Adeno-associated viral vector delivery of a-myoglycans and treatment of muscular dystrophy

Abstract

The invention relates to adeno-associated viral vector delivery of A-myoglycans and treatment of muscular dystrophy. Described herein is a method of treating muscular dystrophy in a subject comprising administering a recombinant AAV vector AAVrh74. TMCK.hSCGA using a systemic administration route and at a dose of from about 1.0 * 10 < 12 > vg / kg to about 5.0 * 10 < 15 > vg / kg. Also disclosed are methods of expressing an alpha-myoglycan gene in a cell or a subject in need thereof, reducing serum CK levels, and increasing alpha-myoglycan positive fibers in muscle tissue of a subject.

Description

[0001] This application is a divisional application of a patent application with an original filing date of August 21, 2020, application number 202080073755.8 (international application number PCT / US2020 / 047339), and invention title "Adeno-associated virus vector delivery of α-sarcoglycan and treatment of muscular dystrophy".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 889,749, filed August 21, 2019, U.S. Provisional Application No. 63 / 014,934, filed April 24, 2020, and U.S. Provisional Application No. 63 / 022,843, filed May 11, 2020, all of which are hereby incorporated by reference in their entirety. Technical field

[0004] Described herein are therapeutic vectors, such as AAV vectors expressing α-sarcoglycan, and methods of using these vectors to treat limb-girdle muscular dystrophy, such as LGMD2D.

[0005] Incorporation of sequence listing by reference

[0006] This application contains a sequence listing in computer-readable form (filename: 54652_SeqListing.txt; 18,768 bytes - ASCII text file; created August 17, 2020) as a separate part of the disclosure, which is hereby incorporated by reference in its entirety. Background of the invention

[0007] Muscular dystrophy (MD) is a group of genetic diseases. The group is characterized by the progressive weakness and degeneration of skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while other forms of MD do not appear until middle age or later. The disorders differ in the distribution and degree of muscle weakness (some forms of MD also affect the heart muscle), age of onset, rate of progression, and genetic pattern.

[0008] One group of MDs is limb-girdle muscular dystrophy (LGMD). LGMD is a rare condition, and different individuals present differently in terms of age of onset, areas of muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or even later. Both sexes are equally affected. LGMD causes weakness in the shoulder and pelvic girdles, and over time, the muscles near the thighs and arms may also weaken. Weakness in the legs usually precedes that in the arms. Facial muscles are generally not affected. As the condition progresses, people may develop problems walking and may require a wheelchair over time. Involvement of the shoulder and arm muscles can make it difficult to raise the arms above the head and lift objects. In some types of LGMD, the heart and respiratory muscles may be involved.

[0009] Specialized tests for LGMD are now available through the National Commissioning Group (NCG) national diagnostic programme.

[0010] The 2D subtype of LGMD (LGMD2D) (commonly known as alpha-dystroglycanopathy) is an autosomal recessive genetic disorder caused by mutations in the alpha-dystroglycan gene (SGCA; alpha-dystroglycan), resulting in complete or reduced absence of the functional protein and absence of other structural components of the dystrophin-associated protein complex. Notably, the absence of the alpha-dystroglycan protein causes progressive muscular dystrophy, with deterioration of muscle function, and an age of onset of 3 to 8 years. Symptoms include: delayed walking, proximal muscle weakness due to fat replacement and fibrosis, elevated creatine kinase, scoliosis, and joint contractures. This debilitating disease often leads to wheelchair dependence and death due to respiratory failure. Accordingly, there remains a need for treatments for LGMD2D. SUMMARY OF THE INVENTION

[0011] Described herein are methods of treating muscular dystrophy in a subject in need thereof, which include the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSGCA, wherein the rAAV uses a systemic administration route and is administered at a dose of about 1.0×10 12 to about 5.0×10 15 based on supercoiled DNA or plasmid as a quantitative standard. In one aspect, the present disclosure relates to rAAV expressing the alpha-dystroglycan gene, and methods of delivering alpha-dystroglycan to muscle to reduce and / or prevent fibrosis; and / or increase muscle strength, and / or treat alpha-dystroglycanopathy in a subject suffering from muscular dystrophy.

[0012] In one aspect, the present disclosure describes recombinant adeno-associated virus (rAAV) that contains a polynucleotide sequence encoding α-dystroglycan protein. In some embodiments, the polynucleotide sequence contains 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 α-dystroglycan activity. In some embodiments, the polynucleotide sequence contains the nucleotide sequence shown in SEQ ID NO:1. In another embodiment, the polynucleotide encodes a protein that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the nucleotide sequence shown in SEQ ID NO:2. In another embodiment, the polynucleotide encodes a protein that contains the amino acid sequence shown in SEQ ID NO:2.

[0013] In some embodiments, the rAAV contains a nucleotide sequence containing the tMCK promoter. For example, the tMCK promoter contains the nucleotide sequence shown in SEQ ID NO:3. In addition, the rAAV contains the 5' inverted terminal repeat of SEQ ID NO:5 and / or the 3' inverted terminal repeat of SEQ ID NO:6. In some embodiments, the rAAV contains the poly A sequence of SEQ ID NO:7. In one embodiment, the disclosed rAAV is serotype AAVrh.74.

[0014] In one embodiment, the polynucleotide 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%, 99%, or 99.5% identical to SEQ ID NO:4. In one embodiment, the polynucleotide contains the nucleotide sequence shown in SEQID NO:4.

[0015] The present disclosure provides methods for treating muscular dystrophy in a subject in need thereof, which include the step of administering any one of the rAAV disclosed herein, wherein the rAAV is administered by a systemic route. In particular, in any of the disclosed methods, the rAAV is AAVrh74.tMCK.hSGCA, wherein the rAAV is administered using a systemic administration route.

[0016] In any of the disclosed methods, based on supercoiled DNA or plasmid as a quantitative standard, rAAV is administered at a dose of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg. For example, based on supercoiled DNA or plasmid as a quantitative standard, rAAV is administered 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 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg or about 5×10 13 vg / kg to about 2×10 14 vg / kg, or rAAV is administered at a dose of about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 vg / kg.

[0017] In another embodiment, in any of the disclosed methods, based on linearized DNA or plasmid as a quantitative standard, rAAV is administered at a dose of about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg. For example, based on linearized DNA or plasmid as a quantitative standard, rAAV is administered at a dose of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg in a dose.

[0018] In addition, in any of the disclosed methods, the systemic administration route is the 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 the intravenous route via a peripheral limb vein.

[0019] 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).

[0020] In one exemplary embodiment, a method of treating muscular dystrophy includes administering rAAV to a subject having limb-girdle muscular dystrophy, and the rAAV is administered in a dose of about 5×10 13 vg / kg to about 2×10 14 vg / kg by intravenous infusion based on supercoiled DNA or plasmid as a quantitative standard, and wherein the rAAV contains the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0021] In an exemplary embodiment, the present disclosure provides a method of treating muscular dystrophy in a subject in need thereof, the method comprising the step of administering rAAV to the subject, wherein the subject has limb-girdle muscular dystrophy, and the rAAV is administered in a dose of about 5×1013 vg / kg to about 2×10 14 vg / kg by intravenous infusion based on supercoiled DNA or plasmid as a quantitative standard, and wherein the rAAV contains the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. For example, in these methods, the α-sarcoglycan gene expression level in the subject's cells increases after administering rAAV as compared to the α-sarcoglycan gene expression level before administering rAAV.

[0022] In any of the disclosed methods, the level of α - dystroglycan gene expression in the subject's cells increases after administration of rAAV as compared to the level of α - dystroglycan gene expression prior to administration of rAAV; and / or wherein the serum CK level in the subject decreases after administration of rAAV as compared to the serum CK level prior to administration of rAAV; and / or wherein voluntary activity and force generation increase; wherein fibrosis decreases; wherein the resistance to contraction - induced injury in the tibialis anterior muscle increases; and / or wherein the number of α - dystroglycan - positive fibers in the muscle tissue of the subject increases after administration of rAAV as compared to the number of α - dystroglycan - positive fibers prior to administration of rAAV; or wherein fibrosis decreases in the subject after administration of rAAV as compared to prior to administration of rAAV; and / or wherein fibrosis decreases in the subject after administration of rAAV as compared to prior to administration of rAAV; and / or wherein the force, fiber diameter size, and / or eccentric contraction in the subject's muscle increase after administration of rAAV as compared to prior to administration of rAAV.

[0023] In some embodiments, α - dystroglycan gene expression is detected by measuring the level of α - dystroglycan protein by Western blot and / or immunohistochemistry.

[0024] In another aspect, the present disclosure provides a method of expressing the α - dystroglycan gene in a cell, which comprises administering to a subject any of the disclosed rAAVs. For example, the present disclosure provides a method of expressing the α - dystroglycan gene in a cell, which comprises administering to a subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. Additionally, in any of the methods, the expression of the α - dystroglycan gene in the subject's cells is detected by measuring the level of α - dystroglycan protein by Western blot in a muscle biopsy. Alternatively, in any of the methods, the expression of the α - dystroglycan gene in the cells is detected by measuring the level of α - dystroglycan protein by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α - dystroglycan gene is measured in a subject by detecting the number of vector genomes per microgram of genomic DNA.

[0025] The present disclosure provides a method of reducing the serum CK level in a subject in need thereof, the method comprising administering to the subject any of the disclosed rAAVs. For example, the present disclosure provides a method of reducing the serum CK level in a subject in need thereof, the method comprising administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0026] In another aspect, the present disclosure provides methods of increasing α - sarcoglycan - positive fibers in the muscle tissue of a subject, which include administering to the subject any one of the disclosed rAAVs. For example, the present disclosure provides a method of increasing α - sarcoglycan - positive fibers in the muscle tissue of a subject in need thereof, the method including administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0027] The present disclosure also provides methods of increasing the expression of α - sarcoglycan in a subject in need thereof, which include administering to the subject any one of the disclosed rAAVs. For example, the present disclosure provides a method of increasing the expression of α - sarcoglycan in a subject in need thereof, the method including administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. Additionally, in any one of the disclosed methods, the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring the α - sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any one of the methods, the expression of the α - sarcoglycan gene in the cells is detected by measuring the α - sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α - sarcoglycan gene is measured in a subject by detecting the vector genome copy number per microgram of genomic DNA.

[0028] The present disclosure provides a composition for treating muscular dystrophy in a subject in need thereof, wherein the composition comprises any one of the rAAVs disclosed herein, and wherein the composition is formulated for administration by a systemic route. In particular, in any one of the compositions, the rAAV is AAVrh74.tMCK.hSGCA.

[0029] Any one of the disclosed compositions 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 dose as a quantitative standard. For example, based on supercoiled DNA or plasmid as a quantitative standard, the dose of rAAV is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg or about 5×10 13vg / kg to about 2×10 14 vg / kg, or the dose of rAAV is about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 vg / kg.

[0030] In another embodiment, in any of the disclosed compositions, based on linearized DNA or plasmid as a quantitative standard, rAAV is administered at a dose of about 1.85×10 13 vg / kg or about 7.41×10 13 vg / kg. For example, based on linearized DNA or plasmid as a quantitative standard, rAAV is administered at a dose of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg.

[0031] In addition, any of the disclosed compositions is formulated for administration by the intravenous route, such as a composition formulated for administration by injection, infusion, or implantation. In some embodiments, the disclosed compositions are formulated for administration by the intravenous route via a peripheral limb vein.

[0032] Any of the disclosed compositions is used to treat limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0033] In one exemplary embodiment, the present disclosure provides a composition for treating a subject with limb-girdle muscular dystrophy, wherein the composition comprises rAAV at a dose of about 5×10 13 vg / kg to about 2×10 14 vg / kg based on the supercoiled DNA or plasmid dose as a quantitative standard, and wherein the composition is formulated for administration by intravenous infusion, and wherein the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0034] In addition, the present disclosure provides a composition for treating limb-girdle muscular dystrophy in a subject in need thereof, wherein the composition comprises rAAV at a dose of about 5×1013 vg / kg to about 2×10 14 vg / kg, and the composition is formulated for administration by intravenous infusion, wherein 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 α-sarcoglycan gene expression in the cells of the subject compared to the level of α-sarcoglycan gene expression prior to administration of rAAV.

[0035] In addition, compared to the α - sarcoglycan gene expression level before administration of the composition, administration of any of the disclosed compositions increases the α - sarcoglycan gene expression level in the cells of the subject; and / or wherein compared to the serum CK level before administration of the composition, administration of the disclosed composition reduces the serum CK level of the subject; and / or wherein voluntary activity and force generation are increased; wherein fibrosis is reduced; wherein the resistance to contraction - induced injury in the tibialis anterior muscle is increased; and / or wherein compared to the number of α - sarcoglycan - positive fibers before administration of the composition, administration of the composition increases the number of α - sarcoglycan - positive fibers in the muscle tissue of the subject; and / or wherein compared to before administration of rAAV, administration of the composition reduces fibrosis in the subject; and / or wherein compared to before administration of the composition, the composition reduces fibrosis; or wherein compared to before administration of the composition, administration of the composition increases the force, fiber diameter size, and / or eccentric contraction in the muscle of the subject. In some embodiments, α - sarcoglycan gene expression is detected by measuring α - sarcoglycan protein levels by Western blot and / or immunohistochemistry.

[0036] In another aspect, the present disclosure provides a composition for expressing the α - sarcoglycan gene in cells, wherein the composition comprises any of the disclosed rAAVs. For example, the present disclosure provides a composition for expressing the α - sarcoglycan gene in cells, which comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. Additionally, in any of the compositions, the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring α - sarcoglycan protein levels by Western blot in a muscle biopsy. Alternatively, in any of the methods, the expression of the α - sarcoglycan gene in cells is detected by measuring α - sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α - sarcoglycan gene is measured by detecting the number of vector genomes per microgram of genomic DNA in the subject.

[0037] The present disclosure provides a composition for reducing the serum CK level of a subject in need thereof, the composition comprising any of the disclosed rAAVs. For example, the present disclosure provides a composition for reducing the serum CK level of a subject in need thereof, the composition comprising the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0038] On the other hand, the present disclosure provides compositions for increasing α - sarcoglycan - positive fibers in the muscle tissue of a subject, wherein the composition comprises any of the disclosed rAAVs. For example, the present disclosure provides a composition for increasing α - sarcoglycan - positive fibers in the muscle tissue of a subject, wherein the composition comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4.

[0039] The present disclosure also provides compositions for increasing the expression of α - sarcoglycan in a subject in need thereof, wherein the composition comprises any of the disclosed rAAVs. For example, the present disclosure provides a composition for increasing the expression of α - sarcoglycan in a subject in need thereof, wherein the composition comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4. Further, after administering any of the disclosed compositions, the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring the α - sarcoglycan protein level by Western blotting in a muscle biopsy. Alternatively, after administering any of the disclosed compositions, the expression of the α - sarcoglycan gene in the cells is detected by measuring the α - sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, after administering any of the disclosed compositions, the expression of the α - sarcoglycan gene is measured in the subject by detecting the vector genome copy number per microgram of genomic DNA.

[0040] The present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein the medicament is formulated for administration by a systemic route. In particular, the present disclosure provides the use of AAVrh74.tMCK.hSGCA for the preparation of a medicament for treating muscular dystrophy, wherein the medicament is formulated for administration by a systemic administration route.

[0041] 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 dose as a quantitative standard. For example, based on supercoiled DNA or plasmid as a quantitative standard, the dose of rAAV is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13vg / kg to about 3.0×10 14 vg / kg or about 5×10 13 vg / kg to about 2×10 14 vg / kg, or the dose of rAAV is about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 [[ID=********]]vg / kg。

[0042] In another embodiment, in any of the disclosed uses, based on linearized DNA or plasmid as a quantitative standard, the drug comprises an rAAV at a dose of about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg. For example, based on linearized DNA or plasmid as a quantitative standard, the drug comprises an rAAV at a dose of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×1x0 13 vg / kg of rAAV.

[0043] It should be noted that there seems to be an incomplete or incorrect expression in the original text at "vg / kg。 ", which is translated as "vg / kg." here. You may want to check and correct the original text for more accurate translation.In addition, in any of the disclosed uses, the drug is formulated for administration by the intravenous route. For example, in any of the disclosed uses, the drug is formulated for administration by injection, infusion, or implantation. In some embodiments, the drug is formulated for administration by the intravenous route via a peripheral limb vein.

[0044] In any of the disclosed uses, the drug is used to treat limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0045] In an exemplary embodiment, the present disclosure provides the use of rAAV for the preparation of a drug for the treatment of limb-girdle muscular dystrophy, wherein the drug is formulated for administration by intravenous infusion, and based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is from about 5×10^13 vg / kg to about 2×10 14 vg / kg, and wherein the rAAV contains the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. For example, compared to the α-sarcoglycan gene expression level before administration of rAAV, administration of the drug to a subject in need results in an increase in α-sarcoglycan gene expression in the subject's cells.

[0046] In any of the disclosed uses, compared to the α-sarcoglycan gene expression level before administration of the drug, administration of the drug to a subject in need results in an increase in the α-sarcoglycan gene expression level in the subject's cells; and / or compared to the serum CK level before administration of the drug, administration of the drug to the subject results in a decrease in the subject's serum CK level; and / or wherein voluntary activity and force generation are increased; wherein fibrosis is reduced; wherein the resistance to contraction-induced injury in the tibialis anterior muscle is increased; and / or compared to the number of α-sarcoglycan-positive fibers before administration of the drug, administration of the drug to the subject results in an increase in the number of α-sarcoglycan-positive fibers in the subject's muscle tissue, and / or compared to before administration of the drug, administration of the drug to a subject in need results in a reduction in fibrosis in the subject; and / or compared to before administration of the drug, administration of the drug results in an increase in force, fiber diameter size, and / or eccentric contraction in the subject's muscle. In some embodiments, α-sarcoglycan gene expression is detected by measuring the α-sarcoglycan protein level by Western blot and / or immunohistochemistry.

[0047] In another aspect, the present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for expressing the α - sarcoglycan gene in cells of a subject in need thereof. For example, the present disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for expressing the α - sarcoglycan gene in cells of a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4. Additionally, in any of the uses, the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring the α - sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any of the uses, the expression of the α - sarcoglycan gene in the cells is detected by measuring the α - sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α - sarcoglycan gene is measured in a subject by detecting the vector genome copy number per microgram of genomic DNA.

