74.

[0011] 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 shown in SEQ ID NO: 4.

[0012] The present disclosure provides a method of doing so in a subject that requires treatment of muscular dystrophy, 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.hSCGA and the rAAV is administered using a systemic administration route.

[0013] In any of the disclosed methods, the rAAV is based on supercoiled DNA or plasmid as a quantitative standard, about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg. For example, the rAAV is based on supercoiled DNA or plasmid as a quantitative standard, 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 2.0×1013 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 It is administered at a dose of vg / kg, or rAAV is approximately 5 × 10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, 2 × 10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, or approximately 5 × 10 14 It is administered at a dose of vg / kg.

[0014] In another embodiment, in one of the disclosed methods, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, resulting in approximately 1.85 × 10⁶ units. 13 vg / kg or 7.41 × 10 13 It is administered at a dose of vg / kg. For example, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, approximately 1.0 × 10⁶ 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 It is administered at a dose of vg / kg.

[0015] In addition, in any of the disclosed methods, the systemic route of administration is an intravenous route. For example, in any of the disclosed methods, rAAV is administered by injection, infusion, or implantation. In some embodiments, rAAV is administered by an intravenous route via peripheral limb veins.

[0016] 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).

[0017] In an exemplary embodiment, a method for treating muscular dystrophy includes administering rAAV to a subject suffering from limb-girdle muscular dystrophy, where rAAV is measured approximately 5 × 10⁻¹⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14 Administered by intravenous infusion at a dose of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0018] In exemplary embodiments, the present disclosure provides a method for treating muscular dystrophy in a subject requiring treatment, the method comprising the step of administering rAAV to a subject, the subject having limb-girdle muscular dystrophy, and the rAAV being approximately 5 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14Administered by intravenous infusion at a dose of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. For example, in these methods, the level of alpha-sarcoglycan gene expression in the target cells increases after rAAV administration compared to the level of alpha-sarcoglycan gene expression before rAAV administration.

[0019] In any of the disclosed methods, the level of alpha-sarcoglycan gene expression in the cells of interest increases after administration of rAAV compared to the level of alpha-sarcoglycan gene expression before administration of rAAV, and / or the serum CK level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV, and / or spontaneous movement and specific force generation increase, fibrosis is reduced, resistance to contraction-induced injury of the tibialis anterior muscle increases, and / or the muscle tissue of the subject The number of alpha-sarcoglycan-positive fibers in the tissue increased after rAAV administration compared to the number of alpha-sarcoglycan-positive fibers before rAAV administration, or fibrosis was reduced in subjects after rAAV administration compared to before rAAV administration, and / or fibrosis was reduced in subjects after rAAV administration compared to before rAAV administration, and / or specific force, fiber diameter size, and / or eccentric contraction in the subject's muscle increased after rAAV administration compared to before rAAV administration.

[0020] In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0021] In another embodiment, the Disclosure provides a method for expressing an alpha-sarcoglycan gene in cells, comprising administering one of the disclosed rAAVs to a target. For example, the Disclosure provides a method for expressing an alpha-sarcoglycan gene in cells, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to a target. In addition, in one of the methods, the expression of an alpha-sarcoglycan gene in cells is detected by measuring alpha-sarcoglycan protein levels by Western blotting in a muscle biopsy. Alternatively, in one of the methods, the expression of an alpha-sarcoglycan gene in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of an alpha-sarcoglycan gene is measured in a target by detecting the number of vector genomes per microgram of genomic DNA.

[0022] This disclosure provides a method for reducing serum CK levels in subjects requiring such reduction, the method comprising administering one of the disclosed rAAVs to a subject. For example, this disclosure provides a method for reducing serum CK levels in subjects requiring such reduction, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to a subject.

[0023] In another aspect, the Disclosure provides a method for increasing alpha-sarcoglycan-positive fibers in target muscle tissue, comprising administering one of the disclosed rAAVs to target. For example, the Disclosure provides a method for doing so in target muscle tissue that requires an increase in alpha-sarcoglycan-positive fibers, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to target.

[0024] This disclosure also provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, including administering one of the disclosed rAAVs to the subject. For example, this disclosure provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to the subject. In addition, in any of the disclosed methods, alpha-sarcoglycan gene expression in the cells of the subject is detected by measuring alpha-sarcoglycan protein levels by Western blotting in a muscle biopsy. Alternatively, in any of the methods, alpha-sarcoglycan gene expression in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, alpha-sarcoglycan gene expression is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0025] This disclosure provides compositions for treating muscular dystrophy in subjects requiring such treatment, comprising one of the rAAVs disclosed herein, and formulated for systemic administration. In particular, in one of the compositions, the rAAV is AAVrh74.tMCK.hSCGA.

[0026] Any of the disclosed compositions, based on superhelical DNA or plasmids as a quantitative standard, yields approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 15 Contains rAAV in doses of vg / kg. For example, rAAV is approximately 1.0 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 The dose of vg / kg, or rAAV, is approximately 5 × 10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, or approximately 5 × 10 14 The dosage is in 1g / kg.

[0027] In another embodiment, in any of the disclosed compositions, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, resulting in approximately 1.85 × 10⁶ units. 13 vg / kg or approximately 7.41 × 10 13 It is administered at a dose of vg / kg. For example, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, approximately 1.0 × 10⁶ 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13vg / kg, or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg and is administered at a dosage of

[0028] In addition, any of the disclosed compositions is formulated for administration by an intravenous route, such as a composition formulated for administration by injection, infusion, or transplantation. In some embodiments, the disclosed compositions are formulated for administration by an intravenous route via a peripheral limb vein.

[0029] Any of the disclosed compositions is for the treatment of limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0030] In an exemplary embodiment, the present disclosure provides a composition for treating a subject suffering from limb-girdle muscular dystrophy, the composition comprising a dosage of rAAV based on supercoiled DNA or plasmid as a quantitative standard, from about 5×10 13 vg / kg to about 2×10 14 vg / kg, the composition is formulated for administration by intravenous infusion, and the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 4.

[0031] In addition, the present disclosure provides a composition for performing it in a subject that requires treating limb-girdle muscular dystrophy, the composition comprising a dosage of rAAV based on supercoiled DNA or plasmid as a quantitative standard, from about 5×10 13 vg / kg to about 2×10 14 vg / kg, the composition is formulated for administration by intravenous infusion, and the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 4. For example, administration of the composition increases the level of alpha-sarcoglycan gene expression in the cells of the subject as compared to the level of alpha-sarcoglycan gene expression prior to administration of the composition.

[0032] In addition, administration of any of the disclosed compositions increases the level of alpha-sarcoglycan gene expression in cells of interest compared to the level of alpha-sarcoglycan gene expression before administration of the composition, and / or administration of the disclosed compositions decreases the serum CK level in the subject compared to the serum CK level before administration of the composition, and / or increases spontaneous movement and specific force generation, reduces fibrosis, increases resistance to contraction-induced injury of the tibialis anterior muscle, and / or administration of the compositions increases the number of alpha-sarcoglycan-positive fibers in the muscle tissue of the subject compared to the number of alpha-sarcoglycan-positive fibers before administration of the composition, and / or administration of the compositions reduces fibrosis in the subject compared to before administration of rAAV, and / or the compositions reduce fibrosis compared to before administration of the composition, or administration of the compositions increases specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject compared to before administration of the composition. In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0033] In another embodiment, the Disclosure provides compositions for expressing alpha-sarcoglycan genes in cells, the compositions comprising any of the disclosed rAAVs. For example, the Disclosure provides compositions for expressing alpha-sarcoglycan genes in cells comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. In addition, in any of the compositions, the expression of alpha-sarcoglycan genes in the cells of interest is detected by measuring alpha-sarcoglycan protein levels by Western blotting in muscle biopsy. Alternatively, in any of the methods, the expression of alpha-sarcoglycan genes in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in muscle biopsy. In other embodiments, the expression of alpha-sarcoglycan genes is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0034] This disclosure provides compositions for achieving a reduction in serum CK levels in subjects requiring such reduction, the compositions comprising one of the disclosed rAAVs. For example, this disclosure provides compositions for achieving a reduction in serum CK levels in subjects requiring such reduction, the compositions comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0035] In another embodiment, the Disclosure provides a composition for increasing alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the composition comprising one of the disclosed rAAVs. For example, the Disclosure provides a composition for increasing alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the composition comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0036] The present disclosure also provides a composition for effecting the same in a subject that requires an increase in the expression of alpha-sarcoglycan, the composition comprising any of the disclosed rAAVs. For example, the present disclosure provides a composition for effecting the same in a subject that requires an increase in the expression of alpha-sarcoglycan, the composition comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. Additionally, following administration of any of the disclosed compositions, the expression of the alpha-sarcoglycan gene in the cells of the subject is detected by measuring the alpha-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, following administration of any of the disclosed compositions, the expression of the alpha-sarcoglycan gene in the cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, following administration of any of the disclosed compositions, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0037] The present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for effecting the same in a subject that requires treatment of muscular dystrophy, the medicament being formulated for administration by the systemic route. In particular, the present disclosure provides the use of AAVrh74.tMCK.hSCGA for the preparation of a medicament for treating muscular dystrophy, the medicament being formulated for administration by the systemic route of administration.

[0038] In any of the disclosed uses, the medicament comprises rAAV at a dose of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg based on supercoiled DNA or plasmid as a quantification standard. For example, the rAAV is at a dose 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 15vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 The dose of vg / kg, or rAAV, is approximately 5 × 10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, or approximately 5 × 10 14 It is located at vg / kg.

[0039] In another embodiment, in any of the disclosed uses, the pharmaceutical is based on linearized DNA or plasmid as a quantitative standard, yielding approximately 1.85 × 10⁻¹⁶ units. 13 vg / kg or 7.41 × 10 13 The drug contains rAAV in a dose of vg / kg. For example, the drug is based on linearized DNA or plasmid as a quantitative standard, approximately 1.0 × 10⁻⁶. 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×1013 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 Includes rAAV at a dose of vg / kg.

[0040] In addition, in any of the disclosed uses, the medicament is formulated for administration via an intravenous route. For example, in any of the disclosed uses, the medicament is formulated for administration by injection, infusion, or implantation. In some embodiments, the medicament is formulated for administration via an intravenous route through peripheral limb veins.

[0041] In any of the disclosed uses, the medicament is for the treatment of limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0042] In exemplary embodiments, the present disclosure provides the use of rAAV for the preparation of a pharmacopoeia for the treatment of limb-girdle muscular dystrophy, the pharmacopoeia being formulated for administration by intravenous infusion, and rAAV being approximately 5 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14 In doses of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. For example, administration of the drug to a subject requiring drug administration results in an increase in alpha-sarcoglycan gene expression in the target cells compared to the level of alpha-sarcoglycan gene expression before rAAV administration.

[0043] In any of the disclosed uses, administration of the pharmacoglycan to a subject requiring pharmacoglycan administration 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 pharmacoglycan, and / or administration of the pharmacoglycan to the subject results in a decrease in serum CK levels in the subject compared to the serum CK levels before administration of the pharmacoglycan, and / or increased spontaneous movement and specific force generation, reduced fibrosis, increased resistance to contraction-induced injury of the tibialis anterior muscle, and / or administration of the pharmacoglycan 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 pharmacoglycan, and / or administration of the pharmacoglycan to a subject requiring pharmacoglycan administration results in reduced fibrosis in the subject compared to before administration of the pharmacoglycan, and / or increased specific force, fiber diameter size, and / or eccentric contraction in the subject's muscle compared to before administration of the pharmacoglycan. In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0044] In another embodiment, the Disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmaceutical for doing so in a subject requiring the expression of an alpha-sarcoglycan gene in cells. For example, the Disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical for doing so in a subject requiring the expression of an alpha-sarcoglycan gene in cells, 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 alpha-sarcoglycan gene in the cells of interest is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. Alternatively, in any of the uses, the expression of the alpha-sarcoglycan gene in cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0045] This disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmacopoeia to achieve a reduction in serum CK levels in subjects requiring such reduction. For example, this disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmacopoeia to achieve a reduction in serum CK levels in subjects requiring such reduction, the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4.

[0046] In another aspect, the Disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmacopoeia to increase alpha-sarcoglycan-positive fibers in a muscle tissue of interest. For example, the Disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmacopoeia to increase alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the scAAVrh74.tMCK.hSGCA construct comprising the nucleotide sequence of SEQ ID NO: 4.

[0047] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a pharmaceutical to do so in subjects requiring increased alpha-sarcoglycan expression. For example, this disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical to do so in subjects requiring increased alpha-sarcoglycan expression, 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 alpha-sarcoglycan gene in the cells of interest is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. Alternatively, in any of the disclosed uses, the expression of the alpha-sarcoglycan gene in cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0048] In any of the disclosed methods, compositions, or uses, the subjects are human subjects aged 4 to 15 years, or human subjects aged 25 to 55 years, or human subjects over 50 years.

[0049] In any of the disclosed methods, compositions, or uses, the subjects are children, adolescents, or young adults. Alternatively, the subjects are middle-aged adults or elderly.

[0050] For example, in any of the disclosed methods, compositions, or uses, the subjects are human subjects aged 4 to 15 years, having alpha-sarcoglycan (SGCA) mutations confirmed in both alleles, being negative for AAVrh74 antibody, and / or having performed a 100-meter walking test of more than 40% or the usual rate.

[0051] In another embodiment, the Disclosure provides a method for generating rAAV administered by any of the disclosed methods, compositions, or uses, the method comprising transferring an AAV vector plasmid into a host cell, the AAV vector plasmid comprising 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: 8, or the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4, or 8.

[0052] In any of the disclosed methods for generating rAAV, the method further comprises transferring a packaging plasmid and / or a helper virus into a host cell. In addition, in any of the disclosed methods for generating rAAV, the packaging cell comprises a stably integrated AAV cap gene and / or the packaging cell comprises a stably integrated AAV rep gene.

