Self-complementary adeno-associated virus vector and its use in treatment of muscular dystrophy

Abstract

To provide therapy vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophies, e.g. limb girdle muscular dystrophy (LGMD) such as LGMD2D.SOLUTION: The present invention provides a polynucleotide sequence, comprising a nucleotide sequence at least 90% identical to a specific nucleotide sequence forming a self complementary recombinant AAV (rAAV) scAAVrh74.tMCK.hSGCA vector, and a recombinant AAV vector comprising the polynucleotide sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 284418, filed on 30 November 2021, which is incorporated herein by reference in its entirety.

[0002] Integration by referencing electronically submitted documents This application includes a computer-readable sequence listing, incorporated herein in its entirety by reference as a separate part of the present disclosure, and identified as 56757_Seqlisting.txt, size: 24,131 bytes, created date: November 9, 2022.

[0003] Therapeutic vectors, such as AAV vectors expressing alpha-sarcoglycans, and methods for using these vectors to mitigate and prevent fibrosis in subjects suffering from muscular dystrophy, such as limb-girdle muscular dystrophy (LGMD) such as LGMD2D, are described herein. [Background technology]

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

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

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

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

[0008] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cellular, and natural infections in humans and other animals are silent and asymptomatic. Furthermore, AAV infects many mammalian cells and allows for the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA within a plasmid, enabling the construction of a recombinant genome. Furthermore, because signals directing AAV replication and genomic capsid formation are contained within the ITR of the AAV genome, some or all of the approximately 4.3 kb of genome (rep-cap, encoding replication and structural capsid proteins) may be replaced with foreign DNA. To generate an AAV vector, the rep and cap proteins may be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. This makes it easy to withstand the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of chilling AAV. AAV can be freeze-dried. Finally, AAV-infected cells do not show resistance to co-infection.

[0009] Improving function in patients with LGMD and other muscular dystrophy requires both gene restoration and fibrosis reduction. There is a need for methods to reduce fibrosis that can be repaired by gene restoration techniques for more effective treatment of LGMD and other muscular dystrophy. [Overview of the Initiative] [Means for solving the problem]

[0010] Gene therapy vectors expressing the alpha-sarcoglycan gene (e.g., AAV), and methods for delivering alpha-sarcoglycan to muscle to alleviate and / or prevent fibrosis, and / or increase muscle strength, and / or treat mammalian subjects suffering from muscular dystrophy are described herein.

[0011] A self-complementary AAV (scAAV) expressing an alpha-sarcoglycan gene is provided herein. For example, the provided scAAV comprises a polynucleotide sequence comprising: i) two nucleotide sequences encoding a self-complementary alpha-sarcoglycan protein; and ii) two polyadenylated sequences that are self-complementary and contain a mutant reverse terminal repeat (ITR) located at the center of the AAV genome sequence (expression cassette).

[0012] Also provided are recombinant AAV (rAAV) vectors containing a polynucleotide sequence, the polynucleotide sequence comprising, in the 5' to 3' direction, (1) a complementary sequence of a polyadenylated sequence, (2) a complementary sequence of the gene of interest, (3) a complementary sequence of an intron, (4) a complementary sequence of a promoter, (5) a 5'ITR sequence, (6) a promoter, (7) an intron, (8) the gene of interest, and (9) a polyadenylated sequence, wherein the polynucleotide sequence is flanked by two 3'ITR sequences, and the two 3'ITR sequences are complementary to each other. In one embodiment, the gene of interest includes the human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, or human ANO5, or calpain-3 (Cap3) gene. In another embodiment, the promoter is a muscle-specific regulatory element. Examples of muscle-specific regulatory elements include human skeletal actin gene elements, cardiac actin gene elements, muscle cell-specific enhancer-binding factor (MEF) elements, muscle creatine kinase (MCK) promoters, truncated MCK (tMCK) promoters, tMCK enhancers, myosin heavy chain (MHC) promoters, MHCK7 promoters, C5-12 promoters, mouse creatine kinase enhancer elements, skeletal fast-twitch muscle troponin c gene elements, slow-twitch muscle cardiac troponin C gene elements, slow-twitch muscle troponin I gene elements, hypoxia-induced nuclear factor-binding elements, or steroid-induced elements, or glucocorticoid-responsive elements (GREs).