[0048] The present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for reducing the serum CK level of a subject in need thereof. For example, the present disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for reducing the serum CK level of a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0049] In another aspect, the present disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for increasing the α - sarcoglycan - positive fibers in the muscle tissue of a subject. For example, the present disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for increasing the α - sarcoglycan - positive fibers in the muscle tissue of a subject, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0050] The present disclosure also provides the use of any one of the disclosed rAAVs for the preparation of a drug for increasing the expression of α-sarcoglycan in a subject in need thereof. For example, the present disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a drug for increasing the expression of α-sarcoglycan in a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4. Additionally, in any one of the disclosed uses, the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy. Alternatively, in any one of the disclosed uses, the expression of the α-sarcoglycan gene in the cells is detected by measuring the α-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the α-sarcoglycan gene is measured in a subject by detecting the vector genome copy number per microgram of genomic DNA.

[0051] In any one of the disclosed methods, compositions or uses, the subject is a human subject between 4 and 15 years of age, or a human subject between 25 and 55 years of age or a human subject over 50 years of age.

[0052] In any one of the disclosed methods, compositions or uses, the subject is a pediatric subject, an adolescent subject or a young adult subject. Alternatively, the subject is a middle-aged adult or an elderly subject.

[0053] For example, in any one of the disclosed methods, compositions or uses, the subject is a human subject between 4 and 15 years of age, having confirmed mutations in α-sarcoglycan (SGCA) in both alleles, being negative for AAVrh74 antibodies and / or having >40% or a normal 100-meter walk test.

[0054] In another aspect, the present disclosure provides a method for producing rAAV to be administered in any one of the methods of the disclosed methods, compositions or uses, the method comprising transferring an AAV vector plasmid to a host cell, wherein the AAV vector plasmid comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8. For example, the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO:8. In some embodiments, the vector plasmid comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, 4 or 8, or the vector plasmid comprises the nucleotide sequence of SEQ ID NO:1, 4 or 8.

[0055] In any of the disclosed methods of producing rAAV, the method further includes transferring a packaging plasmid and / or helper virus to a host cell. Additionally, in any of the disclosed methods of producing rAAV, the packaging cell contains a stably integrated AAV cap gene and / or contains a packaging cell that contains a stably integrated AAV rep gene.

[0056] In another aspect, the present disclosure provides a host cell that contains an AAV vector plasmid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 4, or 8. For example, the host cell contains an AAV vector plasmid that contains the nucleotide sequence of SEQ ID NO: 1, 4, or 8.

[0057] The present disclosure also provides the following.

[0058] 1. A method of treating a subject in need of treatment for muscular dystrophy, the method comprising the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, wherein the rAAV uses a systemic administration route and is administered at a dose of from about 1.0×10 12 to about 5.0×10 15 based on supercoiled DNA or plasmid as a quantification standard.

[0059] 2. The method according to embodiment 1, wherein the rAAV is administered at a dose of from about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, from about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, from about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, from about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or from about 5×10 13 vg / kg to about 2×10 14 vg / kg based on supercoiled DNA or plasmid as a quantification standard.

[0060] 3. The method according to embodiment 1, wherein the rAAV is administered at a dose of from about 5×10 13 vg / kg to about 2×10 14 vg / kg.

[0061] 4. The method according to embodiment 1, wherein the rAAV is based on supercoiled DNA or plasmid as a quantification standard at about 5×1013 vg / kg, about 1×10 14 vg / kg or about 2×10 14 vg / kg.

[0062] 5. A method of treating a subject in need of treatment for muscular dystrophy, comprising the step of administering recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA, wherein the rAAV is administered using a systemic administration route and is based on linearized DNA or plasmid as a quantitative standard at about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0063] 6. The method according to any one of aspects 1 to 5, wherein the level of α-dystroglycan gene expression in the cells of the subject increases after administration of the rAAV as compared to the level of α-dystroglycan gene expression before administration of the rAAV; wherein the serum CK level of the subject decreases after administration of the rAAV as compared to the serum CK level before administration of the rAAV; wherein voluntary activity and force generation increase; wherein fibrosis decreases; wherein the resistance to contraction-induced injury in the tibialis anterior muscle increases; and / or wherein the number of α-dystroglycan-positive fibers in the muscle tissue of the subject increases after administration of the rAAV as compared to the number of α-dystroglycan-positive fibers before administration of the rAAV.

[0064] 7. The method according to any one of aspects 1 to 6, wherein the systemic administration route is an intravenous route.

[0065] 8. The method according to any one of aspects 1 to 7, wherein the rAAV is administered by injection, infusion or implantation.

[0066] 9. The method according to any one of aspects 1 to 8, wherein the rAAV is administered by infusion.

[0067] 10. The method according to any one of aspects 1 to 9, wherein the rAAV is administered by an intravenous route via a peripheral limb vein.

[0068] 11. The method according to any one of aspects 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.

[0069] 12. The method according to aspect 11, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO:1.

[0070] 13. The method according to any one of Aspects 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.

[0071] 14. The method according to any one of Aspects 1 to 12, wherein the rAAV comprises a nucleotide sequence encoding the polypeptide sequence shown in SEQ ID NO:2.

[0072] 15. The method according to any one of Aspects 1 to 14, wherein the rAAV comprises a scAAVrh74.tMCK.hSGCA construct nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0073] 16. The method according to Aspect 15, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4.

[0074] 17. The method according to any one of Aspects 1 to 16, wherein the rAAV comprises a tMCK promoter.

[0075] 18. The method according to any one of Aspects 1 to 16, wherein the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO:3.

[0076] 19. The method according to any one of Aspects 1 to 18, wherein the rAAV comprises the 5' inverted terminal repeat of SEQ ID NO:5.

[0077] 20. The method according to any one of Aspects 1 to 19, wherein the rAAV comprises the 3' inverted terminal repeat of SEQ ID NO:6.

[0078] 21. The method according to any one of Aspects 1 to 20, wherein the rAAV comprises the polyA sequence of SEQ ID NO:7.

[0079] 22. The method according to any one of Aspects 1 to 21, wherein the rAAV is serotype AAVrh.74.

[0080] 23. The method according to any one of Aspects 1 to 22, wherein the muscular dystrophy is limb-girdle muscular dystrophy.

[0081] 24. The method according to any one of aspects 1 to 23, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

[0082] 25. The method according to any one of aspects 1 to 24, wherein the subject has limb-girdle muscular dystrophy, and the rAAV is administered by intravenous infusion at a dose of about 5×10 13 vg / kg to about 2×10 14 vg / kg based on supercoiled DNA or plasmid as a quantitative standard, and wherein the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0083] 26. The method according to any one of aspects 1 to 25, wherein the level of α-sarcoglycan gene expression in the cells of the subject increases after administration of the rAAV as compared to the level of α-sarcoglycan gene expression before administration of the rAAV.

[0084] 27. The method according to any one of aspects 1 to 26, wherein fibrosis is reduced in the subject after administration of the rAAV as compared to before administration of the rAAV.

[0085] 28. The method according to any one of aspects 1 to 26, wherein fibrosis, central nucleation, CK level, and / or collagen deposition in the subject are reduced after administration of the rAAV as compared to before administration of the rAAV.

[0086] 29. The method according to any one of aspects 1 to 26, wherein specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject increase after administration of the rAAV as compared to before administration of the rAAV.

[0087] 30. The method according to aspect 26, wherein the α-sarcoglycan gene expression is detected by measuring the α-sarcoglycan protein level by Western blot and / or immunohistochemistry.

[0088] 31. A method for expressing the α-sarcoglycan gene in a cell, comprising administering to a subject an scAAVrh74.tMCK.hSGCA construct comprising the nucleotide sequence of SEQ ID NO:4.

[0089] 32. The method according to aspect 31, wherein the expression of the α-sarcoglycan gene in the cells of the subject is detected by measuring the α-sarcoglycan protein level by Western blot in a muscle biopsy.

[0090] 33. The method according to embodiment 31, wherein the expression of the α - sarcoglycan gene in the cells is detected by measuring the level of α - sarcoglycan protein in a muscle biopsy by immunohistochemistry.

[0091] 34. The method according to embodiment 31, wherein the expression of the α - sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0092] 35. A method of reducing the serum CK level in a subject in need thereof, the method comprising administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0093] 36. A method of increasing α - sarcoglycan - positive fibers in the muscle tissue of a subject, which comprises administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0094] 37. A method of increasing the expression of α - sarcoglycan in a subject in need thereof, which comprises administering to the subject an effective amount of the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0095] 38. A composition for treating muscular dystrophy in a subject in need thereof, wherein the composition comprises recombinant adeno - associated virus (rAAV) AAVrh74.tMCK.hSCGA 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 quantitative standard, and wherein the composition is formulated for systemic administration.

[0096] 39. A composition for treating muscular dystrophy in a subject in need thereof, wherein the composition comprises recombinant adeno - associated virus (rAAV) AAVrh74.tMCK.hSCGA at a dose of about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg based on linearized DNA or plasmid as a quantitative standard, and wherein the composition is formulated for systemic administration.

[0097] 40. A composition comprising a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a 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.

[0098] 41. The composition according to embodiment 38 or 40, wherein the dose of the rAAV is about 5×10 13 vg / kg to about 2×10 14 vg / kg based on supercoiled DNA or plasmid as a quantification standard.

[0099] 42. The composition according to embodiment 38 or 40, wherein the dose of the rAAV is about 5×10 13 vg / kg, about 1×10 14 vg / kg or about 2×10 14 vg / kg based on supercoiled DNA or plasmid as a quantification standard.

[0100] 43. The composition according to any one of embodiments 38 to 42, wherein the expression level of the α-sarcoglycan gene in the cells of the subject is increased after administration of the composition compared to the α-sarcoglycan gene expression level before administration of the composition; or wherein the serum CK level of the subject is decreased after administration of the composition compared to the serum CK level before administration of the composition; or wherein the number of α-sarcoglycan positive fibers in the muscle tissue of the subject is increased after administration of the composition compared to the number of α-sarcoglycan positive fibers before administration of the composition.

[0101] 44. The composition according to any one of embodiments 38 to 43, wherein the systemic administration route is the intravenous route.

[0102] 45. The composition according to any one of embodiments 38 to 44, wherein the composition is formulated for administration by injection, infusion or implantation.

[0103] 46. The composition according to any one of embodiments 38 to 45, wherein the composition is formulated for administration by infusion.

[0104] 47. The composition according to any one of embodiments 38 to 46, wherein the composition is formulated for administration by the intravenous route through a peripheral limb vein.

[0105] 48. The composition according to any one of aspects 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 to SEQ ID NO: 1.

[0106] 49. The composition according to aspect 48, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 1.

[0107] 50. The composition according to any one of aspects 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.

[0108] 51. The composition according to any one of aspects 38 to 49, wherein the rAAV comprises the nucleotide sequence encoding the polypeptide sequence shown in SEQ ID NO: 2.

[0109] 52. The composition according to any one of aspects 38 to 49, wherein the rAAV comprises a 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.

[0110] 53. The composition according to aspect 52, wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 4.

[0111] 54. The composition according to any one of aspects 38 to 53, wherein the rAAV comprises a tMCK promoter.

[0112] 55. The composition according to aspect 54, wherein the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0113] 56. The composition according to any one of aspects 38 to 55, wherein the rAAV comprises the 5' inverted terminal repeat of SEQ ID NO: 5.

[0114] 57. The composition according to any one of aspects 38 to 56, wherein the rAAV comprises the 3' inverted terminal repeat of SEQ ID NO: 6.

[0115] 58. The composition according to any one of aspects 38 to 57, wherein the rAAV comprises the polyA sequence of SEQ ID NO: 7.

[0116] 59. The composition according to any one of aspects 38 to 58, wherein the rAAV is serotype AAVrh.74.

[0117] 60. The composition according to any one of aspects 38 to 59, wherein the muscular dystrophy is limb-girdle muscular dystrophy.

[0118] 61. The composition according to any one of aspects 38 to 60, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

[0119] 62. The composition according to any one of aspects 38 to 61, wherein the subject has limb-girdle muscular dystrophy, and the composition is formulated for administration by intravenous infusion at a dose of about 5×10 13 vg / kg to about 2×10 14 vg / kg based on supercoiled DNA or plasmid as a quantitative standard, and wherein the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:4.

[0120] 63. The composition according to any one of aspects 38 to 62, wherein the level of α-sarcoglycan gene expression in the cells of the subject increases after administration of the composition as compared to the level of α-sarcoglycan gene expression before administration of the composition.

[0121] 64. The composition according to any one of aspects 38 to 63, wherein fibrosis is reduced in the subject after administration of the composition as compared to before administration of the composition.

[0122] 65. The composition according to any one of aspects 38 to 63, wherein fibrosis, central nucleation, CK level, and / or collagen deposition in the subject are reduced after administration of the composition as compared to before administration of the composition.

[0123] 66. The composition according to any one of aspects 38 to 65, wherein specific force, fiber diameter size, and / or eccentric contraction in the muscle of the subject increase after administration of the composition as compared to before administration of the composition.

[0124] 67. The composition according to aspect 63, wherein the α-sarcoglycan gene expression is detected by measuring the α-sarcoglycan protein level by Western blot and / or immunohistochemistry.

[0125] 68. A composition for expressing the α - sarcoglycan gene in cells, wherein the composition comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0126] 69. The composition according to aspect 68, wherein the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring the α - sarcoglycan protein level by Western blotting in a muscle biopsy.

[0127] 70. The composition according to aspect 68, wherein the expression of the α - sarcoglycan gene in the cells is detected by measuring the α - sarcoglycan protein level by immunohistochemistry in a muscle biopsy.

[0128] 71. The composition according to aspect 68, wherein the expression of the α - sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0129] 72. A composition for reducing the serum CK level in a subject in need thereof, the composition comprising the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO:4.

[0130] 73. A composition for increasing the α - sarcoglycan - positive fibers in the muscle tissue of a subject, which comprises the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO:4.

[0131] 74. A composition for increasing the expression of α - sarcoglycan in a subject in need thereof, which comprises the scAAVrh74.tMCK.hSGCA construct containing the nucleotide sequence of SEQ ID NO:4.

[0132] 75. Use of recombinant adeno - associated virus (rAAV) AAVrh74.tMCK.hSCGA A for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg, and wherein the medicament is formulated for systemic administration routes.

[0133] 76. The use according to aspect 75, wherein based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg or about 5×10 13 vg / kg to about 2×10 14 vg / kg.

[0134] 77. The use according to Scheme 75, wherein based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is about 5×10 13 vg / kg to about 2×10 14 vg / kg.

[0135] 78. The use according to Scheme 75, wherein based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is about 5×10 13 vg / kg, about 1×10 14 vg / kg or about 2×10 14 vg / kg.

[0136] 79. The use of recombinant adeno-associated virus (rAAV) AAVrh74.tMCK.hSCGA A for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein based on linearized DNA or plasmid as a quantitative standard, the dose of the rAAV is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg, and wherein the medicament is formulated for systemic administration route.

[0137] 80. The use according to any one of Schemes 75 to 79, wherein the α-sarcoglycan gene expression level in the cells of the subject increases after administration of the medicament as compared to the α-sarcoglycan gene expression level before administration of the medicament; or wherein the serum CK level of the subject decreases after administration of the medicament as compared to the serum CK level before administration of the medicament; or wherein the number of α-sarcoglycan positive fibers in the muscle tissue of the subject increases after administration of the medicament as compared to the number of α-sarcoglycan positive fibers before administration of the medicament.

[0138] 81. The use according to any one of Schemes 75 to 80, wherein the systemic administration route is the intravenous route.

[0139] 82. The use according to any one of Schemes 75 to 81, wherein the medicament is formulated for administration by injection, infusion or implantation.

[0140] 83. Use according to any one of aspects 75 to 82, wherein the medicament is formulated for administration by infusion.

[0141] 84. Use according to any one of aspects 75 to 83, wherein the composition is formulated for administration by the intravenous route via a peripheral limb vein.

[0142] 85. Use according to any one of aspects 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.

[0143] 86. Use according to aspect 85, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 1.

[0144] 87. Use according to any one of aspects 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.

[0145] 88. Use according to any one of aspects 75 to 86, wherein the rAAV comprises the nucleotide sequence encoding the polypeptide sequence shown in SEQ ID NO: 2.

[0146] 89. Use according to any one of aspects 75 to 86, wherein the rAAV comprises a nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4.

[0147] 90. Use according to aspect 89, wherein the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 4.

[0148] 91. Use according to any one of aspects 75 to 90, wherein the rAAV comprises a tMCK promoter.

[0149] 92. Use according to aspect 91, wherein the tMCK promoter comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0150] 93. Use according to any one of aspects 75 to 92, wherein the rAAV comprises the 5' inverted terminal repeat of SEQ ID NO: 5.

[0151] 94. The use according to any one of aspects 75 to 93, wherein the rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO: 6.

[0152] 95. The use according to any one of aspects 73 to 94, wherein the rAAV comprises the polyA sequence of SEQ ID NO: 7.

[0153] 96. The use according to any one of aspects 73 to 95, wherein the rAAV is serotype AAVrh.74.

[0154] 97. The use according to any one of aspects 73 to 96, wherein the muscular dystrophy is limb-girdle muscular dystrophy.

[0155] 98. The use according to any one of aspects 73 to 97, wherein the muscular dystrophy is limb-girdle muscular dystrophy type 2D (LGMD2D).

[0156] 99. The use according to any one of aspects 73 to 98, wherein the subject has limb-girdle muscular dystrophy, and the medicament is formulated for intravenous infusion, and based on supercoiled DNA or plasmid as a quantitative standard, the dose of the rAAV is about 5×10 13 vg / kg to about 2×10 14 vg / kg, and wherein the rAAV comprises the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 4.

[0157] 100. The use according to any one of aspects 73 to 99, wherein the level of α-dystroglycan gene expression in the cells of the subject increases after administration of the medicament as compared to the level of α-dystroglycan gene expression before administration of the medicament.

[0158] 101. The use according to any one of aspects 73 to 100, wherein fibrosis is reduced in the subject after administration of the medicament as compared to before administration of the medicament.

[0159] 102. The use according to any one of aspects 73 to 100, wherein fibrosis, central nucleation, CK level, and / or collagen deposition in the subject are reduced after administration of the medicament as compared to fibrosis before administration of the medicament.

[0160] 103. The use according to any one of aspects 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 medicament as compared to before administration of the medicament.

[0161] 104. The use as described in Scheme 100, wherein the expression of the α - sarcoglycan gene is detected by measuring the level of α - sarcoglycan protein by Western blot and / or immunohistochemistry.

[0162] 105. The use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for expressing the α - sarcoglycan gene in cells, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0163] 106. The use as described in Scheme 105, wherein the expression of the α - sarcoglycan gene in the cells of the subject is detected by measuring the level of α - sarcoglycan protein by Western blot in a muscle biopsy.

[0164] 107. The use as described in Scheme 105, wherein the expression of the α - sarcoglycan gene in the cells is detected by measuring the level of the α - sarcoglycan protein by immunohistochemistry in a muscle biopsy.