[0053] In another embodiment, the disclosure provides a host cell comprising an AAV vector plasmid containing 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. For example, the host cell comprises an AAV vector plasmid containing the nucleotide sequence of SEQ ID NO: 1, 4, or 8. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, wherein the rAAV is administered via a systemic route, based on superhelical DNA or plasmid as a quantitative standard, to a quantity of approximately 1.0 × 10⁻⁶.12 vg / kg ~ approx. 5.0×10 15 The method involves administering the drug at a dose of vg / kg. (Item 2) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10⁻⁶ 13 vg / kg ~ approx. 2×10 14 The method described in item 1, administered at a dose of vg / kg. (Item 3) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 The method described in item 1, administered at a dose of vg / kg. (Item 4) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 The method described in item 1, administered at a dose of vg / kg. (Item 5) A method for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, wherein the rAAV is administered via a systemic route and is measured in approximately 1.85 × 10⁻¹⁶ units based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 7.41×10 13 The method involves administering the drug at a dose of vg / kg. (Item 6) The method according to any one of items 1 to 5, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of rAAV compared to the level of alpha-sarcoglycan gene expression before administration of rAAV, the serum CK level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV, spontaneous movement and specific force generation increase, fibrosis is reduced, resistance to contraction-induced injury of the tibialis anterior muscle increases, and / or the number of alpha-sarcoglycan-positive fibers in the muscle tissue of the subject increases after administration of rAAV compared to the number of alpha-sarcoglycan-positive fibers before administration of rAAV. (Item 7) The method according to any one of items 1 to 6, wherein the systemic administration route is an intravenous route. (Item 8) The method according to any one of items 1 to 7, wherein the rAAV is administered by injection, infusion, or implantation. (Item 9) The method according to any one of items 1 to 8, wherein the rAAV is administered by infusion. (Item 10) The method according to any one of items 1 to 9, wherein the rAAV is administered via an intravenous route through the veins of the peripheral limbs. (Item 11) The method according to any one of items 1 to 10, wherein the rAAV comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. (Item 12) The method according to item 11, wherein the rAAV comprises the nucleotide sequence of sequence number 1. (Item 13) The method according to any one of items 1 to 12, wherein the rAAV comprises a nucleotide sequence encoding a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. (Item 14) The method according to any one of items 1 to 12, wherein the rAAV comprises a nucleotide sequence encoding the polypeptide sequence shown in Sequence ID No. 2. (Item 15) The method according to any one of items 1 to 14, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. (Item 16) The method according to item 15, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4. (Item 17) The method according to any one of items 1 to 16, wherein the rAAV includes a tMCK promoter. (Item 18) The method according to any one of items 1 to 16, wherein the tMCK promoter includes the nucleotide sequence shown in SEQ ID NO: 3. (Item 19) The method according to any one of items 1 to 18, wherein the rAAV includes the 5' inverted terminal repeat sequence of sequence number 5. (Item 20) The method according to any one of items 1 to 19, wherein the rAAV contains the 3' inverted terminal repeat sequence of sequence number 6. (Item 21) The method according to any one of items 1 to 20, wherein the rAAV includes the polyA sequence of sequence number 7. (Item 22) The method according to any one of items 1 to 21, wherein the rAAV is of serotype AAVrh.74. (Item 23) The method according to any one of items 1 to 22, wherein the muscular dystrophy is limb-girdle muscular dystrophy. (Item 24) The method according to any one of items 1 to 23, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D). (Item 25) The subject suffers from limb-girdle muscular dystrophy, and the rAAV is approximately 5 × 10⁻¹⁶ based on the superhelical DNA or plasmid used as the quantitative standard. 13 vg / kg ~ approx. 2×10 14 The method according to any one of items 1 to 24, wherein the rAAV is administered by intravenous infusion at a dose of vg / kg, and the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. (Item 26) The method according to any one of items 1 to 25, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of rAAV compared to the level of alpha-sarcoglycan gene expression before administration of rAAV. (Item 27) The method according to any one of items 1 to 26, wherein fibrosis is reduced in the subject after administration of rAAV compared to before administration of rAAV. (Item 28) The method according to any one of items 1 to 26, wherein the fibrosis, centronucleation, CK levels, and / or collagen deposition in the subject are reduced after administration of the rAAV compared to before administration of the rAAV. (Item 29) The method according to any one of items 1 to 26, wherein the specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject increase after administration of the rAAV compared to before administration of the rAAV. (Item 30) The method according to item 26, wherein the alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry. (Item 31) A method for expressing an alpha-sarcoglycan gene in cells, comprising administering the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO: 4 to the target. (Item 32) The method according to item 31, wherein the expression of the alpha-sarcoglycan gene in the target cells is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. (Item 33) The method according to item 31, wherein the expression of the alpha-sarcoglycan gene in the cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. (Item 34) The method according to item 31, wherein the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA. (Item 35) A method for reducing serum CK levels in a subject requiring such reduction, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to the subject. (Item 36) A method for increasing alpha-sarcoglycan-positive fibers in target muscle tissue, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4 to the target. (Item 37) A method for increasing the expression of alpha-sarcoglycans in a subject requiring such increase, comprising administering an effective amount of the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to the subject. (Item 38) A composition for treating muscular dystrophy in subjects requiring treatment, wherein the composition contains recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, based on superhelical DNA or plasmid as a quantitative standard, in a quantity of approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 5.0×10 15 A composition comprising a dose of vg, wherein the composition is formulated for systemic administration routes. (Item 39) A composition for treating muscular dystrophy in subjects requiring treatment, wherein the composition contains recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA based on linearized DNA or plasmid as a quantitative standard, in a quantity of approximately 1.85 × 10⁻⁶. 13 vg / kg or 7.41 × 10 13 A composition comprising vg / dose, wherein the composition is formulated for a systemic route of administration. (Item 40) The composition comprises recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an scAAVrh74.tMCK.hSGCA construct nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. (Item 41) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 The composition described in item 38 or 40, in a dose of vg / kg. (Item 42) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 The composition described in item 38 or 40, in a dose of vg / kg. (Item 43) The composition according to any one of items 38 to 42, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the composition compared to the level of alpha-sarcoglycan gene expression before administration of the composition, or the serum CK level in the target of the composition decreases after administration of the composition compared to the serum CK level before administration of the composition, or the number of alpha-sarcoglycan-positive fibers in the muscle tissue of the target increases after administration of the composition compared to the number of alpha-sarcoglycan-positive fibers before administration of the composition. (Item 44) The composition according to any one of items 38 to 43, wherein the systemic administration route is an intravenous route. (Item 45) The composition according to any one of items 38 to 44, wherein the composition is administered by injection, infusion, or implantation. (Item 46) The composition according to any one of items 38 to 45, wherein the composition is formulated for administration by infusion. (Item 47) The composition according to any one of items 38 to 46, wherein the composition is formulated for administration via an intravenous route through peripheral limb veins. (Item 48) The composition according to any one of items 38 to 47, wherein the rAAV comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. (Item 49) The composition according to item 48, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 1. (Item 50) The composition according to any one of items 38 to 49, wherein the rAAV comprises a nucleotide sequence encoding a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. (Item 51) The composition according to any one of items 38 to 49, wherein the rAAV comprises a nucleotide sequence encoding the polypeptide sequence shown in Sequence ID No. 2. (Item 52) The composition according to any one of items 38 to 49, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. (Item 53) The composition according to item 52, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4. (Item 54) The composition according to any one of items 38 to 53, wherein the rAAV comprises a tMCK promoter. (Item 55) The composition according to item 54, wherein the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO: 3. (Item 56) The composition according to any one of items 38 to 55, wherein the rAAV comprises the 5' inverted terminal repeat sequence of SEQ ID NO: 5. (Item 57) The composition according to any one of items 38 to 56, wherein the rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO: 6. (Item 58) The composition according to any one of items 38 to 57, wherein the rAAV comprises the polyA sequence of sequence number 7. (Item 59) The composition according to any one of items 38 to 58, wherein the rAAV is of serotype AAVrh.74. (Item 60) The composition according to any one of items 38 to 59, wherein the muscular dystrophy is limb-girdle muscular dystrophy. (Item 61) The composition according to any one of items 38 to 60, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D). (Item 62) The subject suffers from limb-girdle muscular dystrophy, and the composition, based on the superhelical DNA or plasmid as the quantitative standard, yields approximately 5 × 10⁻¹⁴ units. 13 vg / kg ~ approx. 2×10 14 A composition according to any one of items 38 to 61, formulated for administration by intravenous infusion at a dose of vg / kg, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. (Item 63) The composition according to any one of items 38 to 62, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the composition compared to the level of alpha-sarcoglycan gene expression before administration of the composition. (Item 64) A composition according to any one of items 38 to 63, wherein fibrosis is reduced in the subject after administration of the composition compared to before administration of the composition. (Item 65) The composition according to any one of items 38 to 63, wherein fibrosis, centronucleation, CK levels, and / or collagen deposition in the subject are reduced after administration of the composition compared to before administration of the composition. (Item 66) The composition according to any one of items 38 to 65, wherein the specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject increase after administration of the composition compared to before administration of the composition. (Item 67) The composition according to item 63, wherein the alpha-sarcoglycan gene expression is detected by measuring the alpha-sarcoglycan protein level by Western blotting and / or immunohistochemistry. (Item 68) A composition for expressing an alpha-sarcoglycan gene in cells, wherein the composition comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4. (Item 69) The composition according to item 68, wherein the expression of the alpha-sarcoglycan gene in the target cells is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. (Item 70) The composition according to item 68, wherein the expression of the alpha-sarcoglycan gene in the cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. (Item 71) The composition according to item 68, wherein the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA. (Item 72) A composition for achieving a reduction in serum CK levels in a subject requiring such reduction, comprising the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO: 4. (Item 73) A composition for increasing alpha-sarcoglycan-positive fibers in target muscle tissue, comprising the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of the aforementioned SEQ ID NO: 4. (Item 74) A composition for increasing alpha-sarcoglycan expression in subjects requiring it, comprising the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO: 4. (Item 75) The use of recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA A for the preparation of a pharmaceutical for treating muscular dystrophy in subjects requiring such treatment, wherein the rAAV is measured in approximately 1.0 × 10⁻¹⁶ units based on superhelical DNA or plasmid as the quantitative standard. 12 vg / kg ~ approx. 5.0×10 15 The dosage is vg / kg, and the pharmaceutical product is formulated for use via a systemic route of administration. (Item 76) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10⁻⁶ 13 vg / kg ~ approx. 2×1014 Use as described in item 75 for the VG / kg dosage. (Item 77) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 Use as described in item 75 for the VG / kg dosage. (Item 78) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 Use as described in item 75 for the VG / kg dosage. (Item 79) The use of recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA A for the preparation of a pharmacopoeia for the treatment of muscular dystrophy in subjects requiring such treatment, wherein the rAAV is obtained based on linearized DNA or plasmid as the quantitative standard, resulting in approximately 1.85 × 10⁻⁶ units. 13 vg / kg or 7.41 × 10 13 The dosage is vg / kg, and the pharmaceutical product is formulated for use via a systemic route of administration. (Item 80) The use according to any one of items 75 to 79, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the drug compared to the level of alpha-sarcoglycan gene expression before administration of the drug, or the serum CK level in the subject decreases after administration of the drug compared to the serum CK level before administration of the drug, or the number of alpha-sarcoglycan-positive fibers in the muscle tissue of the subject increases after administration of rAAV compared to the number of alpha-sarcoglycan-positive fibers before administration of the drug. (Item 81) The use described in any one of items 75 to 80, wherein the systemic route of administration is an intravenous route. (Item 82) The use of the medicament as described in any one of items 75 to 81, wherein the medicament is formulated for administration by injection, infusion, or transplantation. (Item 83) The use of the aforementioned pharmaceutical product as described in any one of items 75 to 82, wherein the pharmaceutical product is formulated for administration by infusion. (Item 84) The use of the pharmaceutical product as described in any one of items 75 to 83, wherein the pharmaceutical product is formulated for administration via an intravenous route through the veins of the peripheral limbs. (Item 85) The use according to any one of items 75 to 84, wherein the rAAV comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. (Item 86) The use described in item 85, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 1. (Item 87) The use according to any one of items 75 to 86, wherein the rAAV comprises a nucleotide sequence encoding a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. (Item 88) The use according to any one of items 75 to 86, wherein the rAAV comprises a nucleotide sequence encoding the polypeptide sequence shown in SEQ ID NO: 2. (Item 89) The use described in any one of items 75 to 86, wherein the rAAV comprises an scAAVrh74.tMCK.hSGCA construct nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. (Item 90) The use described in item 89, wherein the rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. (Item 91) The use of the aforementioned rAAV, including the tMCK promoter, as described in any one of items 75 to 90. (Item 92) The use described in item 91, wherein the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO: 3. (Item 93) The use described in any one of items 75 to 92, wherein the rAAV includes the 5' inverted terminal repeat sequence of sequence number 5. (Item 94) The use described in any one of items 75-93, wherein the rAAV contains the 3' inverted terminal repeat sequence of sequence number 6. (Item 95) The use described in any one of items 73 to 94, wherein the rAAV includes the sequence number 7 polyA sequence. (Item 96) The use described in any one of items 73-95, wherein the rAAV is of serotype AAVrh.74. (Item 97) The use described in any one of items 73 to 96, wherein the muscular dystrophy is limb-girdle muscular dystrophy. (Item 98) The use described in any one of items 73 to 97, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D). (Item 99) The subject suffers from limb-girdle muscular dystrophy, the drug is formulated for intravenous infusion, and the rAAV is measured based on superhelical DNA or plasmid as a quantitative standard, approximately 5 × 10⁻¹⁶ 13 vg / kg ~ approx. 2×10 14 The use described in any one of items 73 to 98, wherein the rAAV is in a dose of vg / kg and contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4. (Item 100) The use according to any one of items 73 to 99, wherein the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the drug compared to the level of alpha-sarcoglycan gene expression before administration of the drug. (Item 101) The use described in any one of items 73 to 100, wherein fibrosis is reduced in the subject after administration of the drug compared to before administration of the drug. (Item 102) The use according to any one of items 73 to 100, wherein fibrosis, centronucleation, CK levels, and / or collagen deposition in the subject are reduced after administration of the pharmacopoeia compared to the fibrosis before administration of the pharmacopoeia. (Item 103) The use according to any one of items 72 to 102, wherein the specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject increase after administration of the drug compared to before administration of the drug. (Item 104) The use described in item 100, wherein the alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry. (Item 105) Use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical for expressing the alpha-sarcoglycan gene in cells, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. (Item 106) The use described in item 105, wherein the expression of the alpha-sarcoglycan gene in the target cells is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. (Item 107) The use described in item 105, wherein the expression of the alpha-sarcoglycan gene in the cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. (Item 108) The use described in item 105, wherein the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA. (Item 109) Use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical product for reducing serum CK levels in subjects requiring such reduction, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. (Item 110) Use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical for increasing alpha-sarcoglycan-positive fibers in target muscle tissue, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. (Item 111) Use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical product for increasing alpha-sarcoglycan expression in subjects requiring such increase, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 4. (Item 112) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a human subject aged 4 to 15 years. (Item 113) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a child, an adolescent, or a young adult. (Item 114) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a human subject aged 4 to 15 years, has an alpha-sarcoglycan (SGCA) mutation confirmed in both alleles, is negative for AAVrh74 antibody, and / or has performed a 100-meter walking test of more than 40% or the usual rate. (Item 115) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a middle-aged adult or an elderly person. (Item 116) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a human subject aged 25 to 55 years. (Item 117) The method, composition, or use described in any one of items 1 to 111, wherein the subject is a human being 50 years of age or older. (Item 118) A method for generating the rAAV administered in use by any one of items 1 to 117, comprising transferring an AAV vector plasmid into 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. (Item 119) The method according to item 118, wherein the AAV vector plasmid contains the nucleotide sequence of SEQ ID NO: 8. (Item 120) The method according to item 118 or 119, wherein 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. (Item 121) The method according to item 118 or 119, wherein the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4, or 8. (Item 122) The method according to any one of items 118 to 121, further comprising transferring a packaging plasmid and / or a helper virus into the host cell. (Item 123) The method according to any one of items 118-122, wherein the packaging cells contain a stably incorporated AAVcap gene. (Item 124) The method according to any one of items 118 to 123, wherein the packaging cells contain a stably incorporated AAVrep gene. (Item 125) A host cell containing an AAV vector plasmid containing a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 1, 4, or 8. (Item 126) The host cell described in item 125, wherein the AAV vector plasmid contains the nucleotide sequence of SEQ ID NO: 1, 4, or 8. [Brief explanation of the drawing]