[0013] Single-stranded AAV vectors (ssAAVs), upon entering the nucleus, require cell-mediated synthesis of a second strand before they are ready for replication and transcription. However, since scAAVs bypass the rate-limiting step of cell synthesis of the second strand required in ssAAVs, the scAAVs provided herein are superior to ssAAVs in gene therapy.

[0014] A polynucleotide comprising two self-complementary nucleotide sequences (also referred to as expression cassettes) is provided herein, each nucleotide sequence comprising a tMCK promoter, an hSGCA cDNA sequence, and a polyadenylation sequence, as well as a single 5'ITR located between the two nucleotide sequences. The 5'ITR forms a hairpin when the nucleotide sequences hybridize.

[0015] For example, this disclosure provides the polynucleotide sequence of SEQ ID NO: 1, which is also shown as a schematic diagram in Figure 1. The polynucleotide sequence of SEQ ID NO: 1 is a 4857-nucleotide sequence comprising two hSGCA cDNA sequences (SEQ ID NO: 2 and / or SEQ ID NO: 6) that encode the amino acid sequence of SEQ ID NO: 3 and hybridize with each other, two tMCK promoters (SEQ ID NO: 7 and / or SEQ ID NO: 9) that hybridize with each other, and two polyadenylated sequences (SEQ ID NO: 5 and / or SEQ ID NO: 10) that hybridize with each other. The ITR sequence located at the center of the polynucleotide sequence has a nucleotide sequence shown as SEQ ID NO: 8. Additional ITR sequences are shown as SEQ ID NOs: 4 and 11.

[0016] This disclosure provides a polynucleotide sequence comprising a nucleotide sequence that is at least about 90%, at least about 95%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 1. This disclosure also provides a polynucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1.

[0017] In addition, the Disclosure provides recombinant AAVs (rAAVs) comprising any of the disclosed polynucleotides. For example, the Disclosure provides rAAVs comprising polynucleotide sequences comprising nucleotide sequences that are at least about 90%, at least about 95%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 1. The Disclosure also provides rAAVs comprising polynucleotide sequences comprising the nucleotide sequence of SEQ ID NO: 1.

[0018] The disclosure also provides an rAAV comprising a polynucleotide sequence, the polynucleotide sequence comprising i) two self-complementary nucleotide sequences each encoding a gene of interest, wherein the two self-complementary nucleotide sequences encoding the gene of interest are adjacent to a 5'ITR sequence, and ii) two self-complementary polyadenylated sequences, wherein the polynucleotide sequence is adjacent to two 3'ITR sequences, and the two 3'ITR sequences are complementary. For example, the target genes are GAD, MTM1, LPL, RPE, REP-1, CNGB3, P1ND4, XLRS, FVIII, FIX, FIX19, AAT, NF-κB, IFN-β, ARSA, NGF, hARSB, Neurturin, AADC, SUMF, SUMF1, OTC, FGF-4, ND4, ARSA, REP1, cytosine deaminase, HGF728, HGF723, hGAA, β-globin gene, Gag, MG1MA3, L523S, METRAP, GDNF, AQP1, PG9DP, HBB, ADA, TCR, CAR, filgrastim, IL-12, GM-CSF, ICP34.5, PENK, RB94, SST2, and DCK. These include the P53, HSC, human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, human ANO5, and calpain-3 (Cap3) genes. For example, in a polynucleotide sequence, the first nucleotide sequence is the complement sequence of the target gene, and the second nucleotide sequence encoding the target gene is its sense sequence; therefore, the first and second nucleotide sequences are complementary to each other.