[0165] 108. The use as described in Scheme 105, wherein the expression of the α - sarcoglycan gene is measured by detecting the number of vector genomes per microgram of genomic DNA in the subject.

[0166] 109. The use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for reducing the serum CK level in a subject in need thereof, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0167] 110. The use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for increasing the α - sarcoglycan - positive fibers in the muscle tissue of a subject, wherein the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0168] 111. The use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a medicament for increasing the expression of α - sarcoglycan in a subject in need thereof, the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO:4.

[0169] 112. The method, composition or use as described in any one of Schemes 1 to 111, wherein the subject is a human subject aged 4 to 15 years.

[0170] 113. The method, composition or use according to any one of aspects 1 to 111, wherein the subject is a pediatric subject, an adolescent subject or a young adult subject.

[0171] 114. The method, composition or use according to any one of aspects 1 to 111, wherein the subject is a human subject aged 4 - 15 years, with a confirmed mutation in α - sarcoglycan (SGCA) in both alleles, negative for AAVrh74 antibodies and / or having a > 40% or normal 100 - meter walking test.

[0172] 115. The method, composition or use according to any one of aspects 1 to 111, wherein the subject is a middle - aged adult or an elderly subject.

[0173] 116. The method, composition or use according to any one of aspects 1 to 111, wherein the subject is a human subject aged 25 to 55 years.

[0174] 117. The method, composition or use according to any one of aspects 1 to 111, wherein the subject is a human subject aged 50 years or older.

[0175] 118. A method of producing rAAV to be administered in the method, composition or use according to any one of aspects 1 to 117, which comprises transferring an AAV vector plasmid to a host cell, wherein the AAV vector plasmid comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8.

[0176] 119. The method according to aspect 118, wherein the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO:8.

[0177] 120. The method according to aspect 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.

[0178] 121. The method according to any one of aspects 118 or 119, wherein the vector plasmid comprises the nucleotide sequence of SEQ ID NO:1, 4 or 8.

[0179] 122. The method according to any one of aspects 118 to 121, further comprising transferring a packaging plasmid and / or a helper virus to the host cell.

[0180] 123. The method according to any one of aspects 118 to 122, wherein the packaging cell comprises a stably integrated AAV cap gene.

[0181] 124. The method according to any one of aspects 118 to 123, wherein the packaging cell comprises a stably integrated AAV rep gene.

[0182] 125. A host cell comprising an AAV vector plasmid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 4 or 8.

[0183] 126. The host cell according to aspect 125, wherein the AAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4 or 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1 Shows the scAAVrh74.tMCK.hSGCA gene cassette.

[0185] Figure 2 Shows transgene expression in a dose-escalation study after systemic treatment with scAAVrh74.tMCK.hSGCA. (A) Alpha-dystroglycan immunofluorescence staining of multiple muscles from mice treated systemically (intravenously) with 1×10 12 vg, 3×10 12 vg and 6×10 12 vg (or 5×10 13 vg / kg, 1×10 14 vg / kg and 2×10 14 vg / kg, based on 20-g mice). The range of muscle fibers expressing alpha-dystroglycan was between 70% - 93% 12 weeks after treatment compared to untreated controls. (B) Western blot of muscle from treated sgca - / - mice confirms hSGCA protein expression. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; TRI, triceps; GLUT, gluteus; PSO, psoas major; DIA, diaphragm; HRT, heart; WT, wild type.

[0186] Figure 3 Shows that in sgca- / - mice, the improvement of muscle morphology by scAAVrh74.tMCK.hSGCA is dose-independent. (A) From mice treated with 1×10 12 vg, 3×10 12 vg and 6×10 12 vg (or 5×10 13vg / kg, 1×10 14 vg / kg, and 2×10 14 vg / kg, based on 20-g mice) Hematoxylin and eosin images of various muscles of sgca- / - mice treated with scAAVrh74.tMCK.hSGCA. Representative 20x images show a significant reduction in centralized nuclei and overall normalization of fiber size, independent of treatment dose. (B) Quantification of normalization of myofiber diameter in various muscles in the confirmed treatment groups (n = 6 mice / group) compared to vehicle-treated mice and wild-type controls. (C) Quantification of centrally located nuclei in the muscles of treated mice (n = 6 mice / group) compared to untreated mice and wild-type controls. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteus; PSO, psoas major; TRI, triceps; DIA, diaphragm; WT, wild-type.

[0187] Figure 4 Reduction of fibrosis was shown in sgca - / - mice treated with scAAVrh74.tMCK.hSGCA. (A) Picrosirius red staining showed reduction of fibrosis in scAAVrh74.tMCK.hSGCA-treated mice, as indicated by reduced collagen deposition in representative 20x images of various muscles compared to vehicle-treated sgca - / - mice. (B) Quantification of collagen levels in various muscles confirmed a decrease in collagen levels in all three treatment groups compared to untreated mice and wild-type controls (n = 6 mice / group). Abbreviations: PSO, psoas major; DIA, diaphragm; TRI, triceps; GLUT, gluteus.

[0188] Figure 5 Functional benefits to skeletal muscle after treatment with scAAVrh74.tMCK.hSGCA were shown. (A) After 3 months of treatment, tibialis anterior (TA) muscles (left and right) were harvested to measure specific force and resistance to contraction-induced injury (normalized to TA weight). Specific force and eccentric contraction quantification increased in all treatment groups (minimal differences between doses) compared to untreated controls (n = 6 mice / group). (B) Diaphragm muscle strips were harvested to measure specific force. After 12 weeks of treatment, force was significantly increased in treated mice compared to untreated sgca - / - mice. (C) After 12 weeks of treatment, locomotor and vertical activity of treated mice were improved by open field analysis compared to untreated sgca - / - controls (n = 6 mice / group). (D) Compared to untreated sgca - / -Serum creatine kinase levels were decreased in all treatment groups compared to controls. Data were analyzed by one-way ANOVA followed by Tukey's post hoc analysis for multiple comparisons. Unless otherwise stated, the creatine kinase levels in the sgca treated with vehicle were significantly decreased compared to those in the control groups. - / - *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 compared with mice. Abbreviations: TA, tibialis anterior; DIA, diaphragm; WT, wild type.

[0189] Figure 6 There was no evidence of toxicity as shown by blood chemistry after treatment with scAAVrh74.tMCK.hSGCA. Liver enzymes (ALT, AST, and ALP / K) and blood glucose (GLU) levels were analyzed for toxicity (n=6 / group). As indicated by the dotted line, all chemical values for treated mice were within normal / healthy limits for mice.

[0190] Figure 7A Biodistribution analysis of systemic scAAVrh74.tMCK.hSGCA delivery is shown. 12 vg and 6×10 12 vg(or 1×10 14 vg / kg and 2×10 14 vg / kg, based on 20-g mice) intravenous delivery of scAAVrh74.tMCK.hSGCA - / - Distribution histogram of the average VG copies of transcript per microgram of DNA added to quantitative polymerase chain reaction in various mouse tissues. Figure 7B Display medium (sgca - / - LR (lactate ringer) or 1.0×10 12 vg (left Western blot) or 6×10 12 vg (right Western blot) of scAArh74.tMCK.hSGCA treated WT and sgca - / - Western blot of α-sarcoglycan expression in mouse liver. Each lane represents an individual 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.

[0191] Figure 8 By immunofluorescence staining ( Figure 8 A) and Western blot ( Figure 8 B) shows the expression of scAAVrh74.tMCK.hSGCA in 12-month-old sgca- / - Expression in skeletal muscle of mice. Figure 8 The graph in C provides the percentage of positive muscle fibers in scAAVrh74.tMCK.hSGCAsgca in mice - / - Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteus; TRI, triceps; PSOAS, psoas major; diaphragm; WT, wild type; LRS, lactated ringers solution.

[0192] Figure 9 Shows in 12-month-old SGCA - / - Histological results of administration of scAAVrh74.tMCK.hSGCA in mice. Figure 9 a shows improved muscle pathology. Figure 9 b shows a decrease in central nucleation and an increase in mean fiber size in the gastrocnemius (GAS) and triceps (TRI) muscles. Figure 9 c shows a decrease in the level of fibrosis compared to untreated controls. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteus; PSO, psoas major; TRI, triceps; diaphragm; WT, wild type.

[0193] Figure 10 Shows functional improvement in old mice after administration of scAAVrh74.tMCK.hSGCA. DETAILED DESCRIPTION

[0194] This disclosure is based on the finding that administration of rAAV containing a polynucleotide encoding α-sarcoglycan results in a reduction or complete reversal of muscle fibrosis in an animal model of limb-girdle muscular dystrophy. As demonstrated in the examples herein, administration of the rAAV described herein results in a reversal of dystrophic features, including reduced CK levels, increased muscle strength, improved walking and vertical activity, and other motor functions.

[0195] Unless otherwise indicated, the practice of the present invention will employ conventional methods of virology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art. These techniques are sufficiently explained in the literature. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (current edition); "DNA Cloning: A Practical Approach," Volumes I & II (D. Glover ed.); "Oligonucleotide Synthesis" (N. Gait ed., current edition); "Nucleic Acid Hybridization" (B. Hames & S. Higgins ed., current edition); "Transcription and Translation" (B. Hames & S. Higgins ed., current edition); "CRC Handbook of Parvoviruses," Volumes I & II (P. Tijssen ed.); "Fundamental Virology," 2nd Edition, Volumes I & II (B. N. Fields and D. M. Knipe ed.); "Freshney Culture of Animal Cells, A Manual of Basic Technique" (Wiley-Liss, 3rd Edition); and Ausubel et al. (1991) "Current Protocols in Molecular Biology" (Wiley Interscience, N.Y.).

[0196] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0197] Definitions

[0198] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "a culture" includes reference to one or more cultures and equivalents thereof known to those of ordinary skill in the art, and the like. Reference to "recombinant AAV" includes a mixture of two or more rAAV viral particles, etc. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless clearly indicated otherwise.

[0199] Unless expressly indicated, the term "or" as used in the claims is used to mean "and / or", referring only to alternative embodiments, or the alternative embodiments are mutually exclusive, although the present disclosure supports a definition referring only to alternative embodiments and "and / or".

[0200] Throughout this application, the term "about" is used to indicate that a value includes the statistical experimental error (standard deviation of the error) of the device or method used to determine the value.

[0201] The term "vector" or "expression vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replicating when combined with appropriate control elements and which can transfer gene sequences between cells. In one embodiment, the vector is a viral vector. An expression vector may contain a variety of control sequences, structural genes (e.g., genes of interest), and nucleic acid sequences that also serve other functions.

[0202] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that is generated only in cells, and certain functions are provided by co-infecting helper viruses. Thirteen AAV serotypes have been characterized to date. Basic information and reviews of AAV can be found, for example, in Carter, 1989, The Parvovirus Handbook, Volume 1, pages 169 to 228 and Berns, 1990, Virology, pages 1743 to 1764, Raven Press, (New York). However, it is fully anticipated that these same principles will apply to additional AAV serotypes, since it is well known that the various serotypes are closely related both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pages 165 to 174, edited by J.R. Pattison; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes; and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further demonstrated by heteroduplex analysis, which shows extensive cross-hybridization along the genome length between serotypes; and the presence of similar self-annealing fragments at the termini, which correspond to "inverted terminal repeats" (ITRs). Similar infectious patterns also indicate that the replication functions of each serotype are under similar regulatory control.

[0203] As used herein, an "AAV vector" refers to a vector that contains one or more polynucleotides (or transgenes) of interest, the polynucleotides of interest being flanked by AAV inverted terminal repeats (ITRs). When present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products, this AAV vector can be replicated and packaged into infectious viral particles. In one embodiment, the AAV vector is a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh10, and AAV rh74. The AAV vector can have one or more AAV wild-type genes that are entirely or partially deleted, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are required for the rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein as including at least those sequences necessary for viral replication and packaging in cis (e.g., functional ITRs). The ITRs need not be wild-type nucleotide sequences and can be altered, for example, by nucleotide insertion, deletion, or substitution, provided that the sequences provide functional rescue, replication, and packaging.

[0204] The term "AAV helper function" refers to AAV-derived coding sequences that can be expressed to provide AAV gene products that, in turn, are used in trans for productive AAV replication. Thus, AAV helper functions include the major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to have many functions, including: recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and transcriptional regulation from AAV (or other heterologous) promoters. The Cap expression product provides the necessary packaging function. AAV helper functions are used herein to complement the trans AAV functions that are missing in the AAV vector.

[0205] A "recombinant virus" refers to a virus that has been genetically altered, e.g., by adding or inserting a heterologous nucleic acid sequence into the viral particle.

[0206] "AAV virion", "AAV virus particle", or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and a polynucleotide AAV vector encapsulated by 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 mammalian cells). In some embodiments, the production of AAV vector particles includes the production of the AAV vector, as this vector is contained within the AAV vector particle. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), then it is commonly referred to as an "rAAV vector" or simply an "rAAV particle". Thus, the production of AAV vector particles necessarily includes the production of rAAV, as the rAAV genome is contained within the rAAV vector particle.

[0207] For example, wild-type (wt) AAV virus particles contain a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein coating. AAV virions can be single-stranded (ss) AAV or self-complementary (SC) AAV. In one embodiment, single-stranded AAV nucleic acid molecules with complementary sense (e.g., "sense" or "antisense" strands) can be packaged into AAV virions and both strands are equally infectious.

[0208] The term "recombinant AAV" or "rAAV" is defined herein as an infectious replication-defective virus composed of an AAV protein shell, which encapsidates a heterologous nucleotide sequence of interest flanked by AAV ITRs on both sides. In one embodiment, rAAV is produced in a suitable host cell into which an AAV vector, AAV helper functions, and accessory functions are introduced. In this way, the host cell is capable of encoding the AAV polypeptides required to package the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virus particle particles for subsequent gene delivery.

[0209] The term "transfection" refers to the uptake of exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA is introduced within the cell membrane. Many transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties such as nucleotide integration vectors and other nucleic acid molecules into a suitable host cell.

[0210] The term "transduction" denotes the delivery of a DNA molecule in vivo or in vitro to a recipient cell via a replication-defective viral vector, e.g., via recombinant AAV viral particles.

[0211] The term "host cell" denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of an AAV helper construct, an AAV vector plasmid, a helper function vector, or other transfer DNA. The term includes the progeny of the original cell that has been transfected. Thus, a "host cell" as used herein generally refers to a cell that has been transfected with an exogenous DNA sequence. It should be understood that due to natural, accidental, or deliberate mutations, the progeny of a single parental cell may not be exactly identical in morphology or genomic or total DNA complement to the original parent.

[0212] The term "heterologous" when referring to nucleic acid sequences such as coding sequences and control sequences denotes sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a nucleic acid fragment that is within or attached to another nucleic acid molecule and that is not found in nature to be associated with the other molecule. For example, a heterologous region of a nucleic acid construct can include a coding sequence flanked by sequences that are not found in nature to be associated with the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence having codons different from the native gene). Similarly, for the purposes of the present invention, a cell transformed with a construct that is not normally present in the cell is considered heterologous. As used herein, allelic variations or naturally occurring mutational events do not give rise to heterologous DNA.

[0213] A coding sequence, or a sequence encoding a specific protein, is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) in vitro or in vivo into a polypeptide. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. The coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Transcription termination sequences are generally located at the 3' of the coding sequence.

[0214] A "nucleic acid" sequence refers to a DNA or RNA sequence. Nucleic acids include base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueuosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, pseudouracil, queuosine, 2-thiocytosine, and 2,6-diaminopurine.

[0215] The term DNA "control sequences" generally refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which together provide for the replication, transcription, and translation of the coding sequence in a recipient cell. Not all of these control sequences need to be present all the time as long as the selected coding sequence can be replicated, transcribed, and translated in an appropriate host cell.

[0216] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region that contains a DNA regulatory sequence, where the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcriptional promoters can include "inducible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters". In one embodiment, the promoter is a muscle-specific promoter, including but not limited to the human skeletal actin gene element, cardiac actin gene element, 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 a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-inducible element, and glucocorticoid response element (gre). In another embodiment, the promoter is the MCK promoter, tMCK promoter, or MHCK7 promoter.

[0217] The term "operably linked" refers to the arrangement of elements wherein the components so described are configured to perform their usual function. Thus, a control sequence operably linked to a coding sequence is capable of affecting the expression of the coding sequence. The control sequence need not be adjacent to the coding sequence so long as it functions to direct its expression. Thus, for example, there can be intervening untranslated but transcribed sequences between a promoter sequence and a coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0218] When RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, the promoter "directs transcription" of the coding sequence in the cell, which is then translated into the polypeptide encoded by the coding sequence.

[0219] "Expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence or gene of interest. As described above, an expression cassette includes control elements such as a promoter operably linked (to direct transcription) to the sequence or gene of interest and typically also includes a polyadenylation sequence. In certain embodiments of the invention, the expression cassettes described herein may be contained within plasmid constructs. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, signals that permit the plasmid construct to exist as single-stranded DNA, at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

[0220] When referring to a nucleotide sequence, "isolated" means that the indicated molecule is present substantially free of other biological macromolecules, such as other nucleotide sequences, chromatin material, etc. Thus, an "isolated nucleic acid molecule encoding a particular polypeptide" is a nucleic acid molecule substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or portions that do not deleteriously affect the basic characteristics of the composition.

[0221] For purposes of describing the relative positions of nucleotide sequences within a particular nucleic acid molecule throughout this application, e.g., when a particular nucleotide sequence is described as being "upstream," "downstream," "3'" or "5'" relative to another sequence, it is understood that it is the sequence position in the "sense" or "coding" strand of the DNA molecule, which is conventional in the art.

[0222] In the context of nucleic acid or amino acid sequences, the terms "sequence identity", "percent sequence identity" or "percent identity" refer to residues in two sequences that are the same when aligned to maximize correspondence. The length of the sequence identity comparison can exceed the full length of the genome, with the full length of a gene coding sequence or fragment of at least about 500 to 5000 nucleotides being desired. However, identity between smaller fragments may also be desired, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. The percent identity of sequences can be determined by techniques known in the art. For example, homology can be determined by aligning sequence information and directly comparing the sequence information between two polypeptide molecules using readily available computer programs (such as ALIGN, ClustalW2, and BLAST). In one embodiment, when using BLAST as the alignment tool, the default parameters are as follows: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss-Prot+Spupdate+PIR.

[0223] 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 ape and monkey species; farm animals, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs, etc. In some embodiments, the subject is a human subject in the age range from birth to 2 years, from 1 year to 10 years, or from 4 years to 15 years, or from 10 years to 19 years, or from 20 years to 40 years, or from 15 years to 29 years, or from 25 years to 55 years, or from 40 years 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 old), a human adolescent (10 to 19 years old). In some embodiments, the subject is an adult (18 years old or older). In particular, the subject is a young adult (15 to 29 years old), a middle-aged person (25 to 55 years old) or an elderly person (50 years old or older) or an elderly subject (65 years old or older) or an elderly human subject (70 years old or older).