[0054] [Figure 1] scAAVrh74.tMCK.h shows the SGCA gene cassette. [Figure 2-1] This shows the expression of the transgene in a dose-escalation study after systemic treatment with scAAVrh74.tMCK.hSGCA. (A) Alpha-sarcoglycan immunofluorescence staining (n=6 per group) of multiple muscle cells from mice systemically (intravenously) treated with 1×10¹²vg, 3×10¹²vg, and 6×10¹²vg (or 5×10¹³vg / kg, 1×10¹⁴vg / kg, and 2×10¹⁴vg / kg, respectively, based on 20g mice). At 12 weeks post-treatment, muscle fibers expressing alpha-sarcoglycan ranged from 70% to 93% compared to untreated controls. (B) Western blotting of muscle cells from treated sgca- / - mice confirms the expression of the hSGCA protein. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; TRI, triceps brachii; GLUT, gluteal muscle; PSO, psoas major; DIA, diaphragm; HRT, heart; WT, wild type. [Figure 2-2]This shows the expression of the transgene in a dose-escalation study after systemic treatment with scAAVrh74.tMCK.hSGCA. (A) Alpha-sarcoglycan immunofluorescence staining (n=6 per group) of multiple muscle cells from mice systemically (intravenously) treated with 1×10¹²vg, 3×10¹²vg, and 6×10¹²vg (or 5×10¹³vg / kg, 1×10¹⁴vg / kg, and 2×10¹⁴vg / kg, respectively, based on 20g mice). At 12 weeks post-treatment, muscle fibers expressing alpha-sarcoglycan ranged from 70% to 93% compared to untreated controls. (B) Western blotting of muscle cells from treated sgca- / - mice confirms the expression of the hSGCA protein. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; TRI, triceps brachii; GLUT, gluteal muscle; PSO, psoas major; DIA, diaphragm; HRT, heart; WT, wild type. [Figure 3-1] This shows dose-independent improvement of muscle morphology by scAAVrh74.tMCK.hSGCA in sgca- / - mice. (A) Hematoxylin and eosin images of various muscles from sgca- / - mice treated with scAAVrh74.tMCK.hSGCA at 1×10¹²vg, 3×10¹²vg, and 6×10¹²vg (or 5×10¹³vg / kg, 1×10¹⁴vg / kg, and 2×10¹⁴vg / kg, respectively, based on 20g mice). Representative 20x images show a dramatic reduction in centrifugal nuclei and overall normalization of fiber size, independent of treatment dose. (B) Quantification (n=6 per group) confirming normalization of muscle fiber diameter in various muscles in the treatment group compared to vehicle-treated mice and wild-type controls. (C) Quantification of central muscle nuclei in treated mice compared to untreated mice and wild-type controls (n=6 per group). Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; GLUT, gluteus; PSO, psoas major; TRI, triceps brachii; DIA, diaphragm; WT, wild-type. [Figure 3-2]This shows dose-independent improvement of muscle morphology by scAAVrh74.tMCK.hSGCA in sgca- / - mice. (A) Hematoxylin and eosin images of various muscles from sgca- / - mice treated with scAAVrh74.tMCK.hSGCA at 1×10¹²vg, 3×10¹²vg, and 6×10¹²vg (or 5×10¹³vg / kg, 1×10¹⁴vg / kg, and 2×10¹⁴vg / kg, respectively, based on 20g mice). Representative 20x images show a dramatic reduction in centrifugal nuclei and overall normalization of fiber size, independent of treatment dose. (B) Quantification (n=6 per group) confirming normalization of muscle fiber diameter in various muscles in the treatment group compared to vehicle-treated mice and wild-type controls. (C) Quantification of central muscle nuclei in treated mice compared to untreated mice and wild-type controls (n=6 per group). Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; GLUT, gluteus; PSO, psoas major; TRI, triceps brachii; DIA, diaphragm; WT, wild-type. [Figure 4] This shows the reduction of fibrosis in sgca- / - mice treated with scAAVrh74.tMCK.hSGCA. (a) Piclosirius red staining shows the reduction of fibrosis in mice treated with scAAVrh74.tMCK.hSGCA, indicated by a decrease in collagen deposition in various muscles compared to sgca- / - mice treated with vehicle, in representative 20x images shown. (B) Quantification of collagen levels in various muscles confirms a decrease in collagen levels in all three treatment groups compared to untreated mice and wild-type controls (n=6 per group). Abbreviations: PSO, psoas major; DIA, diaphragm; TRI, triceps brachii; GLUT, gluteal muscle. [Figure 5]This study demonstrates the functional benefits of skeletal muscle after treatment with scAAVrh74.tMCK.hSGCA. (A) After 3 months of treatment, tibialis anterior (TA) muscles were harvested (both left and right) and specific force and resistance to contraction-induced damage were measured (standardized relative to TA weight). Quantification of specific force and eccentric contraction increased in all treatment groups (minimal differences between doses) compared to untreated controls (n=6 per group). (B) Diaphragmatic striae were harvested and specific force was measured. After 12 weeks of treatment, force was significantly increased in treated mice compared to untreated sgca- / - mice. (C) After 12 weeks of treatment, open-field analysis showed improvements in walking and vertical activity in treated mice compared to untreated sgca- / - controls (n=6 per group). (D) Serum creatine kinase levels decreased in all treatment groups compared to untreated sgca- / - controls. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc analysis for multiple comparisons. Unless otherwise noted, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 compared to vehicle-treated sgca- / - mice. Abbreviations: TA, tibialis anterior; DIA, diaphragm; WT, wild-type. [Figure 6] No evidence of toxicity was shown by blood chemistry after treatment with scAAVrh74.tMCK.hSGCA. Toxicity of liver enzymes (ALT, AST, and ALP / K) and blood glucose (GLU) levels was analyzed (n=6 per group). All chemical values ​​of the treated mice were within the normal / healthy limits for mice, as indicated by the dotted line. [Figure 7A] This shows a biodistribution analysis of the whole-body scAAVrh74.tMCK.hSGCA delivery-distribution histogram of the mean vg copies of transcript per microgram of DNA added to quantitative polymerase chain reactions in various tissues from sgca- / - mice after intravenous delivery of scAAVrh74.tMCK at 3×10¹²vg and 6×10¹²vg (or 1×10¹⁴vg / kg and 2×10¹⁴vg / kg, respectively, based on 20g mice) of scAAVrh74.tMCK.hSGCA delivery-distribution histogram. [Figure 7B]Western blots of alpha-sarcoglycan protein expression in the liver of WT and sgca- / - mice treated with either scAArh74.tMCK.hSGCA in vehicle (sgca- / -LR (Lactated Ringer)) or 1.0 × 10¹²vg (left Western blot) or 6 × 10¹²vg (right Western blot) are shown. 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 brachii. [Figure 8] Figure 8B shows the expression of scAAVrh74.tMCK.hSGCA in the skeletal muscle of 12-month-old sgca- / - mice by immunofluorescence (Figure 8A) and Western blotting. The graph in Figure 8C shows the percentage of scAAVrh74.tMCK.hSGCA-positive muscle fibers in sgca- / - mice. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; GLUT, gluteus; TRI, triceps brachii; PSOAS, psoas major, diaphragm; WT, wild-type; LRS, Ringer's lactate solution [Figure 9-1] Histological results of SGCA- / - mice administered scAAVrh74.tMCK.hSGCA are shown. Figure 9a shows improved muscle pathology. Figure 9b shows decreased central nucleus formation and increased mean fiber size in the gastrocnemius (GAS) and triceps brachii (TRI) muscles. Figure 9c shows a reduction in the level of fibrosis compared to untreated controls. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; GLUT, gluteus; PSO, psoas major; TRI, triceps brachii; diaphragm; WT, wild type. [Figure 9-2]Histological results of SGCA- / - mice administered scAAVrh74.tMCK.hSGCA are shown. Figure 9a shows improved muscle pathology. Figure 9b shows decreased central nucleus formation and increased mean fiber size in the gastrocnemius (GAS) and triceps brachii (TRI) muscles. Figure 9c shows a reduction in the level of fibrosis compared to untreated controls. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps femoris; GLUT, gluteus; PSO, psoas major; TRI, triceps brachii; diaphragm; WT, wild type. [Figure 10] scAAVrh74.tMCK.h shows functional improvement in aged mice after administration of SGCA. [Modes for carrying out the invention]

[0055] This disclosure is based on the finding that administration of rAAVs containing polynucleotides expressing alpha-sarcoglycans results in reduction or complete recovery of myofibrosis in an animal model of limb-girdle muscular dystrophy. As demonstrated in the examples herein, administration of the rAAVs described herein resulted in a reversal of dystrophic function, including a decrease in CK levels, an increase in muscle strength, improvements in walking and vertical activity, and other motor functions.

[0056] The implementation of this invention will, unless otherwise indicated, utilize conventional methods of virology, microbiology, molecular biology, and recombinant DNA technology, within the scope of the art of those skilled in the art. Such techniques are fully described in the literature, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Current Edition), DNA Cloning: A Practical Approach, Vol. 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, vol. I&II (P. Tijssen, ed.), Fundamental See Virology, 2nd Edition, vol. I & II (BN Fields and DMKnipe, eds.), Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley-Liss, Third Edition), and Ausubel et al. (1991) Current Protocols in Molecular Biology (Wiley Interscience, NY).

[0057] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0058] definition The singular forms "a," "an," and "the" include plural referents unless otherwise specified in the context. For example, a reference to "cells" includes multiple such cells, and a reference to "culture" includes one or more cultures and their equivalents as known to those skilled in the art. A reference to "recombinant AAV" includes a mixture of two or more rAAV virions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0059] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated to refer only to the alternatives, or unless the alternatives are mutually exclusive; however, this disclosure supports the definition that refers only to the alternatives and “and / or.”

[0060] Throughout this application, the term “approximately” is used to indicate that the value includes the statistical experimental error (standard deviation of the error) to the device or method used to determine the value.

[0061] The terms “vector” or “expression vector” refer to any genetic element that can replicate when associated with appropriate regulatory elements and can transfer gene sequences between cells, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions. In one embodiment, the vector is a viral vector. Expression vectors may contain various regulatory sequences, structural genes (e.g., the gene of interest), and nucleic acid sequences that also perform other functions.

[0062] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells, provided with certain functions by co-infecting helper viruses. Currently, there are 13 serotypes of AAV that have been characterized. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all possessing three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the terminals corresponding to "terminal inversion sequences" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0063] The term “AAV vector” refers to one or more polynucleotides (or transgenes) of interest that are adjacent to an AAV terminal repeat sequence (ITR). Such AAV vectors can be replicated and packaged into infectious viral particles when present in host cells transfected with a vector encoding and expressing rep and cap gene products. 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 is preferably a rep and / or cap gene, in which one or more of the AAV wild-type genes are deleted in whole or in part, but which can retain a functional adjacent ITR sequence. Functional ITR sequences are required for the rescue, replication, and packaging of AAV virions. Therefore, AAV vectors are defined herein to contain at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). ITRs do not need to be wild-type nucleotide sequences and can be modified, for example, by nucleotide insertions, deletions, or substitutions, as long as the sequence provides functional rescue, replication, and packaging.

[0064] The term "AAV helper function" refers to AAV-derived coding sequences that can be expressed to provide the AAV gene product that then functions in trans for productive AAV replication. Thus, AAV helper functions include the major AAV open reading frame (ORF), rep, and cap. Rep expression products have been shown to possess many functions, including, among others, recognition, binding, and nicking of the AAV origin of DNA replication, DNA helicase activity, and regulation of transcription from AAV (or other xenogeneic) promoters. Cap expression products provide the necessary packaging function. AAV helper functions are used herein to complement the trans AAV function lost from the AAV vector.

[0065] The term "recombinant virus" means, for example, a virus that has been genetically modified by the addition or insertion of a heterologous nucleic acid sequence into a virus particle.

[0066] The term "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector encapsulated within the capsid. In one embodiment, the AAV virion 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 one embodiment, the production of AAV virus particles includes the production of an AAV vector, for example, the vector is contained within the AAV vector particle. When 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 typically referred to as an "rAAV vector" or simply an "rAAV particle". Thus, the production of AAV vector particles necessarily includes the production of rAAV, and the rAAV genome is contained within the rAAV vector particle.

[0067] For example, wild-type (wt) AAV virus particles contain a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. The AAV virion can be either single-stranded (ss) AAV or self-complementary (SC) AAV. In one embodiment, a single-stranded AAV nucleic acid molecule of either the complementary sense, e.g., either the "sense" or "antisense" strand, can be packaged into the AAV virion, and both strands are equally infectious.

[0068] The term "recombinant AAV" or "rAAV" is defined herein as an infectious replication-defective virus consisting of an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked by AAV ITRs on both sides. In one embodiment, rAAV is produced in a suitable host cell, which has an AAV vector, AAV helper functions, and accessory functions introduced therein. In this way, the host cell can encode the AAV polypeptides required to package an AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0069] The term "transfection" refers to the uptake of foreign DNA by a cell, and the cell is "transfected" when exogenous DNA is introduced into the cell membrane. 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. Using such techniques, one or more exogenous DNA moieties, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into a suitable host cell.

[0070] The term "transduction" means the delivery of a DNA molecule to a recipient cell either in vivo or in vitro via a replication-defective viral vector, e.g., via a recombinant AAV virion.

[0071] The term “host cell” means, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of AAV helper constructs, AAV vector plasmids, accessory functional vectors, or other transfer DNA. This term includes offspring of the transfected original cell. Thus, as used herein, “host cell” generally refers to a cell transfected with an exogenous DNA sequence. It is understood that offspring of a single parent cell may not necessarily be completely identical in morphology or genomic or whole DNA complement due to natural, accidental, or intentional mutations.

[0072] The term “heterogeneous” refers to sequences that are not normally bound together and / or not normally associated with a particular cell, when relating to nucleic acid sequences such as coding and regulatory sequences. Thus, the “heterogeneous” region of a nucleic acid construct or vector is a segment of nucleic acid that is not found in nature in relation to other molecules, and is located within or attached to another nucleic acid molecule. For example, the heterogeneous region of a nucleic acid construct may include a coding sequence adjacent to a coding sequence that is not found in relation to a coding sequence in nature. Another example of a heterogeneous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from those of a native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterogeneous for the purposes of this invention. As used herein, allelic mutations or naturally occurring mutational events do not produce heterogeneous DNA.

[0073] A "coding sequence," or sequence that "codes" a particular protein, is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into a polypeptide (in the case of DNA) and translated (in the case of mRNA) in vitro or in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxy) end. Coding sequences include, but are 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 will typically be located on the 3' side of the coding sequence.

[0074] Nucleic acid sequences refer to DNA or RNA sequences. Examples of nucleic acids include 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseuduracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, and 5-methylaminomethyluracil. This includes, but is not limited to, base analogs of DNA and RNA, including 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosin, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosin, 2-thiocytosine, and 2,6-diaminopurines.

[0075] The term DNA “regulatory sequences” collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRESs"), enhancers, etc., which collectively provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these regulatory sequences are always necessary, as long as the selected coding sequence can be replicated, transcribed, and translated in a suitable host cell.

[0076] The term “promoter” is used herein in its usual sense to refer to a nucleotide region containing a DNA regulatory sequence, the regulatory sequence being derived from a gene that can bind to RNA polymerase and initiate transcription of a downstream (3'-direction) coding sequence. Transcription promoters may include “inducible promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where 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 alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-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 elements, skeletal fast-twitch muscle troponin c gene elements, slow-twitch muscle cardiac troponin c gene elements, slow-twitch muscle troponin i gene elements, hypoxia-inducible nuclear factor (HIF) response elements (HRE), steroid-inducible elements, and glucocorticoid-inducible elements (gre). In another embodiment, the promoter is the MCK promoter, tMCK promoter, or MHCK7 promoter.

[0077] The term "operably linked" refers to the arrangement of elements configured so that the components described in this way perform their normal functions. Thus, control sequences operably linked to a coding sequence can influence the expression of the coding sequence. Control sequences do not need to be adjacent to the coding sequence as long as they function to direct its expression. For example, an intervening untranslated but transcribed sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0078] When RNA polymerase binds to a promoter sequence, transcribes the coding sequence into mRNA, and then translates it into the polypeptide encoded by the coding sequence, the promoter "directs the transcription" of the coding sequence within the cell.

[0079] An “expression cassette” or “expression construct” refers to an assembly that can direct the expression of a desired sequence or gene. An expression cassette often includes regulatory elements such as promoters that are operablely linked to the desired sequence or gene (to direct transcription), as described above, and often also includes polyadenylated sequences. In certain embodiments of the present invention, the expression cassettes described herein may be contained within a plasmid construct. In addition to the components of an expression cassette, a plasmid construct may also include one or more selectable markers, signals that enable the plasmid construct to exist as single-stranded DNA, at least one multicloning site, and a “mammalian” origin of replication (e.g., SV40 or an adenovirus origin of replication).

[0080] When referring to a nucleotide sequence, "isolated" means that the molecule in question exists in the substantial absence of other nucleotide sequences, chromatin material, or other biological macromolecules. Therefore, an "isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule that substantially contains no other nucleic acid molecules that do not encode the polypeptide in question, however, the molecule may contain some additional bases or parts that do not adversely affect the fundamental properties of the composition.

[0081] When a particular nucleotide sequence is described as being located "upstream," "downstream," "3," or "5" relative to another sequence, for the purpose of describing the relative position of a nucleotide sequence within a particular nucleic acid molecule throughout this application, it should be understood that this refers to the position of the sequence in the "sense" or "coding" strand of the DNA molecule, as is customary in the art.

[0082] In the context of nucleic acid sequences or amino acid sequences, the terms “sequence identity,” “sequence identity ratio,” or “ratio of identity” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison can be the full length of the genome, the full length of the gene coding sequence, or preferably a fragment of at least approximately 500–5000 nucleotides. However, identity between smaller fragments, such as at least approximately 9 nucleotides, typically at least approximately 20–24 nucleotides, at least approximately 28–32 nucleotides, or at least approximately 36 or more nucleotides, may also be desired. The sequence identity ratio can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as an alignment tool, the following default parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; prediction = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swissprotein+Spupdate+PIR.

[0083] 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, domesticated animals such as cattle, sheep, pigs, goats, and horses, domesticated mammals such as dogs and cats, and experimental animals including rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human being in the range of birth to 2 years, 1 to 10 years, or 4 to 15 years, or 10 to 19 years, or 20 to 40 years, or 15 to 29 years, or 25 to 55 years, or 40 to 60 years, or 50 years or older, or 60 years or older, or 65 years or older, or 70 years or older. For example, the subject is a human child (2 to 12 years), or a human adolescent (10 to 19 years). In some embodiments, the subject is an adult (18 years or older). In particular, the target population includes young adults (15-29 years old), middle-aged adults (25-55 years old), adults older than 50 years old, elderly individuals (65 years and older), and elderly individuals (70 years and older).

[0084] "Therapeutic effect" means any therapeutic benefit provided by the treatment described herein. For example, such an effect may be the sustained expression of a protein or enzyme that is incomplete or deficient in the muscular dystrophy of interest in the appropriate target tissue. Furthermore, a therapeutic effect may be any reduction or elimination of one or more clinical or subclinical signs of the disease or disorder of interest. For example, a decrease in CK levels, a reduction in fibrosis, an increase in resistance to contraction-induced injury of the tibialis anterior muscle, and an increase in intramuscular specific force, and an improvement in motor function may provide a therapeutic benefit to a subject having treated LGMD-2D.

[0085] In another embodiment, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding an alpha-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, or the protein retains alpha-sarcoglycan activity. In another embodiment, the alpha-sarcoglycan comprises the polypeptide sequence shown in SEQ ID NO: 2.

[0086] In another embodiment, the herein describes a recombinant AAV vector comprising a polynucleotide sequence encoding a functional alpha-sarcoglycan, or its complement, which comprises a nucleotide sequence that hybridizes 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 under stringent conditions. In another embodiment, the rAAV comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. In yet another embodiment, the rAAV comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

[0087] The term "stringent" is used to refer to conditions that are generally understood as stringent in the art. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65–68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (higher temperature, lower ionic strength, higher formamide, or other denaturing agents, etc.) can also be used, but the rate of hybridization will be affected. When deoxyoligonucleotide hybridization is involved, examples of additional stringent hybridization conditions include washing with 6×SSC, 0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos).

[0088] Where ranges are used herein with respect to physical properties such as molecular weight, concentration, or dosage, it is intended that the range and all combinations and partial combinations of specific embodiments within it are included. The term “approximately” when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variation (or statistical experimental error), and therefore the numerical value or numerical range may vary, for example, between 1% and 15% of the stated numerical value or numerical range.

[0089] To reduce nonspecific and / or background hybridization, other agents may be included in the hybridization and washing buffers. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), Ficol, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, but other suitable agents may also be used. The concentrations and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4, but under typical ionic strength conditions, the rate of hybridization is largely pH-independent. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch.4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to allow DNAs of different sequence similarities to form hybrids, taking these variables into consideration.

[0090] Limb-girdle muscular dystrophy type 2D (LGMD2D) is a progressive muscular dystrophy characterized by muscle weakness, respiratory abnormalities, and, rarely, cardiomyopathy. LGMD2D is caused by mutations in the alpha-sarcoglycan gene, resulting in protein loss and associated loss of the sarcoglycan-dystrophin-associated glycoprotein complex. - / - The mice reproduce the clinical phenotype of LGMD2D patients, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and decreased absolute muscle strength and spontaneous movement. Therefore, sgca - / -Mice provide relevant models for testing the safety and efficacy of gene replacement. Accordingly, 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, the truncated muscle creatine kinase (tMCK). sgca - / - The efficacy and safety of scAAVrh74.tMCK.hSGCA in mice were tested using a dose-escalation design to evaluate single systemic injections of 1×10 12、3 ×10 12 , and 6×10 12 vg compared to vehicle-treated and wild-type mice. sgca - / - In mice, treatment with scAAVrh74.tMCK.hSGCA resulted in strong protein expression of α-SG in the myosheaths of skeletal muscle at all doses tested. Furthermore, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of the limbs and diaphragm muscles of sgca - / - mice, as shown by reduction of fibrosis and decrease of central nucleation, and normalization of muscle fiber size. These molecular changes were associated with a significant increase in specific force generation, protection against loss of eccentric force, and decrease in serum CK in the diaphragm and anterior tibialis muscles. Spontaneous locomotion was improved at all doses of vector-treated mice compared to vehicle-treated sgca - / - mice. Finally, the absence of vector-related toxicity was detected by serum chemistry panels and gross necropsy. Taken together, this study provides support for systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D.

[0091] In another embodiment, the recombinant AAV vectors described herein may be operably linked to muscle-specific regulatory elements. For example, muscle-specific promoters include one or more of the following: human skeletal actin gene elements, cardiac actin gene elements, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-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 promoters, mouse creatine kinase enhancer elements, skeletal fast-twitch muscle troponin c gene elements, slow-twitch muscle cardiac troponin c gene elements, slow-twitch muscle troponin i gene elements, hypoxia-inducible nuclear factor (HIF) response elements (HRE), steroid-inducible elements, and glucocorticoid response elements (gre).

[0092] In one embodiment, the muscle-specific promoter is a tMCK promoter containing the sequence of SEQ ID NO: 3. An exemplary rAAV described herein is AAVrh74.tMCK.hSCGA containing the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide sequence encoding AAVrh74.tMCK.hSCGA includes 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 shown in SEQ ID NO: 4 or to SEQ ID NO: 1.

[0093] In another embodiment, the polynucleotide encoding AAVrh74.tMCK.hSCGA includes a nucleotide sequence encoding 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 at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encodes a protein that retains alpha-sarcoglycan activity.

[0094] In one embodiment, rAAV includes the 5' inverted end repeat sequence of sequence number 5. In another embodiment, rAAV includes the 3' inverted end repeat sequence of sequence number 6. In some embodiments, rAAV includes the polyA sequence of sequence number 7.

[0095] AAV can be any serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74, or variants and derivatives thereof. In one embodiment, rAAV is rAAV of serotype AAVrh.74. The production of pseudotype rAAV is disclosed, for example, in WO2001 / 083692, which is incorporated herein by reference in its entirety. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0096] Compositions comprising any of the rAAV vectors described herein are also intended.

[0097] Provided is a method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, where rAAV is approximately 1.0 × 10⁻⁶ 12vg / kg ~ approx. 5.0×10 15 It is administered using a dose of vg / kg. For example, in any of the methods provided, the dose of rAAV administered is approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×10 13 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The value is vg / kg. In another embodiment, the dose is approximately 5.0 × 10 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10⁻⁶ 14 The dosage is vg / kg. In one embodiment, rAAV is administered via a systemic route, including an intravenous route. In another embodiment, rAAV is administered at approximately 5.0 × 10⁻⁶ 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10⁻⁶ 14 It is administered intravenously at a dose of vg / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy.

[0098] In addition, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 13 vg~approx.3.5×10 16 vg, or approximately 3 × 10 13 vg~approx. 1.0×10 16 vg, or approximately 1.5 × 10 13 vg~approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 14The dose is in vg. In addition, in any of the methods, the dose of rAAV is administered at a concentration of approximately 10 mL / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy type 2D. The doses in this disclosure are expressed in either vg or vg / kg and are based on titration methods by quantitative PCR (qPCR). qPCR-based titration methods are known in the art.

[0099] In addition, provided is a method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, where rAAV is administered via a systemic route at a dose of approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 2.0×10 15When administered at a dose of vg / kg, the level of alpha-sarcoglycan gene expression in the target cells increases after rAAV administration compared to the level of alpha-sarcoglycan gene expression before rAAV administration, the serum CK level in the target decreases after rAAV administration compared to the serum CK level before rAAV administration, and / or spontaneous movement and specific force generation increase, fibrosis is reduced, resistance to contraction-induced injury of the tibialis anterior muscle increases, and / or the number of alpha-sarcoglycan-positive fibers in the target muscle tissue increases after rAAV administration compared to the number of alpha-sarcoglycan-positive fibers before rAAV administration, the fiber diameter size in the target muscle tissue increases after rAAV administration compared to the number of fiber diameters before rAAV administration, or centronucleation in the target muscle tissue decreases after rAAV administration compared to centronucleation before rAAV administration. Muscle tissue includes, but is not limited to, the triceps brachii, tibialis anterior, soleus, gastrocnemius, biceps brachii, trapezius, gluteus, psoas major, deltoid, quadriceps femoris, and diaphragm. In one embodiment, muscle tissue includes the tibialis anterior, gastrocnemius, gluteus, psoas major, and triceps brachii. Alpha-sarcoglycan expression is determined by methods known to those skilled in the art. In one embodiment, expression is determined by Western blotting, immunochemistry in muscle biopsy, and / or by detecting the number of vector genomes per microgram of genomic DNA.

[0100] In some embodiments, the present disclosure includes a method for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein motor function is significantly improved in the subject compared to the motor function of the subject before administration of rAAV.

[0101] Provided is a method for increasing alpha-sarcoglycan expression in subjects requiring it, which involves administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4 to a patient.

[0102] In any of the methods, uses, and compositions for treating muscular dystrophy provided, subjects are 4–15 years of age, have alpha-sarcoglycan (SGCA) mutations confirmed in both alleles, are negative for AAVrh74 antibody, and / or have a 100-meter walk test of more than 40% or normal. In any of the methods, uses, and compositions for treating muscular dystrophy provided, subjects are pediatric subjects. In some embodiments, subjects are pediatric subjects, for example, subjects in the range of 1–21 years. In some embodiments, subjects are 1–10 years, or 2–12 years, 4–15 years, or 10–19 years. In one embodiment, subjects are adolescent subjects, for example, subjects in the range of 12–21 years. In addition, in one embodiment, subjects are young adult subjects, such as subjects in the age range of 15–29 years or 18–39 years. In some embodiments, the subjects are middle-aged adults or elderly subjects, and as a result, middle-aged adults may be in the range of 25 to 55 years, older adult subjects may be in the range of over 50 years, and elderly subjects may be in the range of over 65 years. In some embodiments, rAAV is administered by injection, infusion, or implantation. For example, rAAV is administered by infusion over approximately 1 to 2 hours. In addition, rAAV is administered by an intravenous route via peripheral limb veins.