[0224] "Therapeutic effect" means any therapeutic benefit conferred by the therapies described herein. For example, such an effect can be the sustained expression in a suitable target tissue of a protein or enzyme that is lacking or absent in the muscular dystrophy of interest. In addition, a therapeutic effect can be any reduction or elimination of one or more clinical or sub-clinical manifestations 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 in the tibialis anterior muscle, and / or an increase in specific force and improved motor function in muscle provide a therapeutic benefit to subjects receiving LGMD-2D therapy.

[0225] In another aspect, the recombinant AAV vectors described herein comprise a polynucleotide sequence encoding an α-dystroglycan that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2, and the protein retains α-dystroglycan activity. In another embodiment, the α-dystroglycan comprises the polypeptide sequence shown in SEQ ID NO:2.

[0226] In another aspect, the present disclosure describes a recombinant AAV vector comprising a polynucleotide sequence encoding a functional α-dystroglycan, the polynucleotide sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1 or its complement. In another embodiment, the rAAV comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4. In another embodiment, the rAAV comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4.

[0227] The term "stringent" is used to refer to conditions that are commonly understood in the art as being stringent. The stringency of a hybridization is mainly determined by temperature, ionic strength, and the concentration of a denaturing agent 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, New York, 1989). More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide or other denaturing agents) can also be used; however, the hybridization rate will be affected. In the case of deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37 °C (for 14-base oligomers), 48 °C (for 17-base oligomers), 55 °C (for 20-base oligomers), and 60 °C (for 23-base oligomers).

[0228] When ranges are used herein for physical properties, such as molecular weight, concentration, or dosage, all combinations and subcombinations of the ranges, as well as specific embodiments therein, are intended to be included. When referring to a number or numerical range, the term "about" means that the number or numerical range so designated is an approximation within experimental variability (or statistical experimental error), and thus the number or numerical range may vary, for example, from 1% to 15% of the recited amount or numerical range.

[0229] To reduce non-specific and / or background hybridization, other reagents can be included in the hybridization and wash buffers. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable reagents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically carried out at pH 6.8 - 7.4; however, under typical ionic strength conditions, the hybridization rate is almost independent of pH. See Anderson et al. Nucleic Acid Hybridisation: A Practical Approach 《Nucleic Acid Hybridisation: A Practical Approach》 Chapter 4, IRL Press Limited (Oxford, UK). Those skilled in the art can adjust the hybridization conditions to accommodate these variables and allow DNA of different sequence relatedness to form hybrids.

[0230] Limb-girdle muscular dystrophy type 2D (LGMD2D) is a progressive muscular dystrophy characterized by muscle weakness, abnormal breathing, and, in rare cases, cardiomyopathy. LGMD2D is caused by mutations in the α-sarcoglycan gene, resulting in protein loss and concomitant loss of the sarcoglycan and dystrophin-associated glycoprotein complex. Sgca-null (sgca - / - ) mice recapitulate the clinical phenotypes of patients with LGMD2D, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduced generation of absolute muscle force and spontaneous activity. Thus, sgca - / - mice provide a relevant model to test the safety and efficacy of gene replacement. Accordingly, the present disclosure provides a self-complementary AAVrh74 vector that contains a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter truncated muscle creatine kinase (tMCK). The efficacy and safety of scAAVrh74.tMCK.hSGCA in sgca - / - 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 treatment and wild-type mice. In sgca - / - mice treated with scAAVrh74.tMCK.hSGCA, protein expression of α-SG on the sarcolemma of skeletal muscle was stable at all tested doses. Furthermore, scAAVrh74.tMCK.hSGCA effectively improved the histopathology of the limb and diaphragm muscles of sgca - / - mice, as indicated by reduced fibrosis and central nucleation and normalization of muscle fiber size. These molecular changes were accompanied by a significant increase in specific force generation in the diaphragm and tibialis anterior muscles, prevention of eccentric force loss, and a decrease in serum CK. Spontaneous activity was improved in all doses of vector-treated compared to vehicle-treated sgca - / - mice. Finally, no vector-related toxicity was detected in serum chemistry panels and gross necropsies. Overall, the study provides support for systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D.

[0231] In another aspect, the recombinant AAV vectors described herein can be operably linked to muscle-specific control elements. For example, muscle-specific promoters include one or more of the following: human skeletal muscle 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 a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-inducible element, and glucocorticoid response element (gre).

[0232] In one embodiment, the muscle-specific promoter is the tMCK promoter, which comprises the sequence of SEQ ID NO:3. An exemplary rAAV described herein is AAVrh74.tMCK.hSGCA, which comprises the nucleotide sequence of SEQ ID NO:4. In some embodiments, the polynucleotide sequence encoding AAVrh74.tMCK.hSGCA comprises a sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:4, or 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 of SEQID NO:1.

[0233] In another embodiment, the polynucleotide sequence encoding AAVrh74.tMCK.hSGCA comprises a nucleotide sequence encoding a polynucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ IDNO:1. In another embodiment, the polynucleotide sequence encodes a protein that retains alpha-dystroglycan activity.

[0234] In one embodiment, the rAAV comprises the 5' inverted terminal repeat sequence of SEQ ID NO:5. In another embodiment, the rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO:6. In some embodiments, the rAAV comprises the poly A sequence of SEQ ID NO:7.

[0235] The AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74 or variants or derivatives thereof. In one embodiment, the rAAV is serotype AAV rh.74. The production of pseudotyped rAAV is disclosed, for example, in WO 2001 / 083692, which is incorporated herein by reference in its entirety. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0236] Compositions comprising any of the rAAV vectors described herein are also contemplated.

[0237] Methods of treating a subject in need of treatment for muscular dystrophy are provided, which include the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV uses a systemic administration route and is administered at a dose of about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg. For example, in any of the provided methods, the dose of rAAV administered is between about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg. In another embodiment, the dose is about 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14vg / kg. In one embodiment, rAAV is administered via a systemic route, which includes the intravenous route. In another embodiment, rAAV is administered intravenously at a dose of about 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy.

[0238] In addition, the dose of rAAV administered is from about 1.5×10 13 vg to about 3.5×10 16 vg, or from 3×10 13 vg to 1×10 16 vg, or from about 1.5×10 13 vg to about 2×10 15 vg, or from about 1.5×10 13 vg to about 1×10 15 vg. In addition, in any of the methods, the dose of rAAV is administered at a concentration of about 10 mL / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy type 2D. The doses expressed as vg or vg / kg in the present disclosure are based on a titration assay method using quantitative PCR (qPCR). The qPCR-based titration method is known in the art.

[0239] In addition, a method for treating muscular dystrophy in a subject in need thereof is provided, which includes the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV is administered using a systemic administration route at about 1.0×10 12 vg / kg to about 2.0×10 15administered at a dose of vg / kg; wherein the level of α - sarcoglycan gene expression in the subject's cells increases after administration of rAAV compared to the level of α - sarcoglycan gene expression before administration of rAAV; wherein the serum CK level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV; and / or wherein voluntary activity and force generation increase; wherein fibrosis is reduced; wherein the resistance to contraction - induced injury in the tibialis anterior muscle increases; and / or wherein the number of α - sarcoglycan - positive fibers in the muscle tissue of the subject increases after administration of rAAV compared to the number of α - sarcoglycan - positive fibers before administration of rAAV; wherein the fiber diameter size in the muscle tissue of the subject increases after administration of rAAV compared to the number of fiber diameters before administration of rAAV; or wherein the central nucleation in the muscle tissue of the subject decreases after administration of rAAV compared to the central nucleation before administration of rAAV. Muscle tissue includes, but is not limited to, triceps, tibialis anterior, soleus, gastrocnemius, biceps, trapezius, gluteus, psoas major, deltoid, quadriceps, and diaphragm. In one embodiment, the muscle tissue includes tibialis anterior, gastrocnemius, gluteus, psoas major, and triceps. The expression of α - sarcoglycan is determined by methods known to those of ordinary skill in the art. In one embodiment, the expression is determined by Western blot, immunochemistry in muscle biopsy, and / or by detecting the number of vector genomes per microgram of genomic DNA.

[0240] In some embodiments, the present disclosure includes a method of treating a subject in need thereof for muscular dystrophy, comprising the step of administering recombinant adeno - associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the motor function of the human subject is significantly improved compared to the motor function of the human subject before administration of rAAV.

[0241] A method of increasing α - sarcoglycan in a patient in need thereof is provided, comprising administering to the patient the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0242] In any of the provided methods, uses, and compositions for treating muscular dystrophy, the subject is between 4 and 15 years of age, has a confirmed mutation in both alleles of alpha-sarcoglycan (SGCA), is negative for AAVrh74 antibodies, and / or has a >40% or normal 100-meter walk test. In any of the provided methods, uses, and compositions for treating muscular dystrophy, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject, such as a subject in the age range of 1 to 21 years. In some embodiments, the subject is 1 to 10 years old, or 2 to 12 years old, 4 to 15 years old, or 10 to 19 years old. In one embodiment, the subject is an adolescent subject, such as a subject in the age range of 12 to 21 years. Additionally, in one embodiment, the subject is a young adult subject, such as a subject in the age range of 15 to 29 years or 18 to 39 years. In some embodiments, the subject is a middle-aged adult or an elderly subject, such that the age range of the middle-aged adult can be 25 to 55 years, the age range of the elderly subject can be 50 years or older, and the age range of the elderly subject can be 65 years or older. In some embodiments, rAAV is administered by injection, infusion, or implantation. For example, rAAV is administered by infusion over approximately 1 to 2 hours. Additionally, rAAV is administered by an intravenous route through a peripheral limb vein.

[0243] In a method of treating muscular dystrophy in a human subject in need thereof, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein the rAAV uses a systemic administration route and is at about 1.0×10 12 vg / kg to about 5.0×10 14administered at a dose of vg / kg, and the rAAV comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1. In another embodiment, the rAAV comprises the nucleotide sequence shown in SEQ ID NO:1. In one embodiment, the rAAV encodes a protein comprising a polypeptide sequence that is at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. In another embodiment, the rAAV comprises a nucleotide sequence encoding a protein that comprises the polypeptide sequence shown in SEQ ID NO:2. Additionally, any of the disclosed rAAVs comprises a promoter, such as the tMCK promoter sequence of SEQ ID NO:3. In some embodiments, the rAAV is serotype AAVrh.74. Additionally, the rAAV comprises the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO:3. In one embodiment, the rAAV comprises the 5' inverted terminal repeat of SEQ ID NO:5. In another embodiment, the rAAV comprises the 3' inverted terminal repeat of SEQ ID NO:6. In another embodiment, the rAAV comprises the poly A sequence of SEQ ID NO:7.

[0244] The AAV dose can be determined by a variety of 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 the AAV dose can be determined by measuring the AAV vector genome using quantitative real - time PCR (qPCR). This qPCR method overcomes the inconsistent or arbitrary results of traditional transduction assays. In one embodiment of PCR dose determination, plasmid DNA is used as a calibration standard. The form of the plasmid can affect the dose results of the qPCR method. In one embodiment, circular or supercoiled DNA or plasmid is used as a quantitative standard. In another embodiment, linearized DNA or plasmid is used as a quantitative standard.

[0245] The term "supercoiled DNA" or "supercoiled plasmid" refers to DNA or a plasmid that does not contain free ends. The term "linearized DNA" or "linearized plasmid" refers to DNA or a plasmid that contains free 5' and free 3' ends that are not connected to each other. In one embodiment, linearized DNA or a plasmid is obtained by restriction digestion of circular DNA (such as 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.

[0246] In one exemplary embodiment, a method of treating a subject in need of treatment for muscular dystrophy is provided, which includes the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK7.hSGCA, wherein the rAAV uses a systemic administration route and is administered at a dose of about 1.0×10 12 vg / kg to about 5.0×10 14 vg / kg. In one embodiment, the rAAV is administered by intravenous infusion within about 1 to 2 hours at a dose of about 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg based on supercoiled DNA or a plasmid as a quantification standard, wherein the rAAV contains the nucleotide sequence of the scAAVrh74.tMCK7.hSGCA construct of SEQ ID NO:3. In another embodiment, based on linearized DNA or a plasmid as a quantification standard, the dose is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0247] The present disclosure also provides a method of increasing the expression of sarcoglycan in the muscle tissue of a subject, which includes administering to the subject an scAAVrh74.tMCK.hSGCA construct comprising a nucleotide sequence that is at least 90% identical, at least 95% identical or 99% identical to SEQ ID NO:1 and / or 4.

[0248] The present disclosure further provides a method of improving the muscle function of a subject, which includes administering to the subject a construct comprising a nucleotide sequence that is at least 90% identical, at least 95% identical or 99% identical to SEQ ID NO:1 and / or 4.

[0249] In some aspects, the subject has a gene mutation in the gene encoding the sarcoglycan protein or has muscular dystrophy. In some aspects, the subject has a gene mutation in the gene encoding the α-sarcoglycan protein.

[0250] In any of the provided methods, the level of α - sarcoglycan gene expression in the subject's cells increases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the level of α - sarcoglycan gene expression before administration of the scAAVrh74.tMCK.hSGCA construct.

[0251] Additionally, in any of the provided methods, expression of the α - sarcoglycan gene in cells is detected by measuring α - sarcoglycan protein levels in muscle biopsies by Western blot or immunohistochemistry before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0252] In any of the provided methods, the level of α - sarcoglycan protein increases after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of α - sarcoglycan protein increases by at least 33%, as detected by measuring α - sarcoglycan protein levels in muscle biopsies by Western blot before and after administration of the scAAVrh74.tMCK.hSGCA construct, or by immunohistochemistry in muscle biopsies and / or by measuring the number of vector genomes per microgram of genomic DNA before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0253] In any of the provided methods, the serum CK level of the subject decreases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the serum CK level before administration of the scAAVrh74.tMCK.hSGCA construct.

[0254] In any of the provided methods, the number of α - sarcoglycan - positive fibers in the subject's muscle tissue increases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the number of α - sarcoglycan - positive fibers before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of α - sarcoglycan - positive fibers is detected by measuring α - sarcoglycan protein levels in muscle biopsies by Western blot or immunohistochemistry before and after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of α - sarcoglycan - positive fibers increases in the subject's muscle tissue after administration of the scAAVrh74.tMCK.hSGCA construct.

[0255] In any of the provided methods, the level of α - dystroglycan in the subject increases after administration of rAAV compared to the level of α - dystroglycan before administration of the scAAVrh74.tMCK.hSGCA construct. The level of α - dystroglycan is detected by measuring the α - dystroglycan protein level on muscle biopsies by immunohistochemistry or western blot before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0256] Another embodiment provides a method of expressing the α - dystroglycan gene in a patient's cells, which comprises administering to the patient the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. In any of the provided methods of expressing the α - dystroglycan gene in a patient's cells, the expression of the α - dystroglycan gene in the patient's cells is detected by measuring the α - dystroglycan protein level by western blot or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. In one embodiment, the α - dystroglycan gene is measured in a patient by detecting more than one rAAV vector genome copy per nucleus. In another embodiment, the expression of the α - dystroglycan gene is measured in a subject by detecting the number of vector genomes per microgram of genomic DNA.

[0257] A method of reducing the serum CK level in a patient in need thereof is also provided, the method comprising administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4.

[0258] A method of increasing α - dystroglycan - positive fibers in a patient's muscle tissue is provided, which comprises administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. In any of these methods, the number of α - dystroglycan - positive fibers is detected by measuring the α - dystroglycan protein level by western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0259] Another embodiment provides a method of increasing the expression of α - dystroglycan in a subject in need thereof, which comprises administering to the subject the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO:4. In any of these methods, the level of α - dystroglycan is detected by measuring the α - dystroglycan protein level by western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0260] Also provided are methods of generating recombinant AAV vector particles, which include culturing host cells transfected with any of the recombinant AAV vectors described herein and recovering the recombinant AAV particles from the supernatant of the transfected cells. Viral particles containing any of the recombinant AAV vectors described herein are also contemplated. In one embodiment, the method of generating rAAV includes transferring an AAV vector plasmid into a host cell. In another embodiment, the recombinant AAV vector particles and / or the AAV vector plasmid comprise 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 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO:8. In another aspect, the present disclosure provides a cell comprising an AAV vector plasmid that comprises the nucleotide sequence of SEQ ID NO:8. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. The host cell stably carrying the AAV vector plasmid can be used to generate rAAV. In one embodiment, the AAV vector plasmid is the pAAV.tMCK.hSGCA.KAN plasmid (SEQ ID NO:8).

[0261] Also provided are methods of reducing fibrosis in a mammalian subject in need thereof. In this regard, the methods include administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein). In some embodiments, the mammalian subject has muscular dystrophy. In one embodiment, the muscular dystrophy is LGMD2D. In some embodiments, administering the AAV vector described herein (or a composition comprising an AAV vector described herein) reduces fibrosis in the muscle tissue of the subject. In one embodiment, the muscle tissue includes the psoas major, diaphragm, triceps, and / or gluteus maximus.

[0262] As used herein, the term "muscular dystrophy" refers to a disorder in which there is a progressive decline in strength and muscle mass. Non-limiting examples of muscular dystrophy diseases can include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycanopathy, congenital muscular dystrophy (such as congenital muscular dystrophy due to partial LAMA2 deficiency), merosin-deficient congenital muscular dystrophy, congenital muscular dystrophy type 1D, Fukuyama congenital muscular dystrophy, limb-girdle muscular dystrophy type 1A, limb-girdle muscular dystrophy type 2A, limb-girdle muscular dystrophy type 2B, limb-girdle muscular dystrophy type 2C, limb-girdle muscular dystrophy type 2D, limb-girdle muscular dystrophy type 2E, limb-girdle muscular dystrophy type 2F, limb-girdle muscular dystrophy type 2G, limb-girdle muscular dystrophy type 2H, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2I, limb-girdle muscular dystrophy type 2J, limb-girdle muscular dystrophy type 2K, limb-girdle muscular dystrophy type IC, rigid spine muscular dystrophy with simple epidermolysis bullosa, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy, and Ullrich scleroatonic muscular dystrophy. In some embodiments, the subject has limb-girdle muscular dystrophy. In some embodiments, the subject has limb-girdle muscular dystrophy type 2D (LGMD2D).

[0263] As used herein, the term "fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues (including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas) after injury. The ECM components that are deposited include collagen, such as collagen 1, collagen 2, or collagen 3, and fibronectin.