[0103] A method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein rAAV is administered via a systemic route at a dose of approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 5.0×10 14Administered in a dose of vg / kg, rAAV contains 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, rAAV contains the nucleotide sequence shown in SEQ ID NO: 1. In one embodiment, rAAV encodes a protein containing 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 including the polypeptide sequence shown in SEQ ID NO: 2. In addition, any of the disclosed rAAVs further comprises a promoter, such as the tMCK promoter sequence of SEQ ID NO: 3. In some embodiments, the rAAV is the rAAV of 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 end repeat sequence of SEQ ID NO: 5. In another embodiment, the rAAV comprises the 3' inverted end repeat sequence of SEQ ID NO: 6. In another embodiment, the rAAV comprises the polyA sequence of SEQ ID NO: 7.

[0104] AAV dosage can be determined by several methods, including but not limited to LISA, reverse transcriptase activity assessment, FACS, transduction assays (Northern blotting, 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 with quantitative real-time PCR (qPCR). Such qPCR methods overcome the inconsistent or arbitrary results from conventional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. Plasmid morphology may affect the dosage results by the qPCR method. In one embodiment, circular or superhelical DNA or plasmid is used as a quantitative standard. In another embodiment, linearized DNA or plasmid is used as a quantitative standard.

[0105] The terms “superhelical DNA” or “superhelical plasmid” refer to DNA or plasmids that do not contain free ends. The terms “linearized DNA” or “linearized plasmid” refer to DNA or plasmids that contain free 5' ends and free 3' ends that are not linked to each other. In one embodiment, linearized DNA or plasmids are obtained by restriction digestion of circular DNA (e.g., plasmid DNA) or by restriction digestion of dbDNA. In another embodiment, restriction digestion is performed using an enzyme that produces at least one blunt end.

[0106] In an exemplary embodiment, a method for doing so in a subject requiring treatment for muscular dystrophy includes the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK7.hSGCA, where rAAV is administered via a systemic route in a dose of approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 14 The dose was administered at vg / kg, and the human subjects suffered from limb-girdle muscular dystrophy. In one embodiment, rAAV was measured based on superhelical DNA or plasmid as a quantitative standard, approximately 5.0 × 10⁶ 13 vg / kg, 1.0 × 1014 vg / kg, or 2.0 × 10⁻⁶ 14 The dose is administered by intravenous infusion over approximately 1-2 hours at a dose of vg / kg, and rAAV contains the scAAVrh74.tMCK7.hSGCA construct nucleotide sequence of SEQ ID NO: 3. In another embodiment, the dose is approximately 1.85 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 10 13 It is vg / kg.

[0107] The disclosure also provides a method for increasing sarcoglycan expression in target muscle tissue, the method comprising administering to the target a scAAVrh74.tMCK.hSGCA construct containing a nucleotide sequence that is at least 90% identical, at least 95% identical, or 99% identical to SEQ ID NOs: 1 and / or 4.

[0108] The disclosure also further provides a method for improving muscle function in a subject, the method comprising administering a construct to a subject that contains a nucleotide sequence that is at least 90% identical, at least 95% identical, or 99% identical to SEQ ID NOs: 1 and / or 4.

[0109] In some embodiments, the subjects suffer from a gene mutation in the gene encoding the sarcoglycan protein, or from muscular dystrophy. In some embodiments, the subjects suffer from a gene mutation in the gene encoding the alpha-sarcoglycan protein.

[0110] In any of the provided methods, the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the scAAVrh74.tMCK7.hSGCA construct compared to the level of alpha-sarcoglycan gene expression before administration of the scAAVrh74.tMCK.hSGCA construct.

[0111] In addition, in any of the provided methods, alpha-sarcoglycan gene expression in cells is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0112] In any of the provided methods, the level of alpha-sarcoglycan protein increases after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of alpha-sarcoglycan protein increases by at least 33% when detected by measuring the level of alpha-sarcoglycan protein in Western blots of muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct, or the level of alpha-sarcoglycan protein is measured by detecting immunohistochemistry and / or vector genome count per microgram of genomic DNA in muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0113] In any of the methods provided herein, serum CK levels in the subject decrease after administration of the scAAVrh74.tMCK.hSGCA construct compared to serum CK levels before administration of the scAAVrh74.tMCK.hSGCA construct.

[0114] In any of the methods provided herein, the number of alpha-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the number of alpha-sarcoglycan-positive fibers before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of alpha-sarcoglycan-positive fibers is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of alpha-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of the scAAVrh74.tMCK.hSGCA construct.

[0115] In any of the methods provided herein, the level of alpha-sarcoglycan in the subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of alpha-sarcoglycan is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry or Western blotting in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0116] Another embodiment provides a method for expressing the alpha-sarcoglycan gene in a patient's cells, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4 to a subject. In any of the provided methods for expressing the alpha-sarcoglycan gene in a patient's cells, the expression of the alpha-sarcoglycan gene in the patient's cells is detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. In one embodiment, the alpha-sarcoglycan gene is measured in the patient by detecting more than one rAAV vector genome copy per nucleus. In another embodiment, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0117] A method is also provided for reducing serum CK levels in patients requiring such reduction, which involves administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4.

[0118] A method is provided for increasing alpha-sarcoglycan-positive fibers in a patient's muscle tissue, which involves targeting the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 4. In any of these methods, the number of alpha-sarcoglycan-positive fibers is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0119] Another embodiment provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4 to the subject. In either of these methods, the level of alpha-sarcoglycan is detected by measuring the alpha-sarcoglycan protein level by Western blotting or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0120] A method for producing recombinant AAV vector particles is also provided, comprising culturing cells transfected with any recombinant AAV vector described herein and recovering recombinant AAV particles from the supernatant of the transfected cells. Viral particles comprising any of the recombinant AAV vectors described herein are also contemplated. In one embodiment, a method for generating rAAV comprises transferring an AAV vector plasmid into a host cell. In another embodiment, the recombinant AAV vector particles and / or AAV vector plasmid contain 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 typically about 90%, about 91%, about 92%, about 93%, about 94%, about and 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO: 8. In another embodiment, the disclosure provides a host cell containing an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. rAAV can be generated using the host cell stably harboring the AAV vector plasmid. In one embodiment, the AAV vector plasmid is the pAAV.tMCK.hSGCA.KAN plasmid (SEQ ID NO: 8).

[0121] Methods for alleviating fibrosis in mammalian subjects requiring it are also provided. In this regard, the method comprises administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to a mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In one embodiment, the muscular dystrophy is LGMD2D. In some embodiments, administration of the AAV vector described herein (or a composition comprising the AAV vector described herein) alleviates fibrosis in the muscle tissue of the subject. In one embodiment, the muscle tissue includes the psoas major, diaphragm, triceps brachii, and / or gluteal muscles.

[0122] As used herein, the term “muscular dystrophy” refers to a disorder characterized by a gradual decline in strength and muscle mass. Non-exclusive examples of muscular dystrophy include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycan disorders, congenital muscular dystrophy such as congenital muscular dystrophy due to partial LAMA2 deficiency, merosin-deficient congenital muscular dystrophy, type 1D congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, limb-girdle type 1A muscular dystrophy, limb-girdle type 2A muscular dystrophy, limb-girdle type 2B muscular dystrophy, and limb-girdle type 2C muscular dystrophy. Examples of muscular dystrophy include 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, ankylosing vertebral muscular dystrophy with simple epidermolysis bullosa, oculopharyngeal muscular dystrophy, Ulrich type congenital muscular dystrophy, and Ulrich type scleroatnik muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy type 2D (LGMD2D).

[0123] As used herein, the term “fibrosis” refers to the excessive or uncontrolled deposition of extracellular matrix (ECM) components and the abnormal repair processes in post-injury tissues, including skeletal muscle, cardiac muscle, liver, lungs, kidneys, and pancreas. The deposited ECM components include collagen (e.g., collagen 1, collagen 2, or collagen 3) and fibronectin.

[0124] In another embodiment, the foregoing describes a method for increasing alpha-sarcoglycan-positive fibers, fiber diameter size, eccentric contraction, muscle strength, and / or alpha-sarcoglycan expression in a mammal, comprising administering a therapeutically effective amount of the herein-described AAV vector (or a composition comprising the herein-described AAV vector) to a mammalian subject. Also described herein is a method for reducing fibrosis, centronucleation, CK levels, and / or collage deposition in a subject, comprising administering a therapeutically effective amount of the herein-described AAV vector (or a composition comprising the herein-described AAV vector) to the subject.

[0125] In any of the methods of the present invention, the subject may have a muscular dystrophy such as limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy. In one embodiment, the muscular dystrophy is LGMD-2D.

[0126] A method for treating muscular dystrophy in a mammalian subject is also provided, comprising administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to a mammalian subject. In some embodiments, the muscular dystrophy is limb-girdle muscular dystrophy.

[0127] In any of the methods of the present invention, rAAV is administered by intramuscular or intravenous injection. In addition, in any of the methods of the present invention, rAAV is administered systemically, such as by parenteral administration by injection, infusion, or transplantation.

[0128] The compositions of the present invention are formulated for intramuscular or intravenous injection. In addition, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or transplantation.

[0129] In addition, any of the compositions may be formulated for administration to subjects suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy). In some embodiments, the composition may further comprise a second recombinant AAV vector expressing alpha-sarcoglycan, or a second recombinant AAV vector comprising the polynucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4.

[0130] In any use of the present invention, the pharmacopoeia is formulated for intramuscular or intravenous injection. In addition, in any use of the present invention, the pharmacopoeia is formulated for systemic administration, such as parenteral administration by injection, infusion, or transplantation. In addition, any of the pharmacopoeia may be prepared for administration to subjects suffering from muscular dystrophy (e.g., limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy). In some embodiments, the pharmacopoeia may further comprise a second recombinant AAV vector expressing alpha-sarcoglycan, or a second recombinant AAV vector comprising a polynucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4.

[0131] The preceding paragraphs are not intended to define all aspects of the Invention, and additional aspects are described in other sections, such as the detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein should be considered even if no combination of features is found together in the same sentence, paragraph, or section of this document. The Invention includes, as additional aspects, all embodiments of the Invention that are somewhat narrower in scope than the variations defined in the particular paragraphs above. For example, if a particular aspect of the Invention is described as a genus, each member of the genus should be understood to be an aspect of the Invention individually.

[0132] AAV The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome may be from any AAV serotype from which the recombinant virus may originate, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also intended.

[0133] The DNA plasmid of the present invention comprises an rAAV genome. The DNA plasmid is introduced into a cell tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for the incorporation of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function are provided to the cell, are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAVrep and cap genes isolated from (i.e., not present in) the rAAV genome, and the helper virus function, be present in a single cell (referred to herein as a packaging cell). The AAV rep and cap genes may be from any AAV serotype from which the recombinant virus may originate, and may be from an AAV serotype different from the rAAV genome ITR, 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 AAV rh.74. The generation of pseudotyped rAAV is disclosed, for example, in WO2001 / 083692, which is incorporated herein by reference in its entirety.

[0134] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking the AAVrep and cap genes, the AAVrep and cap genes isolated from the rAAV genome, and selectable markers such as the neomycin resistance gene, is incorporated into the cell genome. The AAV genome is introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0135] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol.Cell.Biol.4:2072(1984), Hermonat et al., Proc.Natl.Acad.Sci.USA,81:6466(1984), Tratschin et al., Mol.Cell.Biol.5:3251(1985), McLaughlin et al., J.Virol.,62:1963(1988), and Lebkowski et al., 1988 Mol.Cell.Biol.,7:349(1988). Samulski et al. (1989, J. Virol., 63:3822-3828), U.S. Patent No. 5,173,414, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. This is described in al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production.

[0136] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0137] The recombinant AAV (i.e., infectious capsidized rAAV particles) of the present invention comprises an rAAV genome. Embodiments include, but are not limited to, an rAAV named pAAV.tMCK.hSCGA, which comprises the polynucleotide sequence shown in SEQ ID NO: 3.

[0138] rAAV can be purified by methods standard in the art, for example, by column chromatography or a cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0139] In another embodiment, the present invention envisions a composition comprising rAAV. The compositions described herein contain rAAV in a pharmaceutically acceptable carrier. The compositions may also include other components such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to the recipient and preferably inactive at the dosage and concentration used, and include buffering agents such as phosphoric acid, citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0140] The titer of rAAV administered by the method of the present invention varies depending, for example, on the specific rAAV, the method of administration, the treatment target, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 This may be in the range of DNase-resistant particles (DRPs) or higher. The dosage may be expressed in units of viral genome (vg) as measured by qPCR.

[0141] The present invention envisions a method for transducing target cells with rAAV in vivo or in vitro. The in vivo method comprises the step of administering an effective dose or multiple effective doses of a composition comprising rAAV of the present invention to an animal (including humans) in need. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition to be treated, a dose that slows or prevents progression to the disorder / disease condition, a dose that reduces the extent of the disease, a dose that results in remission (partial or complete) of the disease, and / or a dose that prolongs survival. An example of a disease envisioned for prevention or treatment by the method of the present invention is muscular dystrophy, e.g., limb-girdle muscular dystrophy. Accordingly, what is provided is a method for transducing target cells with rAAV scAAVrh74.tMCK.hSGCA, comprising the nucleotide sequence of SEQ ID NO: 4.

[0142] In another embodiment, the disclosure provides a method for generating rAAV scAAVrh74.tMCK.hSGCA, which includes transferring an AAV vector plasmid into a host cell. Methods for transferring DNA into a host cell are known in the art and include, but are not limited to, transfection, infection, transformation, electroporation, and transduction. In one embodiment, the vector plasmid contains a nucleotide sequence that is at least 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 typically about 90%, about 91%, about 92%, about 93%, about 94%, about and 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO: 8. In another embodiment, the vector plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8. In another embodiment, the vector plasmid contains the nucleotide sequence of SEQ ID NO: 8. In another embodiment, the disclosure provides a host cell containing an AAV vector plasmid containing the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. rAAV can be generated using a host cell stably harboring the AAV vector plasmid. In one embodiment, the AAV vector plasmid is the pAAV.tMCK.hSGCA.KAN plasmid.

[0143] In one embodiment, the vector plasmid contains a nucleotide sequence that is identical to sequence numbers 1, 4, or 8 by 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 typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more.

[0144] In one embodiment, the vector plasmid contains a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NOs: 1, 4, or 8. In one embodiment, the vector plasmid contains the nucleotide sequence of SEQ ID NO: 8. In one embodiment, the method for generating rAAV further includes transferring a packaging plasmid and / or helper virus into a host cell. In some embodiments, the packaging plasmid contains AAV rep and / or cap genes that are operably ligated to a promoter. In one embodiment, the promoter is an AAV transcription promoter. In one embodiment, the host cell is a packaging cell. In one embodiment, the packaging cell contains a stably integrated AAVcap gene. In another embodiment, the packaging cell contains a stably integrated AAVrep gene.

[0145] As used herein, the term “host cell” refers to a cell that can be used to express an exogenous DNA sequence. Non-limiting examples of host cells include microorganisms, yeast cells, insect cells, and / or mammalian cells. Host cells can be used as recipients of AAV helper constructs, packaging plasmids, AAV vector plasmids, accessory functional vectors, or other DNA. The term used herein encompasses the offspring of the original cell after the original host cell has expressed the exogenous DNA sequence. Non-limiting examples of host cells for AAV production include Sf9 insect cells and HEK293T cells. AAV vector plasmids can be introduced into host cells (e.g., Sf9 or 293T) by infection (virus or baculovirus), transient transfection using reagents (e.g., liposomes, calcium phosphate), or by physical means (e.g., electroporation), or by other means known in the art. In another embodiment, host cell lines are stably incorporated into their genome along with the rAAV plasmid. Such stable cell lines can be established by incorporating a selection marker into the vector plasmid.

[0146] In one embodiment, the host cell is a packaging cell for producing AAV virus particles. Therefore, in another embodiment, the disclosure provides a host cell comprising an AAV vector plasmid comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 8. In one embodiment, the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. In another embodiment, the host cell comprises the nucleotide sequence of SEQ ID NO: 1, 4, or 8.

[0147] Combination therapies are also envisioned by the present invention. Combination therapies as used herein include concurrent or sequential treatments. Combinations of the methods of the present invention with standard medical treatments (e.g., steroids, corticosteroids, and / or glucocorticoids comprising one or more of prednisone, prednisolone, and deflazacort, but not limited to these) are specifically envisioned, as are combinations with novel therapies. In this view, these combinations include administering one or more steroids, corticosteroids, and / or glucocorticoids comprising one or more of prednisone, prednisolone, and deflazacort, to the target before administering the methods of the present invention to the target, simultaneously with or after administering the rAAV to the target.

[0148] In related embodiments of the combination therapy envisioned by the present invention, the glucocorticoid may include, but is not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0149] Antigen-specific T cell responses are recognized as possible in subjects administered with rAAV vectors. This is a response expected 2–4 weeks after gene transfer. One possible consequence of such an antigen-specific T cell response is the clearance of transduced cells and loss of transgene expression. To attenuate the host immune response to rAAV-based therapy, subjects may be initiated with oral prophylactic prezonisone or an equivalent glucocorticoid at approximately 1 mg / kg / day, pretreatment, for example, 24 hours before the treatment procedure, up to a maximum dose of 60 mg / day. If necessary, an equivalent glucocorticoid may be administered intravenously at a dose of approximately 1 mg / kg / day. Treatment lasts for approximately one month. Protocols for tapering the dose of prezonisone or an equivalent glucocorticoid can be implemented based on the individual subject's immune response to gene transfer and evaluated by ELISpot assays and liver function monitoring by GGT.

[0150] The therapeutically effective dose of rAAV vector is approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×10 13 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The dose of rAAV is in the range of vg / kg. In another embodiment, the dose is approximately 5.0 × 10 13 vg / kg, approximately 1.0×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 14 The value is vg / kg. In another embodiment, the dose is 5.0 × 10⁻⁶ 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10⁻⁶ 14 The value is vg / kg. The present invention is also intended to include compositions comprising rAAV vectors within these ranges.

[0151] The dosage may be expressed in units of viral genome (vg). The titer of rAAV may be determined by superhelical DNA or plasmid quantitative standards or linearized DNA or plasmid quantitative standards. The titer or dosage of the AAV vector may vary based on the physical form of the plasmid or DNA as the quantitative standard. For example, the titer or dosage value may vary based on superhelical standard qPCR titration or linearized standard qPCR titration. In one embodiment, the dosage in this disclosure is based on superhelical DNA or plasmid as the quantitative standard. In another embodiment, the dosage in this disclosure is based on linearized DNA or plasmid as the quantitative standard. Thus, in one embodiment, the therapeutically effective dose of rAAV vector is approximately 1.0 × 10⁻¹⁶ based on superhelical DNA or plasmid as the quantitative standard. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×10 13 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The dose of rAAV is in the range of vg / kg. In another embodiment, the dose is approximately 5.0 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg, approximately 1.0×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 14 The dose is vg / kg. In another embodiment, the dose is 5.0 × 10⁻¹⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10⁻⁶ 14 It is vg / kg.

[0152] In another embodiment, the therapeutically effective dose of the rAAV vector is approximately 1.0 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 This refers to the dose of rAAV in the range of vg / kg. For example, the therapeutically effective dose of rAAV vector is approximately 1.85 × 10⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 10 13 The dosage is in 1g / kg.

[0153] In one embodiment, 5.0 × 10⁶ units based on superhelical DNA or plasmid as a quantitative standard are used. 13 The dose of vg / kg is 1.85 × 10⁶ based on linearized DNA or plasmid as a quantitative standard. 13 This is equivalent to a dose of vg / kg. In another embodiment, 2.0 × 10 based on superhelical DNA or plasmids. 14 The dose of vg / kg is 7.41 × 10⁴ based on linearized DNA or plasmid as a quantitative standard. 13 This is equivalent to vg / kg. Therefore, in another embodiment, approximately 1.85 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13vg / kg or 7.41 × 10 13 It is vg / kg.

[0154] The effective dose of the composition may be administered via a standard route in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components of the rAAV of the present invention (specifically, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infectious and / or disease state being treated, as well as the target cells / tissues expressing α-sarcoglycans.

[0155] The present invention provides topical and systemic administration of effective doses of the rAAV and compositions of the present invention. For example, systemic administration means administration to the circulatory system so that the whole body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration by injection, infusion, or transplantation.

[0156] In particular, the practical administration of rAAV according to the present invention can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the liver. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, which is incorporated herein by reference.

[0157] The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation delivered to the muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been developed to date and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. Therefore, in another embodiment, this application relates to a formulation comprising rAAV containing a capsid derived from AAVrh74, a buffer, an ionic strengthening agent, and a surfactant. In one embodiment, the rAAV is approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 14 The concentration is vg / kg. In another embodiment, rAAV is approximately 5.0 × 10⁻⁶. 12 vg / kg~1.0×10 14 The concentration is vg / kg. In another embodiment, rAAV is approximately 5 × 10 13 vg / kg, approx. 1×10 14 vg / kg, and / or approximately 2 × 10 14The concentration is in the range of vg / kg. In one embodiment, the dosage is based on superhelical DNA or plasmid as a quantitative standard. In one embodiment, rAAV is the scAAVrh74.tMCK.hSGCA vector. In one embodiment, the buffer comprises one or more of Tris, Trisine, Bis-Trisine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In another embodiment, the buffer comprises Tris at pH 8.0 at a concentration of about 5 mM to about 40 mM. In one embodiment, the buffer comprises Tris at pH 8.0 at about 20 mM. In one embodiment, the ionic strengthener comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. In one embodiment, the ionic strengthening agent contains MgCl2 at a concentration of about 0.2 mM to about 4 mM. In another embodiment, the ionic strengthening agent contains NaCl at a concentration of about 50 mM to about 500 mM. In yet another embodiment, the ionic strengthening agent contains MgCl2 at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM. In yet another embodiment, the ionic strengthening agent contains MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM. In one embodiment, the surfactant comprises one or more of sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. In one embodiment, the poloxamer comprises one or more of poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. In one embodiment, the surfactant contains poloxamer at a concentration of about 0.00001% to about 1%. In another embodiment, the surfactant contains poloxamer 188 at a concentration of about 0.001%. For intramuscular injection, an adjuvant solution such as sesame oil or peanut oil, or an aqueous propylene glycol solution, and a sterile aqueous solution can be used.Such aqueous solutions can be buffered as needed, and the liquid diluent is first isotonicized with physiological saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacokinetically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0158] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, these forms must be sterile and fluid enough to allow for easy syringe injection. They must be stable under manufacturing and storage conditions and protected against microbial contamination, such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. The prolonged absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0159] Sterile injectable solutions are prepared by combining the required amount of rAAV in a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by mixing the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired components from those solutions that have been previously sterilized and filtered.

[0160] Transduction with rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are isolated from the target, transduced with rAAV, and reintroduced into the target. Alternatively, syngeneic or heterologous muscle cells may be used if those cells do not produce an inappropriate immune response in the target.

[0161] Suitable methods for transduction into a target and reintroduction of transduced cells are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in a suitable medium and screening cells with the desired DNA using conventional techniques such as Southern blotting and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into a target by various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle using a catheter, for example.

[0162] Transduction of cells using rAAV according to the present invention results in sustained expression of α-sarcoglycan. Therefore, the present invention provides a method for administering / delivering rAAV expressing α-sarcoglycan to mammalian subjects, preferably humans. These methods include transducing tissue with one or more rAAVs of the present invention (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands). Transduction may be carried out using a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissue directed by muscle-specific regulatory elements, which include, but are not limited to, those derived from actin and myosin gene families, e.g., the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], regulatory elements derived from the human skeletal muscle actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], cardiac muscle actin gene, muscle creatine kinase sequence elements [Johnson et al.] See al., Mol. Cell. Biol., 9:3393-3399 (1989), as well as regulatory elements derived from mouse creatine kinase enhancer (mCK) elements, skeletal fast-twitch muscle troponin C gene, slow-twitch muscle cardiac troponin C gene, and slow-twitch muscle troponin I gene, hypoxia-induced nuclear factors (Semenza et al., Proc. Natl. Acad. Sci. USA 88:5680-5684 (1991)), glucocorticoid response elements (GRE), steroid-induced elements and promoters (see Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)), and other regulatory elements.

[0163] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. This invention aims to achieve sustained expression of miRNAs derived from plasma-transduced myofibrils.

[0164] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived from the digestive tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, including myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac muscle cells.

[0165] The term "transduction" is used to refer to the administration / delivery of a target polynucleotide (e.g., a polynucleotide sequence encoding α-sarcoglycan) to recipient cells either in vivo or in vitro via the described replication-deficient rAAV, resulting in the expression of alpha-sarcoglycan by the recipient cells.

[0166] Accordingly, this specification also describes a method for administering an effective dose (or a dose essentially administered simultaneously or at intervals) of rAAV encoding alpha-sarcoglycans to a mammalian subject in need thereof.

[0167] All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually incorporated by reference. In the event of any conflict, this application shall prevail, including any definitions herein. The numerical ranges described include each integer value within each range, and include the smallest and largest integers described.

[0168] The present invention is further illustrated in the following embodiments, which do not limit the scope of the invention as described in the claims. [Examples]

[0169] Preclinical studies using AAVrh74.tMCK.hSCGA are described in International Patent Publication WO2013 / 078316, and U.S. Patents 9,434,928 and 10,105,453, which are incorporated herein by reference in their entirety.

[0170] Example 1 material and method Animal models All procedures were approved by the Research Institute at Nationwide Children's Hospital Institutional Animal Care and Use Committee. (SGCA) - / - The mice were bred and maintained as homozygous animals under standard conditions at the Animal Resources Core of the Research Institute at Nationwide Children's Hospital. The mice were maintained on a Teklad Global Rodent Diet (3.8% fiber, 18.8% protein, 5% fat) in a 12:12 dark-to-light cycle. All animals were placed in standard mouse cages and allowed free feeding and watering.

[0171] Genotype Using DNA genotyping, sgca - / -Mice were identified. DNA was isolated from tail clippings 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 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: 5 min at 94°C, 1 min at 94°C, 1 min at 64°C, 2.5 min at 72°C, and 7 min at 72°C.

[0172] α-SG gene construct The scAAVrh74.tMCK.hSGCA transgene cassette was constructed by inserting a tMCK expression cassette driving a codon-optimized human α-SGc DNA sequence (human cDNA, Genbank accession number U08895) into the self-complementary vector backbone pHpa7 using the adeno-associated virus (AAV) vector DNA plasmid pAAV.tMCK.aSG-neo. The only viral sequence contained in this vector is the AAV2 inverted end repeat, which is necessary for both viral DNA replication and packaging of the rAAV vector genome. One of the inverted end repeats (ITRs) has a targeted deletion of a terminal degradation site (TRS) to restrict replication from this ITR and promote the generation of a dimeric replicate for self-complementary vector packaging. The AAVrh74 virus has been proven safe and highly efficient for transduction into muscle across the blood-vascular barrier in mice, non-human primates (NHPs), and humans.

[0173] Vector production Recombinant AAV, (sc)rAAVrh74.tMCK.hSGCA, was constructed by triple transfection. The encapsulated vg titer was determined using a qPCR-based titration method and a Prism 7500 Fast Taqman detector system (PE Applied Biosystems). The construct contains a chimeric intron to promote high levels of expression. The chimeric intron consists of the 5' donor region from the first intron and branch point of the human β-globin gene, and the 3' splice acceptor region from the intron between the leader and body of the immunoglobulin gene's heavy chain variable region. rAAV also contains a synthetic SV40 polyadenylation signal, which is used for efficient transcription termination. A schematic diagram of the expression cassette is shown in Figure 1 below. The vector was produced using a human alpha-sacroglycan (α-SG) gene, flanked by the AAV2 ITR sequence and capsid-formed to the AAVrh74 virion. The construct contains a pre-initial promoter / enhancer for tMCK (GenBank accession number M21390) and uses a β-globin intron for high levels of expression.

[0174] gene delivery Systemic delivery was achieved by injecting the vector into the tail vein of sgca- / - mice. Mice were injected with 1 × 10¹⁶ scAAVrh74.tMCK.hSGCA diluted in 200–250 μL of Ringer's lactate solution using a 30-gauge ultrafine insulin syringe. 12 vg, 3×10 12 vg, or 6×10 12 Total dose of vg (based on mice ranging from 13 to 20 g, each based on 20 g of mice, with dosage based on superhelical DNA or plasmid as a quantitative standard, 5 × 10) 13 vg / kg, 1 × 10 14 vg / kg, and 2 × 10 14 The mice were injected with (vg / kg). All treated mice were injected at 4-5 weeks of age and euthanized 12 weeks after injection. In another embodiment, the dose was approximately 1.85 × 10⁻¹⁰ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 1013 It is vg / kg.