[0264] In another aspect, the present disclosure describes a method of increasing α - sarcoglycan - positive fibers, fiber diameter size, eccentric muscle contraction, muscle strength, and / or the expression of α - sarcoglycan in muscle tissue of a mammalian subject, which comprises administering to the mammalian subject a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein). The present disclosure also describes a method of reducing fibrosis, central nucleation, CK levels, and / or collagen deposition in a subject, which comprises administering to the subject a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein).

[0265] In any of the methods of the present invention, the subject may have muscular dystrophy, such as limb - girdle muscular dystrophy or any other dystrophin - related muscular dystrophy. In one embodiment, the muscular dystrophy is LGMD - 2D.

[0266] There is also provided a method of treating muscular dystrophy in a mammalian subject, which comprises administering to the mammalian subject a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein). In some embodiments, the muscular dystrophy is limb - girdle muscular dystrophy.

[0267] In any of the methods of the present invention, rAAV is administered by intramuscular injection or intravenous injection. Additionally, in any of the methods of the present invention, rAAV is systemically administered, such as by parenteral administration via injection, infusion, or implantation.

[0268] The compositions of the present invention are formulated for intramuscular injection or intravenous injection. Additionally, the compositions of the present invention are formulated for systemic administration, such as by parenteral administration via injection, infusion, or implantation.

[0269] Additionally, any of the compositions is formulated for administration to a subject having muscular dystrophy (such as limb - girdle muscular dystrophy or any other dystrophin - related muscular dystrophy). In some embodiments, the composition may further comprise a second recombinant AAV vector expressing α - sarcoglycan or a second recombinant AAV vector comprising the polynucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:4.

[0270] In any of the uses of the present invention, the drug is formulated for intramuscular injection or intravenous injection. Additionally, in any of the uses of the present invention, the drug is formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation. Additionally, any of the drugs can be prepared for administration to a subject having muscular dystrophy (such as limb-girdle muscular dystrophy or any other dystrophin-related muscular dystrophy). In some embodiments, the drug can further express a second recombinant AAV vector of α-sarcoglycan or a second recombinant AAV vector comprising the polynucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4.

[0271] The foregoing paragraphs are not intended to define every aspect 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 the features described herein are contemplated, even if the combinations of features are not found together in the same sentence or paragraph or section of this document. As an additional aspect, the invention includes all embodiments in which the scope of the invention is narrower in any aspect than that defined by the specific paragraphs above. For example, in cases where certain aspects of the invention are described as a class, it should be understood that each member of the class is individually an aspect of the invention.

[0272] AAV

[0273] The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV having capsid mutations.

[0274] The DNA plasmid of the present invention contains an rAAV genome. The DNA plasmid is transferred into cells that are permissive to infection with an AAV helper virus (such as adenovirus, E1-deleted adenovirus, or herpes virus) to assemble the rAAV genome into infectious virus particles. Techniques for generating rAAV particles are standard in the art, where the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cells. Generation of rAAV requires the following components to be present within a single cell (referred to herein as a packaging cell): an rAAV genome, AAV rep and cap genes separate from (i.e., not within) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome 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. Generation of pseudotyped rAAV is disclosed, for example, in WO 2001 / 083692, which is incorporated herein by reference in its entirety.

[0275] A method for generating packaging cells is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker (such as a neomycin resistance gene) are integrated into the genome of the cell. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene 23:65-73), or by direct blunt-end ligation (Senapathy and Carter, 1984, J. Biol. Chem. 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus rather than a plastid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0276] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777) WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the documents related to rAAV production.

[0277] Accordingly, the present invention provides packaging cells for generating infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (isogenic 293 line). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey embryonic lung cells).

[0278] The recombinant AAV of the present invention (i.e., infectious capsidated rAAV particles) comprises an rAAV genome. Embodiments include, but are not limited to, rAAV designated pAAV.tMCK.hSGCA, which comprises the polynucleotide sequence shown in SEQ ID NO:3.

[0279] rAAV can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper virus are known in the art and are included, for example, in Clark et al., Human Gene Therapy, 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002); U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0280] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The compositions described herein comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to the recipient and are preferably inert at the dosages and concentrations employed and include buffers, such as phosphates, citrates, or other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as 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, pluronic, or polyethylene glycol (PEG).

[0281] In the methods of the present invention, the titer of the rAAV to be administered will vary depending on, for example, the particular rAAV, the mode of administration, the treatment goal, the individual, and the one or more cell types targeted, and can be determined by methods standard in the art. The titer range of the rAAV can be from about 1×10 6 to about 1×10 7 to about 1×10 8 to about 1×10 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 to about 1×10 14 or more DNase-resistant particles (DRP). The dose can also be expressed in terms of viral genomes (vg) as measured by qPCR.

[0282] The present invention contemplates methods of transducing target cells with rAAV in vivo or in vitro. The in vivo method comprises the step of administering to an animal (including a human) in need thereof an effective dose or effective multiple doses of a composition comprising the rAAV of the present invention. If the dose is administered before the development of a disorder / disease, then the administration is prophylactic. If the dose is administered after the development of a disorder / disease, then the administration is therapeutic. In an embodiment of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents the progression of the disorder / disease state, reduces the extent of the disease, causes a remission (partial or complete) of the disease, and / or prolongs survival. Examples of diseases contemplated to be prevented or treated by the methods of the present invention are muscular dystrophies, such as limb-girdle muscular dystrophy. Accordingly, provided is a method of transducing target cells with rAAV scAAVrh74.tMCK.hSGCA, which rAAV scAAVrh74.tMCK.hSGCA comprises the nucleotide sequence of SEQ ID NO:4.

[0283] In another embodiment, the present disclosure provides a method for producing rAAV scAAVrh74.tMCK.hSGCA, which comprises transferring an AAV vector plasmid to a host cell. Methods for transferring DNA to a host cell are well known in the art, including but not limited to transfection, infection, transformation, electroporation, and transduction. In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least 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% identical to SEQ ID NO: 8, more typically about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more identical to SEQ ID NO: 8. In another embodiment, the vector plasmid comprises a nucleotide sequence that is at least 90%, 95% or 99% identical to SEQ ID NO: 8. In another embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 8. On the other hand, the present disclosure provides a cell comprising an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the AAV vector plasmid is stably expressed in the host cell. Host cells stably carrying the AAV vector plasmid can be used to produce rAAV. In one embodiment, the AAV vector plasmid is a pAAV.tMCK.hSGCA.KAN plasmid.

[0284] In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89%, more usually about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identical to SEQ ID NO: 1, 4, or 8.

[0285] In one embodiment, the vector plasmid comprises a nucleotide sequence that is at least about 90%, 95% or 99% identical to SEQ ID NO: 1, 4 or 8. In one embodiment, the vector plasmid comprises the nucleotide sequence of SEQ ID NO: 1, 4 or 8. In one embodiment, the method of producing rAAV further comprises transferring the packaging plasmid and / or helper virus to a host cell. In some embodiments, the packaging plasmid comprises an AAV rep and / or cap gene operably linked to a promoter. In one embodiment, the promoter is an AAV transcriptional promoter. In one embodiment, the host cell is a packaging cell. In one embodiment, the packaging cell comprises a stably integrated AAV cap gene. In another embodiment, the packaging cell comprises a stably integrated AAV rep gene.

[0286] 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, helper function vectors, or other DNA. The term as used herein includes the progeny of the original cell after expression of the exogenous DNA sequence in the original host cell. Non-limiting examples of host cells for AAV production include Sf9 insect cells and HEK 293T cells. The AAV vector plasmid can be introduced into host cells, such as Sf9 or 293T, by infection (viral or baculoviral), transient transfection using reagents (e.g., liposomes, calcium phosphate), or physical means (e.g., electroporation), or other means known in the art. In another embodiment, the host cell line is stably integrated with the rAAV plasmid into its genome. Such stable cell lines can be established by incorporating a selectable marker into the vector plasmid.

[0287] In one embodiment, the host cell is a packaging cell for producing AAV virus particles. Thus, in another aspect, the present disclosure provides a host cell comprising an AAV vector plasmid comprising a nucleotide sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: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.

[0288] The present invention also contemplates combination therapies. Combinations as used herein include both simultaneous and sequential therapies. Combinations of the methods of the present invention with standard medical treatments (e.g., steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort) are specifically contemplated as combinations with the novel therapy. In this regard, the combination includes administering to a subject one or more steroids, corticosteroids, and / or glucocorticoids, including but not limited to one or more of prednisone, prednisolone, and deflazacort, before, simultaneously with, or after administering the rAAV of the method of the present invention to the subject.

[0289] In relevant embodiments of the combination therapies contemplated by the present invention, glucocorticoids include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0290] It has been recognized that antigen-specific T cell responses may occur in subjects administered with rAAV vectors. This is an expected response between 2 to 4 weeks after gene transfer. One possible consequence of such antigen-specific T cell responses is the clearance of transduced cells and the loss of transgene expression. To suppress the host immune response to rAAV-based therapies, prior to treatment, for example, twenty-four hours before the treatment procedure, the subject may begin oral administration of prophylactic prednisone or a similar glucocorticoid at approximately 1 mg / kg / day, with a maximum dose of 60 mg / day. If needed, similar glucocorticoids may also be administered IV at a dose of approximately 1 mg / kg / day. The treatment will continue for approximately one month. A tapering regimen of prednisone or a similar glucocorticoid may be implemented based on the individual subject's immune response to gene transfer, as assessed by ELISpot assays and by monitoring liver function with GGT.

[0291] A therapeutically effective amount of the rAAV vector is about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg of rAAV dose. In another embodiment, the dose is about 5.0×10 13 vg / kg, about 1.0×10 14 vg / kg or about 2.0×10 14 vg / kg. In another embodiment, the dose is 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14vg / kg. The present invention also contemplates compositions comprising these rAAV vectors within these ranges.

[0292] The dose can also be expressed in terms of viral genomes (vg). The titer of rAAV can be determined by supercoiled DNA or plasmid quantification standards or linearized DNA or plasmid quantification standards. The titer or dose of the AAV vector can vary based on the physical form of the plasmid DNA used as the quantification standard. For example, the value of the titer or dose may vary depending on the supercoiled standard qPCR titration method or the linear standard qPCR titration method. In one embodiment, the dose in the present disclosure is based on supercoiled DNA or plasmid used as the quantification standard. In another embodiment, the dose in the present disclosure is based on linearized DNA or plasmid used as the quantification standard. Thus, in one embodiment, based on supercoiled DNA or plasmid used as the quantification standard, the therapeutically effective amount of the rAAV vector is in the range of about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 5×10 13 vg / kg to about 2×10 14 vg / kg or about 2.0×10 13 vg / kg to about 3.0×10 14 vg / kg of the rAAV dose. In another embodiment, based on supercoiled DNA or plasmid used as the quantification standard, the dose is about 5.0×10 13 vg / kg, about 1.0×10 14 vg / kg or about 2.0×10 14 vg / kg. In another embodiment, based on supercoiled DNA or plasmid used as the quantification standard, the dose is 5.0×10 13 vg / kg, 1.0×10 14 vg / kg or 2.0×10 14 vg / kg.

[0293] In another embodiment, based on linearized DNA or plasmid used as the quantification standard, the therapeutically effective amount of the rAAV vector is in the range of about 1.0×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.5×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.6×10 13 vg / kg to about 8.0×1013 vg / kg, about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg, about 1.2×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg, about 1.4×10 13 vg / kg to about 7.4×10 13 vg / kg, about 1.9×10 13 vg / kg to about 7.5×10 13 vg / kg or about 1.8×10 13 vg / kg to about 8.0×10 13 vg / kg of the rAAV dose. For example, based on linearized DNA or plasmid as a quantitative standard, a therapeutically effective amount of the rAAV vector is about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg of the dose.

[0294] In one embodiment, a dose of 5.0×1013 vg / kg based on supercoiled DNA or plasmid as a quantitative standard is equivalent to a dose of 1.85×10 13 vg / kg based on linearized DNA or plasmid as a quantitative standard. In another embodiment, a dose of 2.0×10 14 vg / kg based on supercoiled DNA or plasmid is equivalent to a dose of 7.41×10 13 vg / kg based on linearized DNA or plasmid as a quantitative standard. Thus, in another embodiment, based on linearized DNA or plasmid as a quantitative standard, about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0295] An effective dose of the composition can be administered by standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Considering the state of infection and / or disease being treated and one or more target cells / tissues in which α - dystroglycan is to be expressed, one of ordinary skill in the art can select and / or match one or more administration routes and one or more serotypes of the AAV components (especially AAV ITR and capsid proteins) of the rAAV of the present invention.

[0296] The present invention provides for local and systemic administration of an effective dose of rAAV and the compositions of the present invention. By way of example, systemic administration is administration to the circulatory system such that the entire body is affected. Systemic administration includes enteral administration, such as by intestinal absorption and parenteral administration by injection, infusion, or implantation.

[0297] In particular, the actual administration of the rAAV of the present invention can be achieved by any physical method that transports the rAAV recombinant vector to the target tissue of an animal. According to the present invention, administration includes, but is not limited to, injection into muscle, the bloodstream, and / or direct injection into the liver. Simply resuspending rAAV in phosphate buffered solution has been shown to be sufficient to provide a vehicle for expression in muscle tissue, and there are no known limitations on the vectors or other components that can be co-administered with rAAV (although compositions that degrade DNA should generally be avoided in combination with rAAV). The capsid protein of rAAV can be modified such that rAAV targets a specific target tissue of interest, such as muscle. See, e.g., WO 02 / 053703, the disclosure of which is incorporated herein by reference.

[0298] The pharmaceutical composition can be formulated as an injectable formulation or a topical formulation that can be delivered to muscle by transdermal transport. Many formulations for both intramuscular injection and transdermal transport have been developed previously 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. Thus, in another aspect, the present application relates to a formulation that comprises rAAV containing an AAVrh74-derived capsid, a buffering agent, an ionic strength agent, and a surfactant. In one embodiment, the concentration of rAAV is about 1.0×10 12 vg / kg to about 5.0×10 14 vg / kg. In one embodiment, the concentration of rAAV is about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg. In another embodiment, the concentration of rAAV is about 5×10 13 vg / kg, about 1×10 14 vg / kg, and / or about 2×10 14vg / kg. In one embodiment, the dose is based on supercoiled DNA or plasmid as a quantitative standard. In one embodiment, the rAAV is the scAAVrh74.tMCK.hSGCA vector. In one embodiment, the buffer comprises one or more of tris, tricine, Bis-tricine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In another embodiment, the buffer comprises tris having a pH of 8.0 at a concentration of about 5 mM to about 40 mM. In one embodiment, the buffer comprises tris having a pH of 8.0 at a concentration of about 20 mM. In one embodiment, the ionic strength agent comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. In one embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM. In another embodiment, the ionic strength agent comprises NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strength agent comprises MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM. In one embodiment, the surfactant comprises one or more of sulfonates, sulfates, phosphonates, phosphates, Poloxamer, and cationic surfactants. In one embodiment, the Poloxamer includes one or more of Poloxamer 124, Poloxamer 181, Poloxamer 184, Poloxamer 188, Poloxamer 237, Poloxamer 331, Poloxamer 338, and Poloxamer 407. In one embodiment, the surfactant comprises Poloxamer at a concentration of about 0.00001% to about 1%. In another embodiment, the surfactant comprises Poloxamer 188 at a concentration of about 0.001%. For intramuscular injection purposes, adjuvants such as sesame oil or peanut oil or aqueous propylene glycol solution, as well as sterile aqueous solutions, can be used. If desired, such aqueous solutions can be buffered and first diluted to isotonicity with saline or glucose using a liquid diluent. The rAAV solution as the free acid (DNA contains acidic phosphate groups) or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and their mixtures and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed can be readily obtained by standard techniques well-known to those skilled in the art.

[0299] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. By way of example, proper fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents such as sugars or sodium chloride will be preferably included. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0300] Sterile injectable solutions are prepared by incorporating rAAV in the required amount into a suitable solvent along with the various other ingredients enumerated above, and then filtering the solution sterilize. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0301] Transduction with rAAV can also be carried out in vitro. In one embodiment, desired target muscle cells are removed from a subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or allogeneic muscle cells can be used, where these cells will not produce an inappropriate immune response in the subject.

[0302] Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells (e.g., in a suitable culture medium), and screening for those cells with the DNA of interest using conventional techniques (such as Southern blotting and / or PCR), or by using selectable markers. The transduced cells can then be formulated into a pharmaceutical composition and introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth and cardiac muscle using, for example, a catheter.

[0303] Transduction of cells with the rAAV of the present invention results in sustained expression of α - dystroglycan. Accordingly, the present invention provides methods for administering / delivering rAAV expressing α - dystroglycan to a mammalian subject (preferably human). These methods include transducing tissues (including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more of the rAAV of the present invention. Transduction can be carried out using a gene cassette containing tissue - specific control elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissues directed by a muscle - specific control element, said control elements including but not limited to those derived from the actin and myosin gene families, such as control elements derived from the myoD gene family [see Weintraub et al., Science, 251:761 - 766 (1991)]; the muscle - cell - specific enhancer - binding factor MEF - 2 [Cserjesi and Olson, Molecular and Cellular Biology, 11:4854 - 4862 (1991)]; those derived from the human skeletal actin gene [Muscat et al., Molecular and Cellular Biology, 7:4089 - 4099 (1987)]; control elements of the cardiac actin gene; the muscle creatine kinase sequence element [see Johnson et al., Molecular and Cellular Biology, 9:3393 - 3399 (1989)] and the murine creatine kinase enhancer (mCK) element; control elements derived from the skeletal fast - twitch troponin C gene, the slow - twitch cardiac troponin C gene, and the slow - twitch troponin I gene: hypoxia - inducible nuclear factor (Semenza et al., Proceedings of the National Academy of Sciences of the United States of America, 88:5680 - 5684 (1991)); steroid - inducible elements and promoters including glucocorticoid response elements (GRE) (see Mader and White, Proceedings of the National Academy of Sciences of the United States of America 90:5603 - 5607 (1993)); and other control elements.

[0304] Muscle tissue is an attractive target for in - vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention contemplates sustained expression of miRNA from transduced muscle fibers.

[0305] "Muscle cell" or "muscle tissue" refers to a cell or population of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, such as from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardioblasts.

[0306] The term "transduction" is used to refer to the in - vivo or in - vitro administration / delivery of a polynucleotide of interest (e.g., a polynucleotide sequence encoding α - dystroglycan) to a recipient cell via the replication - defective rAAV, resulting in the recipient cell expressing α - dystroglycan.

[0307] Accordingly, the present disclosure also describes methods of administering an effective dose (or multiple doses administered substantially simultaneously or multiple doses administered at intervals) of rAAV encoding α - sarcoglycan to mammalian subjects in need thereof.

[0308] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application (including any definitions herein) shall prevail. The described numerical ranges include every integer value within each range and include the lowest and highest of the stated integers.