[0175] Serum creatine kinase measurement Creatine kinase levels were measured in wild-type C57BL / 6 mice (n=6) treated with a vehicle (Lactated Ringer's solution) sgca - / - Mice (n=6) and sgca treated with scAAVrh74.tMCK.hSGCA - / - Creatine kinase levels were measured in the serum of mice (n=6 per dose) using the creatine kinase SL assay and the corresponding manufacturer's protocol (Sekisui Diagnostics, Charlottetown, PE, Canada) (catalog number 326-10). Briefly, 25 μL of serum was mixed with 1 mL of working reagent and added to a cuvette. A kinetic assay was set up on a spectrophotometer, and absorbance at 340 nm was measured every 30 seconds for 180 seconds. Creatine kinase levels were calculated using the absorbance readings and the formulas listed below. U / L=[ΔAbs. / min)*1.025*1000] / [1*6.22*0.025]=(ΔAbs. / min)*6592

[0176] Diaphragmatic tetanus for functional assessment Mice were euthanized, and the diaphragm (DIA) was incised intact at the rib attachment and central tendon, and placed in KH buffer. Sections of the DIA 2-4 mm wide were isolated. The DIA sections were securely tied at the tendon center with braided surgical silk (6 / 0, Surgical Specialties, Reading, PA) and sutured through a portion of the rib attached to the distal end of the section. Each muscle was transferred to a water bath filled with oxygenated KH solution maintained at 37°C. The muscles were aligned horizontally and directly connected between a fixing pin and a dual-mode force transducer servo motor (305C, Aurora Scientific, Aurora, Ontario, Canada). Two platinum plate electrodes were positioned in the organ bath adjacent to the length of the muscle. The muscles were stretched to the optimal length for measuring spasmodic contraction, and then allowed to stand for 10 minutes before initiating the tetanic contraction protocol. Once the muscle stabilized, it was set to an optimal length of 1g and subjected to a warm-up consisting of three 1Hz contractions every 30 seconds, followed by three 150Hz contractions every minute. After a 3-minute rest period, the DIA was stimulated at 20, 50, 80, 120, 150, and 180Hz for 250 milliseconds each, with a 2-minute rest period between each stimulus, to determine the maximum tetanic force. Muscle length and weight were measured. The force was normalized against muscle weight and length.

[0177] Tibialis anterior (TA) tetanus for functional assessment The TA evaluation 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 dissection scope, the skin of the hind limbs was removed to expose the TA muscle and patella. A double square was tied around the patellar tendon with 4-0 sutures. Next, the distal TA tendon was incised, and the double square was tied around the tendon as close to the muscle as possible with 4-0 sutures, and then the tendon was cut. The exposed muscle was kept moist with saline at all times. The mice were then transferred to a thermal control platform and maintained at 37°C. The knee was fixed to a metal pin at the patellar tendon suture and to the level arm of a force transducer (Aurora Scientific, Aurora, Canada) at the distal TA tendon suture. Electrodes were placed near the sciatic nerve to stimulate it. Once the muscle stabilized, the resting tension was set to the length at which spasmodic contraction was maximized (optimal length). After a 3-minute rest period, the TA was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute rest period between each stimulus. After a 5-minute rest, the muscle was subjected to a series of 10 isometric contractions, occurring at 1-minute intervals using a 10% stretch-re-extension procedure. After eccentric contractions, the mice were euthanized, the TA muscle was dissected, weighed, and frozen for analysis.

[0178] Immunofluorescence Frozen sections (12 μm thick) of muscle tissue from the TA, gastrocnemius (GAS), quadriceps femoris (QD), psoas major (PSOAS), gluteus maximus (GLUT), triceps brachii (TRI), DIA, and heart (HRT) were subjected to immunofluorescence staining for hSGCA transgenes. Sections were incubated with rabbit monoclonal α-SG primary antibody (Abcam, Cambridge, UK, catalog number ab189254) at a 1:100 dilution. Four random 20x images covering four different quadrants of the muscle sections were acquired using a Zeiss (Germany) AxioCam MRC5 camera. The percentage of positive fibers to α-SG staining compared to the control was determined for each image and averaged for each muscle. Positive α-SG fiber expression was defined as at least 30% brighter fiber staining than the vehicle-treated sgca- / - control.

[0179] Western blot analysis Samples from wild-type C57BL / 6 mice, vehicle-treated sgca- / - mice, and vector-administered sgca- / - mice were used for each Western blot. hSGCA blots were prepared using a 1:10,000 dilution of rabbit monoclonal α-SG antibody (Abcam, catalog no. ab189254) and a 1:5,000 dilution of mouse monoclonal α-actinin antibody (Sigma-Aldrich, catalog no. A7811). A 1:1,000 dilution of rabbit monoclonal mouse vinculin antibody (Invitrogen, catalog no. 70062) was also used. Chemiluminescent immunodetection was enhanced using anti-mouse (Millipore, catalog no. AP308P) and anti-rabbit (Life Technologies, catalog no. 656120) secondary horseradish peroxidase antibodies. Western blot quantification was performed by densitometry using ImageQuantTL 1D 8.1.0 (GE Healthcare Life Sciences).

[0180] Morphometric analysis Hematoxylin and eosin (H&E) staining was performed on 12 μm thick frozen muscle sections from 16-17 week old wild-type C57BL / 6 mice (n=6), vehicle-treated sgca- / - mice (n=6), and 16-17 week old treated sgca- / - mice (n=6 per dose) of scAAVrh74.tMCK.hSGCA for analysis. The percentage of muscle fibers with nuclei was determined in TA, GAS, QD, GLUT, PSOAS, and TRI muscles. Furthermore, muscle fiber diameter was measured in TA, GAS, QD, TRI, and PSOAS muscles. Four random 20x magnification images per muscle of each animal were acquired using a Zeiss AxioCam MRC5 camera. Nucleated fibers were quantified using National Institutes of Health's ImageJ software, and fiber diameter was measured using Zeiss Axiovision LE4 software.

[0181] Quantitative polymerase chain reaction (PCR) analysis of biological distribution Taqman quantitative PCR was performed to quantify the number of vector genome copies present in the target and non-target contralateral muscles, as well as in non-target organs, as described above. A vector-specific primer-probe set was used to amplify the sequence of the intron region immediately downstream of the unique tMCK promoter located within the scAAVrh.74.tMCK.hSGCA transgene cassette. The following primers and probes were used in this study: tMCK intron forward primer 5'-ACC CGA GAT GCC TGG TTA TAA TT-3' (Sequence ID 12) tMCK Intron Reverse Primer 5'-TCC ATG GTG TAC AGA GCC TAA GAC-3' (Sequence ID 13) , and tMCK intron probe 5'-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C- IABkFQ-3' (Sequence ID 14) (Integrated DNA Technologies). Copy number is reported as vector genome per microgram of genomic DNA.

[0182] Piclosilius red staining and collagen quantification Piclosilius red staining was performed to determine the level of collagen deposition in muscle tissue. Staining was performed on 12 μm thick frozen sections from GLUT, PSOAS, TRI, and DIA muscle of 16-17 week old wild-type C57BL / 6 (n=6), vehicle-treated sgca- / - (n=6), and scAAVrh74.tMCK.hSGCA treated sgca- / - mice (n=6 per dose). Four 20x images were taken per muscle of each mouse, and the amount of collagen deposition was measured using the ImageJ software program. The mean collagen percentage for each muscle was calculated for all groups.

[0183] Laser monitoring of open field cage activities The overall activity of experimental mice was determined using an open-field activity chamber. The mice included wild-type C57BL / 6 (n=6) and untreated SGCA mice. - / - (n=6) 16-17 week old mice from the control group were treated with scAAVrh74.tMCK.hSGCA. - / -Mice (n=6 per dose) were included in the analysis. All mice were tested at the same time each day in a cycle from early morning to near the end of the night, when mice are most active. All mice were tested in isolation rooms under dim light each time and by the same handler. In addition, mice that were not individually housed were tested to reduce anxiety and minimize variable behavior that could affect the normal behavior of the mice and, consequently, the assay results. Mouse behavior was monitored using a Photobeam Activity System (San Diego Instruments, San Diego, CA). This system monitors the position and movement of mice in the xyz plane using an invisible infrared light grid that crosses the anterior, posterior and lateral sides of the animal chamber. Activity was recorded in 1-hour cycles with 5-minute intervals. Mice were acclimated to the activity laboratory in 1-hour sessions several days prior to the start of data acquisition. Mice were tested in groups of four in individual chambers. Test equipment was cleaned after each use to reduce reactive variable behavior in the mice that could alter the results. The data was converted to a Microsoft Excel worksheet, and all calculations were performed within the Excel program. The total gait was obtained by adding the individual ray interruptions of each mouse's movement in the x and y planes, and the vertical activity within a one-hour interval was obtained by adding the ray interruptions in the z plane.

[0184] Safety testing hematology Whole blood was collected from cardiac puncture for hemochemistry. Blood was collected in serum separation tubes and centrifuged at 15,000 rpm for 10 minutes. Serum was collected, frozen, and sent to Charles River Laboratories for chemical testing. Hematological analysis prioritized liver enzymes and glucose chemistry.

[0185] histopathology During the autopsy, the muscles were freshly frozen in methylbutane cooled with liquid nitrogen, and all other organs were harvested, fixed in formalin, and embedded in paraffin. After processing, the tissues were stained with H&E, and the slides and all tissues were sent to GEMPath, Inc. for formal review by a veterinary pathologist.

[0186] statistical analysis Data are presented as mean ± SEM (error bars), and unless otherwise specified, analysis was performed using one-way ANOVA with multiple comparisons between groups, evaluated by Tukey's post-hoc analysis using GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

[0187] Example 2 Efficiency of whole-body delivery of scAAVrh74.tMCK.hSGCA We started a small-scale pilot study, 1 × 10 12 VG dosage (based on 20g mice: 5 × 10 13 (vg / kg, n=4) sgca - / - The effectiveness of gene delivery by intravenous injection into the lateral tail vein of mice was observed. Immunofluorescence analysis was performed on muscle tissue collected 4 weeks after gene transfer. The levels of hSGCA transgene expression in seven different limb skeletal muscles were measured: TA, GAS, GLUT, QD, PSOAS, TRI, and DIA. Mice lacking α-SG showed complete protein deficiency when analyzed by immunofluorescence (Figure 2A; representative images of TA, GAS, TRI, and DIA). 1 × 10⁻⁶ 12 The total therapeutic dose of vg resulted in a mean 54 ± 23.81% vector transfer across all skeletal muscle, including DIA 4 weeks after gene delivery.

[0188] sgca - / - Dose escalation study of scAAVrh74.tMCK.hSGCA delivered systemically to mice. To determine the safest and most effective dose, the delivery of three separate doses of vectors was studied in dose-escalation trials in 4-week-old SGCAs. - / -The lateral tail vein of the mouse scAAVrh74.tMCK.hSGCA 1×10 12 vg(5×10 13 Total dose (vg / kg), 3 × 10 12 Total dose of vg (1 x 10) 14 (vg / kg), or 6 × 10 12 Total dose of vg (2 × 10 14 Mice were treated with (vg / kg). To evaluate the expression of the hSGCA transgene in TA, GAS, QD, GLUT, PSOAS, TRI, DIA, and HRT muscles using immunofluorescence, mice were euthanized 12 weeks after gene delivery. 12 Total dose of vg (5 x 10 13 In mice treated with (vg / kg), the mean hSGCA expression was 70.07 ± 3.71% overall in skeletal muscle, including DIA. 3 × 10 12 Total dose of vg (1 x 10) 14 The mean hSGCA expression in mice treated with the intermediate dose (vg / kg) was 85.35 ± 2.36% in all skeletal muscle. 12 Total dose of vg (2 × 10 14 The mean hSGCA expression in mice treated with (vg / kg) was 93.86 ± 2.02% in all skeletal muscle. For clarity, doses were calculated based on superhelical DNA or plasmids as a quantitative standard. hSGCA expression in HRT muscle remained dose-independent at 75%. Representative tissue images are shown in Figure 2A. Potent hSGCA expression demonstrates the efficacy of gene delivery at all three doses. Gene delivery targeted multiple muscles in both the forelimbs and hindlimbs and showed exceptional α-SG expression in mice at all three doses. Most importantly, key muscles of the diaphragm also showed α-SG gene expression after delivery. Western blots shown in Figure 2B confirm protein expression in all muscles of all three dose cohorts of treated mice. Mouse cardiomyopathy also showed α-SG expression after treatment.

[0189] Histopathological features in both humans and mice lacking α-SG protein include centronucleation, irregular fiber size distribution, necrosis, and fibrosis. H&E staining was used to visualize muscle morphology, including fiber size and centronucleation (Figure 3). As shown in Figures 3A and 3B, normalization of fiber size distribution similar to that observed in wild-type controls was observed in sgca treated with the vehicle. - / - Compared to the control, SGCA treated with scAAVrh74.tMCK.hSGCA - / - This was observed in mouse TA, QD, and TRI. The mean fiber diameter size was significantly increased at all doses of TA, QD, and TRI muscle compared to sgca- / - control mice treated with the vehicle (Table 1). [Table 1]

[0190] scAAVrh74.tMCK.hSGCA treated sgca - / - In mice, a decrease in central nucleation was also observed. SGCA treated with a vehicle. - / - Mouse skeletal muscle contained 68.72 ± 3.01% of fibers with centrally located nuclei. After treatment with scAAVrh74.tMCK.hSGCA, the overall value of central nucleation across all muscle tissue decreased at the lowest dose of scAAVrh74.tMCK.hSGCA, resulting in 55.60 ± 3.25% of skeletal muscle fibers exhibiting centrally located nuclei (Table 2). [Table 2]

[0191] Mice treated with the intermediate dose had 61.85 ± 4.00% of muscle fibers containing a central nucleus, while nucleation in muscle fibers treated with the highest dose was reduced to 37.93 ± 12.46% (Figure 3C).

[0192] Fibrosis, in which tissue is overcome by collagen, frequently occurs in the muscles of LGMD patients and leads to the formation of scar tissue. Fibrosis was evaluated using picrosilius red staining to detect the content of collagen I and III as a marker of fibrosis. As shown in Figure 4, a strong decrease in red staining was observed in sgca- / - mice after treatment with scAAVrh74.tMCK.hSGCA. Quantification revealed that in sgca- / - mice treated with scAAVrh74.tMCK.hSGCA, sgca staining was significantly reduced compared to sgca treated with vehicle. - / - Compared to control mice, a significant decrease in collagen content was observed throughout all muscles (Figure 4B). Together, these data suggest strong expression and SGCA in muscle tissue. - / - We demonstrate the success of systemic delivery of the hSGCA transgene, as shown by the improvement of histopathological characteristics associated with α-SG protein deficiency in mice.

[0193] Example 3 scAAVrh74.tMCK.hSGCA rAAV improves the function of the diaphragm and tibialis anterior muscle and enhances athletic performance. Proximal muscle weakness and loss of function are major symptoms of LGMD2D, and respiratory failure is the leading cause of death in LGMD2D; therefore, improving the function and strength of TA and DIA is essential to increasing the length and quality of life of controls with LGMD2D. Using striae of DIA and whole TA muscles, we examined the correlation between hSGCA expression and muscle strength. As shown in Figures 5A and 5B, compared to wild-type mice, hSGCA expression was improved. - / - Deficiencies in specific force and resistance to contraction-induced injury were observed in the specific force of TA and DIA muscles of untreated mice.