[0309] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0310] Examples

[0311] Pre - clinical studies using AAVrh74.tMCK.hSGCA are described in International Patent Publication No. WO 2013 / 078316 and U.S. Patents Nos. 9,434,928 and 10,105,453, which are incorporated herein by reference in their entirety.

[0312] Example 1

[0313] Materials and Methods

[0314] Animal Model

[0315] All procedures were approved by the research institution's Institutional Animal Care and Use Committee at Nationwide Children's Hospital. Under standardized conditions in the Animal Resources Core at the Research Institute of Nationwide Children's Hospital, gene - knockout (sgca - / - ) mice were bred and maintained as homozygous animals. The mice were maintained on a 12:12 hour dark:light cycle in Teklad Global Rodent Diet (3.8% fiber, 18.8% protein, 5% fat diet). All animals were housed in standard mouse cages with free access to food and water.

[0316] Genotyping

[0317] DNA genotyping was used to identify sgca - / -Mice. DNA was isolated and analyzed from tail clips by polymerase chain reaction (PCR) using OneTaq DNA polymerase (New England Biolabs, Ipswich, MA). A series of primers were used in the PCR analysis to determine the α-SG gene knockout status. The following primers and conditions were used: Intron 1 (CAGGGCTGGGAGCTGGGTTCTG; SEQ ID NO:9); Mutation primer - Intron 3 (CCCAGGGCCTTGATGCCT; SEQ ID NO:10); and NEOTR (GCTATCAGGACATAGCGTTGGCTA; SEQ ID NO:11). Genomic DNA was subjected to 30 cycles of reaction under the following conditions: 94 °C, 5 min; 94 °C, 1 min; 64 °C, 1 min; 72 °C, 2.5 min; and 72 °C, 7 min.

[0318] α-SG gene construct

[0319] The scAAVrh74.tMCK.hSGCA transgenic cassette was made by inserting the tMCK expression cassette driving the codon-optimized human α-SG cDNA 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 sequences included in the vector are the inverted terminal repeats of AAV2, which are required for viral DNA replication and packaging of the rAAV vector genome. One of the inverted terminal repeats (ITRs) lacks a targeting terminal resolution site (TRS) to restrict R replication from this ITR, thus facilitating the production of a dimeric replication form for self-complementary vector packaging. The AAVrh74 virus has been shown to be safe and efficient in transducing muscle across the vascular barrier in mice, non-human primates (NHPs), and humans.

[0320] Vector production

[0321] Recombinant AAV, (sc)rAAVrh74.tMCK.hSGCA, was prepared by triple transfection. A qPCR-based titration method was used to determine the encapsulated vg titer using a Prism 7500 Fast Taqman detector system (PE Applied Biosystems). The construct contains a chimeric intron that promotes high-level expression. The chimeric intron consists of the 5' donor site of the first intron of the human β-globin gene and the branch point and 3' splice acceptor site of the intron between the leader sequence and the body of the variable region of the immunoglobulin heavy chain gene. rAAV also contains a synthetic SV40 polyadenylation signal for efficient transcriptional termination. A schematic diagram of the expression cassette is shown below Figure 1 In. The vector was generated using the human α-sarcoglycan (α-SG) gene, which is flanked by AAV2 ITR sequences and encapsulated in AAVrh74 virus particles. The construct contains the tMCK immediate early promoter / enhancer (GenBank accession number M21390) and uses the β-globin intron for high-level expression.

[0322] Gene delivery

[0323] Systemic delivery to mice was achieved by injecting the vector into the tail vein of sgca- / - mice. Mice were injected with 1×10 12 vg, 3×10 12 vg or 6×10 12 vg total dose (mice ranged from 13 - 20 g; 5×10 13 vg / kg, 1×10 14 vg / kg and 2×10 14 vg / kg - doses based on supercoiled DNA or plasmid as a quantitative standard, respectively, for 20-g mice) of scAAVrh74.tMCK.hSGCA diluted in 200 to 250 μL volume of lactated Ringer's solution. All treated mice were injected at 4 to 5 weeks of age and euthanized 12 weeks after injection. In another embodiment, based on linearized DNA or plasmid as a quantitative standard, the doses were approximately 1.85×10 13 vg / kg or 7.41×10 13 vg / kg.

[0324] Serum creatine kinase measurement

[0325] In wild-type C57BL / 6 mice (n = 6), vehicle (lactated Ringer's solution)-treated sgca - / - mice (n = 6) and scAAVrh74.tMCK.hSGCA-treated sgca - / -In the sera of mice (n = 6 per dose), creatine kinase levels were measured using creatine kinase SL assay and the corresponding manufacturer's protocol (Sekisui Diagnostics; Charlottetown, Ontario, Canada) (Catalog No. 326-10). Briefly, 25 μL of serum was mixed with 1 mL of working reagent and added to a cuvette. 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 following formula:

[0326] U / L = [(ΔAbs. / min)*1.025*1000] / [1*6.22*0.025] = (ΔAbs. / min)*6592.

[0327] Diaphragm tetanic contraction for functional assessment

[0328] Mice were euthanized, the diaphragm (DIA) was dissected with the costal attachments and central tendon left intact, and placed in K-H buffer. DIA slices 2-4 mm wide were isolated. The DIA strips were firmly tied at the central tendon with braided surgical silk (6 / 0; Surgical Specialties, Reading, Pennsylvania), and sutured through a portion of rib fixed to the distal end of the strip. Each muscle was transferred to a water bath filled with oxygenated KH solution, and the water bath temperature was maintained at 37 °C. The muscle was arranged horizontally and directly tied between a fixed pin and a dual-mode force sensor-servo motor (305C; Aurora Scientific, Aurora, Ontario, Canada). Two platinum plate electrodes were placed in the organ bath to flank the sides of the muscle length. The muscle was stretched to optimal length to measure twitch contractions and then allowed to rest for 10 minutes before starting the tetanic protocol. Once the muscle was stable, it was set to optimal length at 1 g and warmed up, which consisted of 3 1-Hz twitches every 30 seconds, then 3 150-Hz twitches every minute. After a 3-min rest, the DIA was stimulated at frequencies of 20, 50, 80, 120, 150, 180 Hz, with a 2-min rest between each stimulation, and the duration of each stimulation was 250 ms to determine the maximum tetanic force. Muscle length and weight were measured. Force was normalized against muscle weight and length.

[0329] Tibialis anterior (TA) tetanic contraction for functional assessment

[0330] The TA assessment procedure follows the protocol listed in Hakim et al., Methods Mol Biol; 709:75–89 (2011). Mice were anesthetized intraperitoneally with a ketamine / xylazine mixture (100 mg / kg and 10 mg / kg, respectively). Under a dissecting microscope, the hindlimb skin was removed to expose the TA muscle and patella. A double square knot was tied around the patellar tendon with 4-0 suture. Then the distal TA tendon was dissected out, and a double square knot was tied around the tendon with 4-0 suture as close to the muscle as possible, and then the tendon was cut. The exposed muscle was continuously moistened with saline. Then the mice were transferred to a thermo-controlled platform and maintained at 37 °C. The knee was fixed to a metal pin with the patellar tendon suture, and the distal TA tendon was sutured to the horizontal arm of a force sensor (Aurora Scientific, Aurora, Canada). An electrode was placed near the sciatic nerve to stimulate it. Once the muscle was stable, the resting tension was set to the length at which twitch contractions were maximal (optimal length). After a 3-min rest, the TA was stimulated at frequencies of 50, 100, 150, and 200 Hz, with a 1-min rest between each stimulation. After a 5-min rest, the muscle underwent a series of 10 isometric contractions with a 10% stretch-recoil procedure at 1-min intervals. After the eccentric contractions, the mice were then euthanized, and the TA muscles were dissected, weighed, and frozen for histology.

[0331] Immunofluorescence

[0332] Immunofluorescence staining for the hSGCA transgene was performed on cryosections (12-μm thick) from TA, gastrocnemius (GAS), quadriceps (QD), psoas (PSOAS), gluteus (GLUT), triceps (TRI), DIA, and heart (HRT) muscles. 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 taken using a Zeiss (Germany) AxioCam MRC5 camera. The percentage of fibers positive for α-SG staining in each image compared to controls was determined, and the mean was taken for each muscle. Positive α-SG fiber expression was defined as having at least 30% brighter fiber staining than vehicle-treated sgca- / - controls.

[0333] Western blot analysis

[0334] Samples from wild-type C57BL / 6 mice, vehicle-treated sgca- / - mice, and vector-administered sgca- / - mice were used for each western blot. Rabbit monoclonal α-SG antibody (Abcam, catalog number ab189254) at a 1:10,000 dilution and mouse monoclonal α-actin antibody (Sigma-Aldrich, catalog number A7811) at a 1:5,000 dilution were used for hSGCA blots. Rabbit monoclonal vinculin antibody (Invitrogen, catalog number 70062) at a 1:1,000 dilution was also used. Anti-mouse (Millipore, catalog number AP308P) and anti-rabbit (Life Technologies, catalog number 656120) secondary horseradish peroxidase antibodies were used for enhanced chemiluminescence immunodetection. Western blot quantification was performed by densitometry using ImageQuantTL 1D 8.1.0 (GE Healthcare Life Sciences).

[0335] Morphological analysis

[0336] Twelve-μm thick cryosections of muscle from 16- to 17-week-old wild-type C57BL / 6 mice (n = 6), vehicle-treated sgca- / - mice (n = 6), and scAAVrh74.tMCK.hSGCA-treated 16- to 17-week-old sgca- / - mice (n = 6 per dose) were stained with hematoxylin and eosin (H&E) for analysis. The percentage of myofibers with central nuclei was determined in TA, GAS, QD, GLUT, PSOAS, and TRI. In addition, myofiber diameters were measured in TA, GAS, QD, TRI, and PSOAS muscles. Four random 20x images of each muscle per animal were taken using a Zeiss AxioCam MRC5 camera. Central nucleated fibers were quantified using NIH's ImageJ software, and fiber diameters were measured using Zeiss Axiovision LE4 software.

[0337] Biodistribution quantitative polymerase chain reaction (PCR) analysis

[0338] As described above, Taqman quantitative PCR was performed to quantify the vector genome copy numbers present in the targeted and non-targeted contralateral muscles and non-targeted organs. A vector-specific primer-probe set was used to amplify a sequence of the intron region directly downstream of the tMCK promoter, which is unique and located within the scAAVrh.74.tMCK.hSGCA transgene cassette. The following primers and probe were used in this study: tMCK intron forward primer 5′-ACC CGA GAT GCC TGG TTATAA TT-3′; tMCK intron reverse primer 5′-TCC ATG GTG TAC AGA GCC TAA GAC-3′; and tMCK intron probe 5′-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C-IABkFQ-3’ (Integrated DNA Technologies). Copy numbers were reported as vector genomes per microgram of genomic DNA.

[0339] Sirius red staining and collagen quantification

[0340] Sirius red staining was performed to determine the level of collagen deposition in muscle tissue. 12-μm cryosections of GLUT, PSOAS, TRI, and DIA muscles from 16- to 17-week-old wild-type C57BL / 6 (n = 6), vehicle-treated sgca− / − (n = 6), and scAAVrh74.tMCK.hSGCA-treated 16- to 17-week-old sgca− / − (n = 6 per dose) were stained. Four 20x images were taken per muscle per mouse, and the amount of collagen deposition was determined using the ImageJ software program. The mean percentage of collagen per muscle was calculated for all groups.

[0341] Laser monitoring of open-field cage activity

[0342] An open-field activity chamber was used to determine the overall activity of experimental mice. 16- to 17-week-old mice from wild-type C57BL / 6 (n = 6) and untreated sgca - / - (n = 6) controls and 16- to 17-week-old scAAVrh74.tMCK.hSGCA-treated sgca - / -Mice (n = 6 per dose) were analyzed. All mice were tested at the same time of day, early in the morning near the end of the nocturnal cycle when mice are most active. All mice were tested in isolated rooms under dim light, with the same handling procedure used each time. In addition, to reduce anxiety and keep behavioral variables that could affect normal mouse activity and thus assay results to a minimum, we tested mice that were not singly housed. Mouse activity was monitored using a Photobeam activity system (San Diego Instruments, San Diego, CA). This system uses a grid of invisible infrared beams that pass through the animal room from front to back and from left to right to monitor the position and movement of mice in the x-y-z plane. Activity was recorded at 5-min intervals for a 1-hour period. Mice were acclimated to the activity test room with an initial 1-hour training session several days before starting data collection. Mice were tested in individual chambers, four per group. The test equipment was cleaned between each use to reduce mouse reactive behavioral variables that could alter results. Data were converted to a Microsoft Excel worksheet, and all calculations were done in the Excel program. The individual beam breaks for each mouse's movement in the x and y planes were summed to represent total locomotion during the 1-hour time interval, and beam breaks in the z plane were summed to obtain vertical activity.

[0343] Safety study

[0344] Hematology

[0345] Whole blood was taken from cardiac puncture for blood chemistry. Blood was collected in serum separator tubes and centrifuged at 15,000 rpm for 10 min. Serum was collected, frozen, and sent to Charles River Laboratory for chemical testing. Liver enzymes and glucose chemistry were prioritized in the hematology analysis.

[0346] Histopathology

[0347] At autopsy, muscle was freshly frozen in liquid nitrogen-cooled methylbutane; all other organs were harvested and fixed in formalin and embedded in paraffin. After treatment, tissues were stained with H&E, and slides and all tissues were sent to GEMPath, Inc. for formal review by a veterinary pathologist.

[0348] Statistical analysis

[0349] Unless otherwise stated, data are represented as mean ± SEM (error bars) and analyzed 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).

[0350] Example 2

[0351] Efficiency of systemic delivery of scAAVrh74.tMCK.hSGCA

[0352] A small pilot study was initiated to observe the efficacy of gene delivery by intravenous injection into the lateral tail vein of sgca 12 mice at a total dose of 1×10 13vg / kg vg (5×10 - / - vg / kg, based on 20-g mice, n = 4). Immunofluorescence analysis of harvested muscle was performed 4 weeks after gene transfer. Expression levels of the hSGCA transgene in seven different limb skeletal muscles: TA, GAS, GLUT, QD, PSOAS, and TRI and DIA. When analyzed by immunofluorescence, mice lacking α-SG showed complete absence of the protein ( Figure 2 A; representative images of TA, GAS, TRI, and DIA). A therapeutic dose of 1×10 12 vg total dose resulted in an average of 54 ± 23.81% vector transduction in all skeletal muscles, including DIA, 4 weeks after gene delivery.

[0353] Systemic delivery to sgca - / - Dose escalation study of scAAVrh74.tMCK.hSGCA in sgca

[0354] To determine the safest and most effective dose, delivery of three different doses of vector was investigated in a dose escalation study, in which 4-week-old sgca - / - mice were treated via the lateral tail vein with 1×10 12 vg total dose (5×10 13 vg / kg), 3×10 12 vg total dose (1×10 14 vg / kg), or 6×10 12 vg total dose (2×10[[ID=40]] 14 vg / kg) of scAAVrh74.tMCK.hSGCA. Mice were euthanized 12 weeks after gene delivery to assess hSGCA transgene expression in TA, GAS, QD, GLUT, PSOAS, TRI, DIA, and HRT muscles using immunofluorescence. With a 1×10 12 vg total dose (5×1013vg / kg ) The average hSGCA expression in mice treated with the lowest dose was 70.07 ± 3.71% in skeletal muscles including the DIA. With 3×10 12 vg total dose (1×10 14 vg / kg), the average hSGCA expression in mice treated with the intermediate dose was 85.35 ± 2.36% in all skeletal muscles. With 6×10 12 vg total dose (2×10 14 vg / kg), the average hSGCA expression in mice treated with the highest dose was 93.86 ± 2.02% in all skeletal muscles. For clarity, the dose was calculated based on supercoiled DNA or plasmid as a quantitative standard. The hSGCA expression in the HRT muscle remained at 75%, independent of the dose. Representative images of the tissues are shown in Figure 2 A. Stable hSGCA expression demonstrated the gene delivery effect at all three doses. Gene delivery targeted multiple muscles in the forelimbs and hindlimbs, showing abnormal α-SG expression in mice at all three doses. Most importantly, the important diaphragm muscle also showed α-SG gene expression after delivery. Figure 2 The western blot shown in

[0355] B confirmed the protein expression in all muscles of all three dosing groups of treated mice. Mouse myocardium also showed α-SG expression after treatment. Figure 3 Figure 3 A and 3B, compared with the vehicle-treated sgca - / - control, normalization of the fiber size distribution was observed in the TA, QD, and TRI of scAAVrh74.tMCK.hSGCA-treated sgca - / - mice, similar to that observed in wild-type controls. Compared with vehicle-treated sgca- / - control mice, the average fiber diameter size was significantly increased in the TA, QD, and TRI muscles at all doses (Table 1).

[0356] Table 1

[0357]

[0358] * = p < 0.05, **** = p < 0.0001. Abbreviations: TA, tibialis anterior muscle; AD, quadriceps; and TRI, triceps. Lowest dose: 1.0×10 12 vg; intermediate dose: 3.0×10 12 vg; highest dose: 6.0×10​12 vg.

[0359] In sgca mice treated with scAAVrh74.tMCK.hSGCA, a decrease in central nucleation was also observed. - / - In the skeletal muscle of vehicle-treated sgca mice, 68.72 ± 3.01% of the fibers had nuclei located centrally. After treatment with scAAVrh74.tMCK.hSGCA, the overall value of central nucleation in all muscle tissues decreased at the lowest dose of scAAVrh74.tMCK.hSGCA, resulting in 55.60 ± 3.25% of the skeletal muscle fibers showing nuclei located centrally (Table 2). - / - In the skeletal muscle of vehicle-treated sgca mice, 68.72 ± 3.01% of the fibers had nuclei located centrally. After treatment with scAAVrh74.tMCK.hSGCA, the overall value of central nucleation in all muscle tissues decreased at the lowest dose of scAAVrh74.tMCK.hSGCA, resulting in 55.60 ± 3.25% of the skeletal muscle fibers showing nuclei located centrally (Table 2).

[0360] Table 2

[0361]

[0362] **** = p < 0.0001. Abbreviations: TA, tibialis anterior; GAS, gastrocnemius; QD, quadriceps; GLUT, gluteus; PSO, psoas major; AVG, average; and TRI, triceps.

[0363] Mice treated with the medium dose had 61.85 ± 4.00% of the muscle fibers with centralized nuclei, while nucleation in the muscle fibers treated with the highest dose decreased to 37.93 ± 12.46% ( Figure 3 C).

[0364] Fibrosis, in which the tissue is overcome by collagen, usually occurs in the muscles of LGMD patients, leading to the formation of scar tissue. Sirius red staining was used to evaluate fibrosis to detect the content of collagen I and III as markers of fibrosis. As Figure 4 shown, a steady decrease in red staining was observed in sgca− / − mice after treatment with scAAVrh74.tMCK.hSGCA. Quantification showed that the collagen content in all muscles was significantly reduced in scAAVrh74.tMCK.hSGCA-treated sgca− / − mice compared to vehicle-treated sgca - / - control mice ( Figure 4 B). Collectively, these data demonstrate the successful systemic delivery of the hSGCA transgene, as indicated by stable expression in muscle tissue and improvement of the histopathological features associated with α-SG protein deficiency in sgca - / - mice.

[0365] Example 3

[0366] scAAVrh74.tMCK.hSGCA rAAV Improves Diaphragm and Tibialis Anterior Muscle Function and Increases Locomotor Ability

[0367] Since proximal muscle weakness and loss of function are the main symptoms of LGMD2D, and respiratory failure is the main cause of death in LGMD2D, improving the function and strength of TA and DIA is crucial for increasing the lifespan and quality of life of LGMD2D subjects. Strips of DIA and the entire TA muscle were used to confirm the correlation between hSGCA expression and muscle strength. As Figure 5 shown in A and 5B, compared with wild-type mice, deficits in specific force and resistance to contraction-induced damage were identified in the specific force of TA and DIA muscles of untreated sgca - / - mice.

[0368] Compared with wild-type mice, the TA muscle of sgca - / - mice showed a significant 44% functional deficit in reduced specific force output (161.6 ± 8.20 mN / mm 2 versus 291.7 ± 6.17 mN / mm 2 ; p < 0.0001); and a greater loss of force generated after a rigorous eccentric contraction protocol (sgca - / - mice lost 44.0 ± 6.0%; wild-type mice lost 18.0 ± 1.0%; p < 0.0001) ( Figure 5 A). Twelve weeks after tail vein delivery, the applicant noted a significant increase in specific force output after treatment with low, medium, and high doses of scAAVrh74.tMCK.hSGCA, which increased to 218 ± 11.94 mN / mm 2 , 227 ± 11.7 mN / mm 2 and 255 ± 11.7 mN / mm 2 . Resistance to damage after the eccentric contraction protocol was also improved compared with vehicle-treated sgca - / - mice, with mice treated with low, medium, and high doses losing only 22.0 ± 4.0%, 22.0 ± 3.0%, and 12.0 ± 1.0% respectively (p < 0.0001 compared with vehicle-treated sgca - / - mice) ( Figure 5 A).

[0369] In the DIA of vehicle-treated sgca - / - mice, the specific force generated showed a 41% decrease in intensity compared with wild-type mice (131.5 ± 12.07 mN / mm 2 versus 223.8 ± 15.85 mN / mm 2 ). Improvement in force was observed after treatment with scAAVrh74.tMCK.hSGCA at all three doses, with the specific force of the DIA in low-dose mice increasing to 179.2 ± 21.03 mN / mm2 , increased to 201.2 ± 22.94 mN / mm in mice at medium dose 2 and increased to 261.46 ± 9.73 mN / mm in mice at high dose 2 ( Figure 5 B). These data show that sgca - / -- mice have insufficient strength in TA and DIA muscles and are depleted faster than wild-type mice. However, after delivery of scAAVrh74.tMCK.hSGCA, functional recovery was achieved.

[0370] Other symptoms of LGMD2D include exercise intolerance and reduced activity and walking, possibly due to muscle damage, resulting in pain and muscle fatigue. To evaluate the level of physical activity, sgca - / - and wild-type C57BL / 6 mice received an open-field activity protocol similar to that used in previous reports. Walking-related activities of the mice were monitored to determine whether the lack of α-SG in sgca - / - mice leads to reduced walking compared to wild-type mice. Figure 5 The graph in C depicts the reduction in walking and vertical rearing in the sgca - / - mouse model compared to wild-type controls. The average horizontal movement beam breaks recorded in sgca - / - mice were 2000 ± 159 beam breaks / hour, a 77.5% reduction in movement compared to 8911 ± 1193 beam breaks / hour in wild-type controls. The average vertical rearing beam breaks recorded in sgca - / - mice were 24.75 ± 11.47 beam breaks / hour, a 97% reduction in vertical rearing compared to 803.3 ± 55.03 beam breaks / hour in wild-type mice. After treatment with scAAVrh74.tMCK.hSGCA, the walking and vertical rearing activities of the mice increased 12 weeks after gene delivery. The average horizontal walking increased to 3595 ± 55.03 beam breaks / hour in mice treated with a total dose of 1 × 10 12 vg, increased to 5238 ± 861.9 beam breaks / hour in mice treated with a total dose of 3 × 10 12 vg, and increased to 6487 ± 467.9 beam breaks / hour in mice treated with a total dose of 6 × 10 12 vg. The average vertical rearing activity increased to 377 ± 146.1 beam breaks / hour in mice treated with a total dose of 1 × 10 12 vg, increased to 321 ± 126.1 beam breaks / hour in mice treated with a total dose of 3 × 10 12 vg, and increased to [value not provided] beam breaks / hour in mice treated with a total dose of 6 × 10 12In the mice treated with the total vg dose, it increased to 448.8 ± 53.43 DNA strand breaks per hour ( Figure 5 C). Compared with the vehicle-treated sgca - / - mice, the physical activity of the treated mice showed improvements in walking by 44% - 69% and vertical rearing by 92% - 94%. In addition, compared with the untreated mice, the serum creatine kinase levels were significantly reduced in all treatment groups ( Figure 5 D). In summary, these data show that the delivery of α-SG restores physical activity and prevents muscle breakdown in sgca - / - mice.

[0371] Safety and biodistribution analysis of scAAVrh74.tMCK.hSGCA

[0372] As a safety measure, blood chemistry and hematology studies were performed on the vehicle-administered sgca - / - and wild-type mice. All values were within the normal reference ranges for mice ( Figure 6 ). In addition, tissue sections of all muscles and organs stained with H&E from the sgca - / - and wild-type mice administered scAAVrh74.tMCK.hSGCA were sent to a veterinary pathologist for formal review. No adverse effects were found in any of the samples from either the sgca - / - or wild-type mice administered scAAVrh74.tMCK.hSGCA. In addition to efficacy, these data demonstrate that the systemic delivery of all three doses of scAAVrh74.tMCK.hSGCA is well tolerated, safe, and non-toxic to sgca - / - and wild-type mice.

[0373] To test for potential toxicity or safety issues of delivering scAAVrh74.tMCK.hSGCA, vector biodistribution quantitative PCR was performed to quantify the presence of the vector genome ( Figure 7A ). A vector-specific tMCK.hSGCA primer-probe set was used to detect the vector genome in all muscles and organs tested from the sgca - / - mice administered scAAVrh74.tMCK / hSGCA. As expected, the vector genome was present in the tissues tested, with the highest copy number in the liver, followed by muscle. Western blots of α-sarcoglycan protein in the liver of WT and sgca- / - mice treated with vehicle (sgca - / - LR) or scAArh74.tMCK.hSGCA are shown in Figure 7B .

[0374] To improve the efficiency of α-SG expression, in one embodiment, the hSGCA cDNA sequence was packaged into a self-complementary vector. Self-complementary AAV vectors contain inverted repeat genomes that facilitate the formation of dsDNA, allowing replication and transcription to occur without the need for multiple vector genomes to promote these processes. Thus, the use of self-complementary vectors eliminates the rate-limiting step to allow for more rapid expression of the transgene. The Applicant has shown that intravascular delivery of scAAVrh74.tMCK.hSGCA in LGMD2D patients was associated with increased α-SG expression 180 days after gene transfer at doses of 1×10 12 and 3×10 12 .

[0375] Intravenous delivery of scAAVrh74.tMCK.hSGCA provided enhanced strength to muscle and resistance to contraction-induced injury in the tibialis anterior and diaphragm muscles in all three vector-treated groups compared to vehicle-treated controls. In addition, treatment with scAAVrh74.tMCK.hSGCA resulted in a significant decrease in CK. Further, after treatment with scAAVrh74.tMCK.hSGCA, mice were able to move around more frequently and lean on their hind limbs compared to vehicle-treated mice.

[0376] Significant histopathology including centrally located nuclei, extensive variability in fiber size, inflammation, necrosis, and fibrosis was commonly observed by muscle biopsy in patients with LGMD2D. After hSGCA delivery, the CN in mice decreased, the distribution of muscle fiber size became more uniform, and the collagen content decreased, and the muscle had an overall healthier appearance compared to vehicle-treated mice. The overall reduction in histopathology and scar tissue was accompanied by an improvement in the overall normal function and physiology of the muscle in vector-treated mice.

[0377] Finally, safety studies by quantitative PCR, serum chemistry analysis, and histopathology did not detect signs of toxicity. The tMCK promoter was detected only in the targeted tissue (all muscle) and was absent in other (non-muscle) organs except the liver. Detection of tMCK in the liver is not uncommon or concerning as it is a clearance organ. Histopathological review of all tissues (including the liver) by a board-certified veterinary pathologist concluded that systemic delivery of scAAVrh74.tMCK.hSGCA was not only safe in all tissues but also gene delivery significantly reduced the amount of dystrophic pathology found in the skeletal muscle of vehicle-treated sgca - / - mice. Chemical analysis of blood samples from vector-treated mice also supported the lack of toxicity.

[0378] As in the present disclosure, the dose escalation study provided preclinical data to support the lowest dose tested in the systems herein, namely 1×10 12 vg total (5×10 13 vg / kg) was sufficient to reduce signs and symptoms associated with α-SG protein loss. At the lowest dose tested, improvement in the function of all muscles was observed in vehicle-treated mice, as demonstrated by increased strength and motor behavior (walking and rearing). Even at the highest delivery dose of 6×10 12 vg total (2×10 14 vg / kg), the safety study showed no signs of toxicity.

[0379] Example 4

[0380] Older patients and durability

[0381] rAAVrh74.tMCK.hSGCA-mediated gene replacement has shown positive results in the treatment of LGMD-2D and other related diseases. This study was designed to test the ability of rAAVrh74.tMCK.hSGCA to treat older, more severely affected muscles and to determine the long-term durability of the AAV viral vector.

[0382] All procedures were conducted in accordance with the approval of the Research Institute at the Nationwide Children’s Hospital Institutional Animal Care and Use Committee. Mice were maintained on a 12:12 hour light:dark cycle under standard conditions with food and water provided ad libitum. First, rAAVrh74.tMCK.hSGCA was administered systemically via tail vein injection at three doses (1.0×10 12 、3.0×10 12 and 6.0×10 12 vg) to 12-month-old sgca - / - mice (n = 5) that exhibited severe muscle histopathology. Controls included sgca - / -Mice (n = 5) and BL6 wild-type mice injected with LRS (n = 4). At the endpoint of 6 months after treatment, SGCA protein expression, histological rescue, and functional improvement were evaluated in the muscles from treated mice. Compared to the control, all three doses showed stable protein expression of α-SG at the sarcolemma, increased histopathology, increased spontaneous activity and specific force generation, prevention of centrifugal force loss, and reduced serum CK. No vector toxicity was detected. In aged mice, treatment led to widespread high-level protein expression in the muscles, reduced fibrosis, and increased resistance to contraction-induced injury in the tibialis anterior muscle.

[0383] In particular, in 12-month-old sgca - / - IV injection of rAAVrh74.tMCK.hSGCA into mice resulted in widespread high-level protein expression in the muscles of the entire lower limb, upper limb, and proximal muscles of the trunk, including the diaphragm and heart ( Figure 8 ).

[0384] Overall improvement in muscle pathology was observed after administration of scAAVrh74.tMCK.hSGCA ( Figure 9 a) and a reduction in central nucleation. In addition, after administration, the average fiber size increased to a level similar to that of WT fibers in the gastrocnemius (GAS) and triceps (TRI) muscles ( Figure 9 b).

[0385] The level of collagen deposition was quantified as a measure of fibrosis. Compared to untreated controls, administration of scAAVrh74.tMCK.hSGCA resulted in a reduction in the level of fibrosis ( Figure 9 c). Functional improvement after administration of scAAVrh74.tMCK.hSGCA was demonstrated by improved force output (specific force) in the tibialis anterior (TA) and diaphragm (DIA) muscles and increased resistance to contraction-induced injury in the TA muscle ( Figure 10 ).

[0386] To further investigate the long-term durability of gene therapy, 4-week-old sgca - / - mice were systemically administered rAAVrh74.tMCK.hSGCA. More than 24 months after treatment, vector genome copy numbers were detected by qPCR in all transduced muscles tested (TA, TRI, DIA, GLUT, PSOAS, GAS, and QUAD). Protein expression and localization were studied by immunofluorescence staining of the treated muscles.

[0387] Although the present disclosure has been described in terms of specific embodiments, it should be understood that variations and modifications will occur to those skilled in the art. Therefore, it is only these limitations that appear in the claims that define the present disclosure.

[0388] All documents mentioned in this application are hereby incorporated by reference in their entirety.

[0389] SEQ ID NO:1

[0390] SGCA cDNA codon-optimized sequence:

[0391] ATGGCCGAGACACTGTTCTGGACTCCTCTGCTGGTGGTGCTGCTGGCTGGACTGGGA

[0392] GATACCGAGGCTCAGCAGACCACACTGCACCCACTGGTGGGCCGGGTGTTCGTGCA

[0393] CACCCTGGACCATGAGACATTTCTGAGTCTGCCAGAACACGTGGCTGTGCCACCTG

[0394] CTGTGCATATCACTTACCACGCCCATCTGCAGGGCCATCCTGATCTGCCACGGTGGC

[0395] TGAGATACACCCAGAGATCACCCCACCATCCTGGATTCCTGTATGGAAGCGCTACC

[0396] CCAGAGGACAGGGGACTGCAGGTGATCGAAGTGACAGCTTACAACCGCGACAGTTT

[0397] TGATACTACCAGGCAGCGCCTGGTGCTGGAGATTGGGGATCCAGAAGGACCCCTGC

[0398] TGCCTTATCAGGCCGAGTTCCTGGTGCGGTCACACGACGCTGAGGAAGTGCTGCCA

[0399] TCAACACCCGCCAGCAGATTTCTGTCCGCTCTGGGAGGACTGTGGGAGCCAGGAGA

[0400] ACTGCAGCTGCTGAATGTGACTAGCGCTCTGGATAGGGGAGGAAGGGTGCCACTGC

[0401] CAATCGAGGGAAGGAAGGAAGGGGTGTACATTAAAGTGGGAAGCGCTTCCCCATT

[0402] CTCCACCTGCCTGAAGATGGTGGCTTCTCCTGATAGTCACGCTAGGTGCGCTCAGGG

[0403] ACAGCCACCACTGCTGTCCTGTTATGACACACTGGCCCCCCATTTTCGCGTGGACTG

[0404] GTGCAACGTGACTCTGGTGGATAAATCTGTGCCTGAGCCAGCTGACGAAGTGCCAA

[0405] CCCCTGGAGACGGAATCCTGGAGCACGATCCTTTCTTTTGTCCTCCAACAGAAGCCC

[0406] CAGACAGGGATTTCCTGGTGGACGCTCTGGTGACTCTGCTGGTGCCTCTGCTGGTGG

[0407] CTCTGCTGCTGACCCTGCTGCTGGCTTATGTGATGTGCTGTCGGAGAGAGGGACGGC

[0408] TGAAGAGAGACCTGGCCACATCTGATATCCAGATGGTGCACCATTGTACTATTCAC

[0409] GGCAACACCGAGGAACTGCGCCAGATGGCTGCTTCTAGGGAGGTGCCAAGGCCACT

[0410] GAGTACACTGCCTATGTTTAATGTGCACACTGGCGAACGGCTGCCCCCTAGAGTGG

[0411] ATAGCGCCCAGGTGCCACTGATTCTGGACCAGCATTGA

[0412] SEQ ID NO:2

[0413] Human SGCA protein sequence:

[0414] MAETLFWTPLLVVLLAGLGDTEAQQTTLHPLVGRVFVHTLDHETFLSLPEHVAVPPAV

[0415] HITYHAHLQGHPDLPRWLRYTQRSPHHPGFLYGSATPEDRGLQVIEVTAYNRDSFDTTR

[0416] QRLVLEIGDPEGPLLPYQAEFLVRSHDAEEVLPSTPASRFLSALGGLWEPGELQLLNVTS

[0417] ALDRGGRVPLPIEGRKEGVYIKVGSASPFSTCLKMVASPDSHARCAQGQPPLLSCYDTL

[0418] APHFRVDWCNVTLVDKSVPEPADEVPTPGDGILEHDPFFCPPTEAPDRDFLVDALVTLL

[0419] VPLLVALLLTLLLAYVMCCRREGRLKRDLATSDIQMVHHCTIHGNTEELRQMAASREVPRPLSTLPMFNVHTGERLPPRVDSAQVPLILDQH*

[0420] SEQ ID NO:3

[0421] tMCK promoter sequence:

[0422] CCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGAT

[0423] GCCTGGTTATAATTAACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCC

[0424] TGAGCCTGAGCGGTTACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGG

[0425] AGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGATCCACTACGGGTCTAT

[0426] GCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA

[0427] ACCCCAACACCTGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTT

[0428] ACCCCACCCCGGTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCT

[0429] CTAAAAATAACCCTGTCCCTGGTGGACCACTACGGGTCTAGGCTGCCCATGTAAGG

[0430] AGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCCAACACCTGCT

[0431] GCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTGAGCGGTTACCCCACCCCGGTGCC

[0432] TGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGT

[0433] CCCTGGTCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCT

[0434] ATATAACCCAGGGGCACAGGGGCTGCCCCCGGGTCAC

[0435] SEQ ID NO:4

[0436] AAVrh74-tMCK-SGCA:

[0437] CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG

[0438] GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG

[0439] GAGTGGGGTT AACCAATTGG CGGCCGCAAA CTTGCATGCC CCACTACGGG

[0440] TCTAGGCTGC CCATGTAAGG AGGCAAGGCC TGGGGACACC CGAGATGCCT

[0441] GGTTATAATT AACCCCAACA CCTGCTGCCC CCCCCCCCCC AACACCTGCT

[0442] GCCTGAGCCT GAGCGGTTAC CCCACCCCGG TGCCTGGGTC TTAGGCTCTG

[0443] TACACCATGG AGGAGAAGCT CGCTCTAAAA ATAACCCTGT CCCTGGTGGA

[0444] TCCACTACGG GTCTATGCTG CCCATGTAAG GAGGCAAGGC CTGGGGACAC

[0445] CCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCC CCCCCCCCCC

[0446] CAACACCTGC TGCCTGAGCC TGAGCGGTTA CCCCACCCCG GTGCCTGGGT

[0447] CTTAGGCTCT GTACACCATG GAGGAGAAGC TCGCTCTAAA AATAACCCTG

[0448] TCCCTGGTGG ACCACTACGG GTCTAGGCTG CCCATGTAAG GAGGCAAGGC

[0449] CTGGGGACAC CCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCC

[0450] CCCCCCCCCC AACACCTGCT GCCTGAGCCT GAGCGGTTAC CCCACCCCGG

[0451] TGCCTGGGTC TTAGGCTCTG TACACCATGG AGGAGAAGCT CGCTCTAAAA

[0452] ATAACCCTGT CCCTGGTCCT CCCTGGGGAC AGCCCCTCCT GGCTAGTCAC

[0453] ACCCTGTAGG CTCCTCTATA TAACCCAGGG GCACAGGGGC TGCCCCCGGG

[0454] TCACCTGCAG AAGTTGGTCG TGAGGCACTG GGCAGGTAAG TATCAAGGTT

[0455] ACAAGACAGG TTTAAGGAGA CCAATAGAAA CTGGGCTTGT CGAGACAGAG

[0456] AAGACTCTTG CGTTTCTGAT AGGCACCTAT TGGTCTTACT GACATCCACT

[0457] TTGCCTTTCT CTCCACAGGT GTCCACTCCC AGTTCAATTA CAGCGCGTGG

[0458] TACCACCATG GCCGAGACAC TGTTCTGGAC TCCTCTGCTG GTGGTGCTGC

[0459] TGGCTGGACT GGGAGATACC GAGGCTCAGC AGACCACACT GCACCCACTG

[0460] GTGGGCCGGG TGTTCGTGCA CACCCTGGAC CATGAGACAT TTCTGAGTCT

[0461] GCCAGAACAC GTGGCTGTGC CACCTGCTGT GCATATCACT TACCACGCCC

[0462] ATCTGCAGGG CCATCCTGAT CTGCCACGGT GGCTGAGATA CACCCAGAGA

[0463] TCACCCCACC ATCCTGGATT CCTGTATGGA AGCGCTACCC CAGAGGACAG

[0464] GGGACTGCAG GTGATCGAAG TGACAGCTTA CAACCGCGAC AGTTTTGATA

[0465] CTACCAGGCA GCGCCTGGTG CTGGAGATTG GGGATCCAGA AGGACCCCTG

[0466] CTGCCTTATC AGGCCGAGTT CCTGGTGCGG TCACACGACG CTGAGGAAGT

[0467] GCTGCCATCA ACACCCGCCA GCAGATTTCT GTCCGCTCTG GGAGGACTGT

[0468] GGGAGCCAGG AGAACTGCAG CTGCTGAATG TGACTAGCGC TCTGGATAGG

[0469] GGAGGAAGGG TGCCACTGCC AATCGAGGGA AGGAAGGAAG GGGTGTACAT

[0470] TAAAGTGGGA AGCGCTTCCC CATTCTCCAC CTGCCTGAAG ATGGTGGCTT

[0471] CTCCTGATAG TCACGCTAGG TGCGCTCAGG GACAGCCACC ACTGCTGTCC

[0472] TGTTATGACA CACTGGCCCC CCATTTTCGC GTGGACTGGT GCAACGTGAC

[0473] TCTGGTGGAT AAATCTGTGC CTGAGCCAGC TGACGAAGTG CCAACCCCTG

[0474] GAGACGGAAT CCTGGAGCAC GATCCTTTCT TTTGTCCTCC AACAGAAGCC

[0475] CCAGACAGGG ATTTCCTGGT GGACGCTCTG GTGACTCTGC TGGTGCCTCT

[0476] GCTGGTGGCT CTGCTGCTGA CCCTGCTGCT GGCTTATGTG ATGTGCTGTC

[0477] GGAGAGAGGG ACGGCTGAAG AGAGACCTGG CCACATCTGA TATCCAGATG

[0478] GTGCACCATT GTACTATTCA CGGCAACACC GAGGAACTGC GCCAGATGGC

[0479] TGCTTCTAGG GAGGTGCCAA GGCCACTGAG TACACTGCCT ATGTTTAATG

[0480] TGCACACTGG CGAACGGCTG CCCCCTAGAG TGGATAGCGC CCAGGTGCCA

[0481] CTGATTCTGG ACCAGCATTG AGGCCGCAAT AAAAGATCTT TATTTTCATT

[0482] AGATCTGTGT GTTGGTTTTT TGTGTGTCCT GCAGGGGCGC GCCTCTAGAG

[0483] CATGGCTACG TAGATAAGTA GCATGGCGGG TTAATCATTA ACTACAAGGA

[0484] ACCCCTAGTG ATGGAGTTGG CCACTCCCTC TCTGCGCGCT CGCTCGCTCA

[0485] CTGAGGCCGG GCGACCAAAG GTCGCCCGAC GCCCGGGCTT TGCCCGGGCG

[0486] GCCTCAGTGA GCGAGCGAGC GCGCAG

[0487] SEQ ID NO:5

[0488] 5’ITR

[0489] CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG

[0490] GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG

[0491] GAGTGGGGTT

[0492] SEQ ID NO:6

[0493] 3’ITR

[0494] CCACTCCCTC TCTGCGCGCT CGCTCGCTCA CTGAGGCCGG GCGACCAAAG

[0495] GTCGCCCGAC GCCCGGGCTT TGCCCGGGCG GCCTCAGTGA GCGAGCGAGC GCGCSEQ ID NO:7

[0496] PolyA

[0497] GGCCGCAAT AAAAGATCTT TATTTTCATT AGATCTGTGT GTTGGTTTTT TGTG

[0498] SEQ ID NO:8

[0499] ATGCAGCTGC GCGCTCGCTC GCTCACTGAG GCCGCCCGGG CAAAGCCCGGGCGTCGGGCG 60

[0500] ACCTTTGGTC GCCCGGCCTC AGTGAGCGAG CGAGCGCGCA GAGAGGGAGTGGGGTTAACC 120

[0501] AATTGGCGGC CGCAAACTTG CATGCCCCAC TACGGGTCTA GGCTGCCCATGTAAGGAGGC 180

[0502] AAGGCCTGGG GACACCCGAG ATGCCTGGTT ATAATTAACC CCAACACCTGCTGCCCCCCC 240

[0503] CCCCCCAACA CCTGCTGCCT GAGCCTGAGC GGTTACCCCA CCCCGGTGCCTGGGTCTTAG 300

[0504] GCTCTGTACA CCATGGAGGA GAAGCTCGCT CTAAAAATAA CCCTGTCCCTGGTGGATCCA 360

[0505] CTACGGGTCT ATGCTGCCCA TGTAAGGAGG CAAGGCCTGG GGACACCCGAGATGCCTGGT 420

[0506] TATAATTAAC CCCAACACCT GCTGCCCCCC CCCCCCCAAC ACCTGCTGCCTGAGCCTGAG 480

[0507] CGGTTACCCC ACCCCGGTGC CTGGGTCTTA GGCTCTGTAC ACCATGGAGGAGAAGCTCGC 540

[0508] TCTAAAAATA ACCCTGTCCC TGGTGGACCA CTACGGGTCT AGGCTGCCCATGTAAGGAGG 600

[0509] CAAGGCCTGG GGACACCCGA GATGCCTGGT TATAATTAAC CCCAACACCTGCTGCCCCCC 660

[0510] CCCCCCAACA CCTGCTGCCT GAGCCTGAGC GGTTACCCCA CCCCGGTGCCTGGGTCTTAG 720

[0511] GCTCTGTACA CCATGGAGGA GAAGCTCGCT CTAAAAATAA CCCTGTCCCTGGTCCTCCCT 780

[0512] GGGGACAGCC CCTCCTGGCT AGTCACACCC TGTAGGCTCC TCTATATAACCCAGGGGCAC 840

[0513] AGGGGCTGCC CCCGGGTCAC CTGCAGAAGT TGGTCGTGAG GCACTGGGCAGGTAAGTATC 900

[0514] AAGGTTACAA GACAGGTTTA AGGAGACCAA TAGAAACTGG GCTTGTCGAGACAGAGAAGA 960

[0515] CTCTTGCGTT TCTGATAGGC ACCTATTGGT CTTACTGACA TCCACTTTGCCTTTCTCTCC 1020

[0516] ACAGGTGTCC ACTCCCAGTT CAATTACAGC GCGTGGTACC ACCATGGCCGAGACACTGTT 1080

[0517] CTGGACTCCT CTGCTGGTGG TGCTGCTGGC TGGACTGGGA GATACCGAGGCTCAGCAGAC 1140

[0518] CACACTGCAC CCACTGGTGG GCCGGGTGTT CGTGCACACC CTGGACCATGAGACATTTCT 1200

[0519] GAGTCTGCCA GAACACGTGG CTGTGCCACC TGCTGTGCAT ATCACTTACCACGCCCATCT 1260

[0520] GCAGGGCCAT CCTGATCTGC CACGGTGGCT GAGATACACC CAGAGATCACCCCACCATCC 1320

[0521] TGGATTCCTG TATGGAAGCG CTACCCCAGA GGACAGGGGA CTGCAGGTGATCGAAGTGAC 1380

[0522] AGCTTACAAC CGCGACAGTT TTGATACTAC CAGGCAGCGC CTGGTGCTGGAGATTGGGGA 1440

[0523] TCCAGAAGGA CCCCTGCTGC CTTATCAGGC CGAGTTCCTG GTGCGGTCACACGACGCTGA 1500

[0524] GGAAGTGCTG CCATCAACAC CCGCCAGCAG ATTTCTGTCC GCTCTGGGAGGACTGTGGGA 1560

[0525] GCCAGGAGAA CTGCAGCTGC TGAATGTGAC TAGCGCTCTG GATAGGGGAGGAAGGGTGCC 1620

[0526] ACTGCCAATC GAGGGAAGGA AGGAAGGGGT GTACATTAAA GTGGGAAGCGCTTCCCCATT 1680

[0527] CTCCACCTGC CTGAAGATGG TGGCTTCTCC TGATAGTCAC GCTAGGTGCGCTCAGGGACA 1740

[0528] GCCACCACTG CTGTCCTGTT ATGACACACT GGCCCCCCAT TTTCGCGTGGACTGGTGCAA 1800

[0529] CGTGACTCTG GTGGATAAAT CTGTGCCTGA GCCAGCTGAC GAAGTGCCAACCCCTGGAGA 1860

[0530] CGGAATCCTG GAGCACGATC CTTTCTTTTG TCCTCCAACA GAAGCCCCAGACAGGGATTT 1920

[0531] CCTGGTGGAC GCTCTGGTGA CTCTGCTGGT GCCTCTGCTG GTGGCTCTGCTGCTGACCCT 1980

[0532] GCTGCTGGCT TATGTGATGT GCTGTCGGAG AGAGGGACGG CTGAAGAGAGACCTGGCCAC 2040

[0533] ATCTGATATC CAGATGGTGC ACCATTGTAC TATTCACGGC AACACCGAGGAACTGCGCCA 2100

[0534] GATGGCTGCT TCTAGGGAGG TGCCAAGGCC ACTGAGTACA CTGCCTATGTTTAATGTGCA 2160

[0535] CACTGGCGAA CGGCTGCCCC CTAGAGTGGA TAGCGCCCAG GTGCCACTGATTCTGGACCA 2220

[0536] GCATTGAGGC CGCAATAAAA GATCTTTATT TTCATTAGAT CTGTGTGTTGGTTTTTTGTG 2280

[0537] TGTCCTGCAG GGGCGCGCCT CTAGAGCATG GCTACGTAGA TAAGTAGCATGGCGGGTTAA 2340

[0538] TCATTAACTA CAAGGAACCC CTAGTGATGG AGTTGGCCAC TCCCTCTCTGCGCGCTCGCT 2400

[0539] CGCTCACTGA GGCCGGGCGA CCAAAGGTCG CCCGACGCCC GGGCTTTGCCCGGGCGGCCT 2460

[0540] CAGTGAGCGA GCGAGCGCGC AGCTGGCGTA ATAGCGAAGA GGCCCGCACCGATCGCCCTT 2520

[0541] CCCAACAGTT GCGCAGCCTG AATGGCGAAT GGCGATTCCG TTGCAATGGCTGGCGGTAAT 2580

[0542] ATTGTTCTGG ATATTACCAG CAAGGCCGAT AGTTTGAGTT CTTCTACTCAGGCAAGTGAT 2640

[0543] GTTATTACTA ATCAAAGAAG TATTGCGACA ACGGTTAATT TGCGTGATGGACAGACTCTT 2700

[0544] TTACTCGGTG GCCTCACTGA TTATAAAAAC ACTTCTCAGG ATTCTGGCGTACCGTTCCTG 2760

[0545] TCTAAAATCC CTTTAATCGG CCTCCTGTTT AGCTCCCGCT CTGATTCTAACGAGGAAAGC 2820

[0546] ACGTTATACG TGCTCGTCAA AGCAACCATA GTACGCGCCC TGTAGCGGCGCATTAAGCGC 2880

[0547] GGCGGGTGTG GTGGTTACGC GCAGCGTGAC CGCTACACTT GCCAGCGCCCTAGCGCCCGC 2940

[0548] TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC CACGTTCGCC ATCTTCAAATATGTATCCGC 3000

[0549] TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAGAGTCCTGAGG 3060

[0550] CGGAAAGAAC CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT GGAAAGTCCCCAGGCTCCCC 3120

[0551] AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGTGTGGAAAGTC 3180

[0552] CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGTCAGCAACCAT 3240

[0553] AGTCCCGCCC CTAACTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATGCAGAGGCCGA 3300

[0554] GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTGGAGGCCTAGG 3360

[0555] CTTTTGCAAA GATCGATCAA GAGACAGGAT GAGGATCGTT TCGCATGATTGAACAAGATG 3420

[0556] GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT ATTCGGCTATGACTGGGCAC 3480

[0557] AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT GTCAGCGCAGGGGCGCCCGG 3540

[0558] TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA ACTGCAAGACGAGGCAGCGC 3600

[0559] GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC TGTGCTCGACGTTGTCACTG 3660

[0560] AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG GCAGGATCTCCTGTCATCTC 3720

[0561] ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC AATGCGGCGGCTGCATACGC 3780

[0562] TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA TCGCATCGAGCGAGCACGTA 3840

[0563] CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA CGAAGAGCATCAGGGGCTCG 3900

[0564] CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGAGCATGCC CGACGGCGAGGATCTCGTCG 3960

[0565] TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA AAATGGCCGCTTTTCTGGAT 4020

[0566] TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA GGACATAGCGTTGGCTACCC 4080

[0567] GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG CTTCCTCGTGCTTTACGGTA 4140

[0568] TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT TCTTGACGAGTTCTTCTGAG 4200

[0569] CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC AACCTGCCATCACGAGATTT 4260

[0570] CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCCGGGACGCCGG 4320

[0571] CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACCCTAGGGGGAG 4380

[0572] GCTAACTGAA ACACGGAAGG AGACAATACC GGAAGGAACC CGCGCTATGACGGCAATAAA 4440

[0573] AAGACAGAAT AAAAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACTCTAGCTTCCC 4500

[0574] GGCAACAATT AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTTCTGCGCTCGG 4560

[0575] CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGTGGGTCTCGCG 4620

[0576] GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTTATCTACACGA 4680

[0577] CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATAGGTGCCTCAC 4740

[0578] TGATTAAGCA TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAGATTGATTTAA 4800

[0579] AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAATCTCATGACCA 4860

[0580] AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAAAAGATCAAAG 4920

[0581] GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACAAAAAAACCAC 4980

[0582] CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTTCCGAAGGTAA 5040

[0583] CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCGTAGTTAGGCC 5100

[0584] ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATCCTGTTACCAG 5160

[0585] TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGACGATAGTTAC 5220

[0586] CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCCAGCTTGGAGC 5280

[0587] GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGCGCCACGCTTC 5340

[0588] CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACAGGAGAGCGCA 5400

[0589] CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGGTTTCGCCACC 5460

[0590] TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTATGGAAAAACG 5520

[0591] CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCTCACATGTTCT 5580

[0592] TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCTTTGAGTGAGCTGATA 5640

[0593] CCGCTCGCCG CAGCCGAACG ACCGAGCGCA GCGAGTCAGT GAGCGAGGAAGCGGAAGAGC 5700

[0594] GCCCAATACG CAAACCGCCT CTCCCCGCGC GTTGGCCGAT TCATTAATG 5749

Claims

1. A nucleic acid molecule comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 or SEQ ID NO:8, and wherein said nucleotide sequence encodes a protein that retains α - sarcoglycan activity.

2. The nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:

4.

3. The nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:

8.

4. A plasmid comprising the nucleic acid molecule according to any one of claims 1 to 3.

5. A recombinant adeno - associated virus (rAAV) vector comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO:4, and wherein said nucleotide sequence encodes a protein that retains α - sarcoglycan activity.

6. A recombinant adeno - associated virus (rAAV) vector comprising the nucleotide sequence shown in SEQ ID NO:

4.

7. The rAAV vector according to claim 5 or 6, wherein said rAAV belongs to serotype AAV rh.

74.

8. A host cell comprising the nucleic acid molecule according to any one of claims 1 to 3, the plasmid according to claim 4, or the rAAV vector according to any one of claims 5 to 7.

9. The host cell according to claim 8, wherein said cell is a yeast cell, an insect cell, a mammalian cell or a bacterial cell.

10. The host cell according to claim 9, wherein said cell is a packaging cell selected from the following: HeLa cell, HEK 293 cell, HEK 293T cell, PerC.6 cell, MRC - 5 cell, WI - 38 cell, Vero cell, FRhL - 2 cell or a Sf9 cell.

11. A composition comprising the nucleic acid molecule according to any one of claims 1 to 3, the plasmid according to claim 4, the rAAV vector according to any one of claims 5 to 7, or the cell according to any one of claims 8 to 10.

12. An in vitro method for producing recombinant adeno-associated virus (rAAV) that encodes a protein retaining α-dystroglycan activity, the method comprising: Transfer the plasmid according to claim 4, or the nucleic acid molecule according to any one of claims 1 to 3, or the rAAV according to any one of claims 5 to 7 to a host cell, culture the host cell, and recover the rAAV from the supernatant of the culture.

13. An in vitro method for producing recombinant adeno-associated virus (rAAV) particles, wherein the recombinant adeno-associated virus (rAAV) particles encode a protein that retains the activity of α-sarcoglycan, the method comprising: Transfer the nucleic acid molecule according to any one of claims 1 to 3, the plasmid according to claim 4, or the rAAV according to any one of claims 5 to 7 to a packaging cell, culture the host cell, and recover the rAAV particles from the supernatant of the culture.

14. An rAAV particle produced by the method according to claim 13.

Images