[0194] sgca - / - The TA muscle of mice showed a significant 44% decrease in specific force output compared to wild-type mice (161.6 ± 8.20 mN / mm², respectively). 2 Relative to 291.7 ± 6.17 mN / mm 2(p<0.0001), as well as greater force loss from forces produced according to a strict eccentric contraction protocol (sgca - / - Mice showed a loss of 44.0 ± 6.0%, and wild-type mice showed a loss of 18.0 ± 1.0% (p < 0.0001) (Figure 5A). Twelve weeks after tail vein delivery, the applicant noticed dramatic improvements in specific force output after treatment with low, intermediate, and high doses of scAAVrh74.tMCK.hSGCA, which were 218 ± 11.94 mN / mm², respectively. 2 , 227±11.7mN / mm 2 , and 255±11.7mN / mm 2 It increased. Resistance to injury after eccentric contraction protocol also increased with vehicle-treated SGCA - / - Compared to mice, those treated with low, intermediate, and high doses showed improvement, and mice treated with the vehicle showed improvement. - / - Compared to mice, only 22.0±4.0%, 22.0±3.0%, and 12.0±1.0% (p<0.0001) were lost, respectively (Figure 5A).

[0195] SGCA treated with a vehicle - / - In mouse DIA, the specific force generated showed a 41% decrease in strength compared to wild-type mice (131.5 ± 12.07 mN / mm²). 2 Relative to 223.8 ± 15.85 mN / mm 2 In all three administrations, an improvement in force was observed after treatment with scAAVrh74.tMCK.hSGCA, and the specific force of DIA in low-dose mice was 179.2 ± 21.03 mN / mm². 2 It increased to 201.2±22.94 mN / mm² in mice receiving an intermediate dose. 2 It increased to 261.46±9.73 mN / mm² in high-dose mice. 2 It increased to (Figure 65B). These data were collected by SGCA - / - The TA and DIA muscles of mice exhibited strength deficiencies and showed faster attenuation than those of wild-type mice. However, functional recovery was achieved after delivery of scAAVrh74.tMCK.hSGCA.

[0196] Additional symptoms of LGMD2D include exercise intolerance and decreased activity and walking ability, likely due to muscle damage, causing pain and muscle fatigue. To assess the level of physical activity, SGCA - / - Wild-type C57BL / 6 mice were subjected to an open-field activity protocol similar to that used in previous reports. The mice's walking-related activity was monitored to assess SGCA. - / - We determined whether the absence of α-SG in mice resulted in reduced walking ability compared to wild-type mice. The graph in Figure 5C shows the difference between sgca and wild-type controls. - / - This shows a decrease in walking and vertical standing in the mouse model. sgca - / - The average horizontal walking ray interruption recorded in mice was 2000 ± 159 ray interruptions / hour, compared to 8911 ± 1193 ray interruptions / hour in wild-type controls, representing a 77.5% reduction in walking. (sgca) - / - The average vertical standing ray interruption recorded in mice was 24.75 ± 11.47 ray interruptions / hour, compared to 803.3 ± 55.03 ray interruptions / hour in wild-type mice, representing a 97% reduction in vertical standing. Following treatment with scAAVrh74.tMCK.hSGCA, walking and vertical standing activity in mice increased 12 weeks after gene delivery. The average horizontal walking time was 1 × 10⁻⁶. 12 Mice treated with the total dose measured 3595 ± 55.03 light interruptions / hour, 3 × 10⁻¹⁴. 12 Mice treated with a total dose of VG showed 5238 ± 861.9 light interruptions / hour, 6 × 10⁶ 12 In mice treated with the total dose of vg, the amount of light interruption / hour increased to 6487 ± 467.9. Mean vertical standing activity was 1 × 10⁻⁶. 12 Mice treated with a total dose of 377 ± 146.1 light interruptions / hour, 3 × 10⁻¹⁴ 12 Mice treated with a total dose of vg showed 321 ± 126.1 light interruptions / hour, 6 × 10⁶ 12 In mice treated with a total dose of vg, the total number of photointerruptions / hour increased to 448.8 ± 53.43 (Figure 5C). Physical activity in treated mice was increased compared to SGCA treated with a vehicle. - / -Compared to mice, the treated mice showed improvements of 44%–69% in walking and 92%–94% in standing upright. Furthermore, serum creatine kinase levels were significantly reduced in all treated groups compared to untreated mice (Figure 5D). Together, these data suggest that α-SG delivery restores physical activity and SGCA - / - This demonstrates protection against muscle damage in mice.

[0197] Safety and biodistribution analysis of scAAVrh74.tMCK.hSGCA As a safety measure, blood chemistry and hematology studies were conducted on SGCA administered with a vector. - / - The tests were also performed on wild-type mice. All values ​​were within the normal reference range for mice (Figure 6). Furthermore, sgca administered with scAAVrh74.tMCK.hSGCA - / - All muscle and organ tissue sections stained with H&E from wild-type mice were sent to a veterinary pathologist for formal review. (scAAVrh74.tMCK.hSGCA administered to sgca) - / - No adverse effects were observed in any of the samples from either the mice or the wild-type mice. In addition to efficacy, these data indicate that systemic delivery of all three doses of scAAVrh74.tMCK.hSGCA was well-tolerated, safe, and effective. - / - Furthermore, it was demonstrated that the study was non-toxic to wild-type mice.

[0198] To test potential toxicity or safety concerns regarding the delivery of scAAVrh74.tMCK.hSGCA, vector biodistribution quantitative PCR was performed to quantify the presence of the vector genome (Figure 7A). Using a vector-specific tMCK.hSGCA primer probe set, sgca administered with scAAVrh74.tMCK / hSGCA were analyzed. - / - Vector genomes were detected in all muscle and organ tissues tested from mice. As expected, the vector genomes were present in the tested tissues, showing the highest copy number in the liver, followed by muscle. - / -Western blots of alpha-sarcoglycan proteins in the livers of WT and sgca- / - mice treated with either LR) or scAArh74.tMCK.hSGCA are shown in Figure 7B.

[0199] To improve the efficiency of α-SG expression, in one embodiment, the hSGCA cDNA sequence is packaged in a self-complementary vector. The self-complementary AAV vector contains an inverted repeat genome that facilitates dsDNA formation, thus enabling replication and transcription without requiring multiple vector genomes to facilitate these processes. Thus, the use of a self-complementary vector eliminates the rate-limiting step, enabling faster expression of the transgene. The applicant claims that intravascular delivery of scAAVrh74.tMCK.hSGCA in LGMD2D patients is 1 × 10⁻⁶. 12 and 3×10 12 This indicates an increase in α-SG expression 180 days after gene transfer at this dose.

[0200] Intravenous delivery of scAAVrh74.tMCK.hSGCA provided increased muscle strength and resistance to contraction-induced injury of the tibialis anterior and diaphragmatic muscles in all three vector-treated cohorts compared to vehicle-treated controls. In addition, treatment with scAAVrh74.tMCK.hSGCA resulted in a significant reduction in CK levels. Furthermore, after treatment with scAAVrh74.tMCK.hSGCA, mice were able to walk more frequently and stand on their hind limbs than vehicle-treated mice.

[0201] Significant histopathology, including centrally located nuclei, broad variation in fiber size, inflammation, necrosis, and fibrosis, is typically observed through muscle biopsies of LGMD2D patients. Following hSGCA delivery, mice exhibited reduced CN, a more uniform distribution of muscle fiber size, and decreased collagen content, and the muscles had an overall healthier appearance compared to mice treated with the vehicle. The overall reduction in histopathology and scar tissue was associated with an improvement in the overall normal function and physiology of the muscles of vector-treated mice.

[0202] Finally, safety studies conducted through quantitative PCR, serological analysis, and histopathology revealed no signs of toxicity. The tMCK promoter was detected only in target tissues (all muscle) and not in other (non-muscle) organs except the liver. The detection of tMCK in the liver is neither unusual nor a concern, as it is a clearance organ. Histopathological reviews of all tissues (including liver) by certified veterinary pathologists confirmed that systemic delivery of scAAVrh74.tMCK.hSGCA is safe in all tissues, as well as that gene delivery is safe for SGCA treated in the vehicle. - / - The study concluded that it dramatically reduced the amount of dystrophy pathology observed in mouse skeletal muscle. Chemistry performed on blood samples from vector-treated mice also supported the lack of toxicity.

[0203] As disclosed herein, the dose escalation study was conducted using the lowest dose tested systemically, i.e., a total of 1 × 10⁻⁶ doses. 12 vg(5×10 13 Preclinical data support that vg / kg is 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 vector-treated mice, as demonstrated by increased strength and motor behavior (walking and standing on hind legs). In safety studies, a total of 6 × 10⁶ doses were observed. 12 vg(2×10 14 Even at the highest administered dose (vg / kg), no signs of toxicity were observed.

[0204] Example 4 Elderly patients and persistent Genetic substitution via rAAVrh74.tMCK.hSGCA has shown positive results in the treatment of LGMD-2D and other related diseases. This study was designed to test rAAVrh74.tMCK.hSGCA's ability to treat more severely affected old muscle and to determine the long-term persistence of the AAV viral vector.

[0205] All procedures were carried out 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 standardized conditions with a 12:12 light-dark cycle and were given free access to food and water. First, rAAVrh74.tMCK.hSGCA was administered in three doses (1.0 × 10⁻¹). 12 , 3.0×10 12 , and 6.0×10 12 A 12-month-old SGCA patient presenting with severe muscle tissue pathology (vg) - / - 5 mice (n=5) were administered systemically by tail vein injection. A control group received sgca injected with Ringer's lactate solution (LRS). - / - The study included mice (n=5) and BL6 wild-type mice (n=4) injected with LRS. At the 6-month post-treatment endpoint, muscle tissue from treated mice was evaluated for SCGA protein expression, histological salvage, and functional improvement. All three doses showed potent α-SG protein expression in the muscular sheath, improved histopathology, increased spontaneous movement and specific force generation, protection against eccentric force loss, and reduced serum CK compared to controls. No vector toxicity was detected. In aged mice, treatment resulted in widespread high levels of protein expression in the analyzed muscle, reduced fibrosis, and increased resistance to contraction-induced injury of the tibialis anterior muscle.

[0206] In particular, 12-month-old SGCA - / - Intravenous administration of rAAVrh74.tMCK.hSGCA to mice resulted in widespread high levels of protein expression throughout the muscles of the lower limbs, upper limbs, and proximal torso, including the diaphragm and heart (Figure 8).

[0207] Following administration of scAAVrh74.tMCK.hSGCA, an overall improvement in muscle lesions (Figure 9a) and a decrease in central nucleus formation were observed. In addition, mean fiber size increased to levels similar to those of WT fibers in the gastrocnemius (GAS) and triceps brachii (TRI) muscles after administration (Figure 9b).

[0208] The level of collagen deposition was quantified as a measure of fibrosis. Administration of scAAVrh74.tMCK.hSGCA resulted in a reduction in the level of fibrosis compared to untreated controls (Figure 9c). Functional improvements after administration of scAAVrh74.tMCK.hSGCA were demonstrated by improved force output (specific force) of the tibialis anterior (TA) and diaphragmatic (DIA) muscles, and increased resistance to contraction-induced injury in the TA muscle (Figure 10).

[0209] To further investigate the long-term sustainability of gene therapy, 4-week-old SGCA - / - Mice are systemically administered rAAVrh74.tMCK.hSGCA. For at least 24 months post-treatment, the copy number of the vector genome is detected by qPCR across all transduced muscles tested (TA, TRI, DIA, GLUT, PSOAS, GAS, and QUAD). Protein expression and localization are studied by immunofluorescence staining of the treated muscles.

[0210] While this disclosure describes specific embodiments, it will be understood by those skilled in the art that changes and modifications may occur. Therefore, only such limitations as those found in the claims should be imposed on this disclosure.

[0211] All documents referenced in this application are incorporated herein by reference in their entirety. Sequence ID 1 SGCA cDNA codon-optimized sequence: Sequence ID 2 Human SGCA protein sequence: MAETLFWTPLLVVLLAGLGDTEAQQTTLHPLVGRVFVHTLDHETFLSLPEHVAVPPAVHITYHAHLQGHPDLPRWLRYTQRSPHHPGFLYGSATPEDRGLQVIEVTAYNRDSFDTTRQRLVLEIGDPEGPLLPYQAEFLVRSHDAEEVLPSTPASRFLSALGGLWEPGELQLLNVTSALDRGGRVPLPIEGRKE GVYIKVGSASPFSTCLKMVASPDSHARCAQGQPPLSCYDTLAPHFRVDWCNVTLVDKSVPEPADEVPTPGDGILEHDPFFCPPTEAPDRDFLVDALVTLLVPLLVALLLTLLLAYVMCCRREGRLKRDLATSDIQMVHHCTIHGNTEELRQMAASREVPRPLSTLPMFNVHTGERLPPRVDSAQVPLILDQH* Sequence ID 3 tMCK promoter sequence: CCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGATCCACTACGGGTCTATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGACCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCCCGGGTCAC Sequence number 4 AAVrh74-tMCK-SGCA:

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Claims

1. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) having a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, wherein the percentage identity is determined by BLAST, and the pharmaceutical composition further comprises poloxamer at a concentration of about 0.00001% to about 1%, wherein the nucleotide sequence encodes a protein that maintains alpha-sarcoglycan activity.

2. The pharmaceutical composition according to claim 1, wherein the nucleotide sequence includes the sequence of Sequence ID No.

4.

3. The pharmaceutical composition according to claim 1 or claim 2, wherein the rAAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh. 10, AAV rh. 74, or their variants.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the rAAV has the serotype AAV rh.

74.

5. A composition for treating muscular dystrophy in a person requiring treatment for muscular dystrophy, comprising the pharmaceutical composition described in claim 1.

6. The composition, based on linearized DNA or plasmids as a quantitative standard, yields 1.0 × 10⁻⁶ １３ vg / kg~8.0×10 １３ The composition according to claim 5, comprising rAAV in a dose of vg / kg.

7. The composition according to claim 5 or 6, wherein the composition is formulated for a systemic route of administration, and the composition further comprises a buffer and an ionic strengthener.

8. The composition according to claim 5 or 6, wherein the composition is formulated for injection, infusion, or implantation.

9. The composition according to claim 5 or 6, wherein the composition is formulated for intravenous, intramuscular, or parenteral administration.

10. The composition according to any one of claims 5 to 9, wherein the composition is formulated for administration via an intravenous route through peripheral limb veins.

11. A composition according to any one of claims 5 to 10, The expression level of the alpha-sarcoglycan gene in the target cells increases after administration of the composition compared to the expression level of the alpha-sarcoglycan gene before administration of the composition; or The serum creatine kinase (CK) level in the subject decreases after administration of the composition compared to the serum CK level before administration of the composition; or The number of alpha-sarcoglycan-positive fibers in the target muscle tissue increases after administration of the composition compared to the number of alpha-sarcoglycan-positive fibers before administration of the composition. composition.

12. The composition according to any one of claims 5 to 11, wherein the muscular dystrophy is limb-girdle muscular dystrophy.

13. The composition according to any one of claims 5 to 12, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

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