[0019] This disclosure also provides a polynucleotide-containing rAAV, the polynucleotide sequence comprising: i) two self-complementary nucleotide sequences, each encoding a human alpha-sarcoglycan (hSGCA) protein, such as the amino acid sequence of SEQ ID NO: 3; and ii) two self-complementary polyadenylated sequences. In some embodiments, the nucleotide sequence encoding the hSGCA protein is at least about 90%, at least about 95%, or at least about 99% identical to the nucleotide sequence of SEQ ID NO: 2, or the nucleotide sequence encoding the hSGCA protein comprises the nucleotide sequence of SEQ ID NO: 2. For example, the polyadenylated sequence comprises the nucleotide sequence of SEQ ID NO: 6.

[0020] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding alpha-sarcoglycan are described herein. In some embodiments, the polynucleotide sequence encoding alpha-sarcoglycan comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to, for example, the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 7, and encodes a protein that retains alpha-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding alpha-sarcoglycan comprises the nucleotide sequence described in SEQ ID NO: 2. In some embodiments, the polynucleotide sequence encoding alpha-sarcoglycan comprises the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 7.

[0021] In another embodiment, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding an alpha-sarcoglycan that has sequence identity with at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, or 99%, to the amino acid sequence of SEQ ID NO: 3, and the protein retains alpha-sarcoglycan activity.

[0022] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding a functional alpha-sarcoglycan, or its complement, which includes a nucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 under stringent conditions, are described herein.

[0023] The term "stringent" is used to refer to conditions that are generally understood as stringent in the art. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65–68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (higher temperature, lower ionic strength, higher formamide, or other denaturing agents, etc.) can also be used, but the rate of hybridization will be affected. When deoxyoligonucleotide hybridization is involved, examples of additional stringent hybridization conditions include washing with 6×SSC 0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos).

[0024] Where ranges are used herein with respect to physical properties such as molecular weight, concentration, or dosage, it is intended that the range and all combinations and partial combinations of specific embodiments within that range are included. The term “approximately” when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variation (or statistical experimental error), and therefore the numerical value or numerical range may vary, for example, by 1% to 15% of the stated numerical value or numerical range.

[0025] To reduce nonspecific and / or background hybridization, other agents may be included in the hybridization and washing buffers. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), Ficol, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, but other suitable agents may also be used. The concentrations and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4, but under typical ionic strength conditions, the rate of hybridization is largely pH-independent. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch.4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to allow DNAs of different sequence similarities to form hybrids, taking these variables into consideration.

[0026] In addition, any of the rAAVs provided contain polynucleotides, each of which has two self-complementary nucleotide sequences operably linked to a muscle-specific regulatory element, and the two muscle-specific regulatory elements are self-complementary. For example, muscle-specific regulatory elements include human skeletal actin gene elements, cardiac actin gene elements, muscle cell-specific enhancer-binding factor (MEF) elements, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, MHCK7 promoter (a hybrid version of MHC and MCK), C5-12 (synthetic promoter), mouse creatine kinase enhancer elements, skeletal fast-twitch muscle troponin C gene elements, slow-twitch muscle cardiac troponin C gene elements, slow-twitch muscle troponin I gene elements, hypoxia-induced nuclear factor-binding elements, steroid-induced elements, or glucocorticoid response elements (GREs).

[0027] In some embodiments, the disclosed rAAV comprises a polynucleotide sequence, each of which two complementary nucleotide sequences is operably linked to a muscle-specific regulatory element MCK (tMCK) comprising the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 10.

[0028] In additional embodiments, the disclosed rAAV comprises three reverse terminal repeats (ITRs), one of which is flanked by two complementary muscle-specific regulatory elements. For example, the ITRs may include SEQ ID NO: 5 and / or SEQ ID NO: 9 and / or SEQ ID NO: 12. In a particular example, the ITR flanked by two self-complementary muscle-specific regulatory elements comprises the nucleotide sequence of SEQ ID NO: 8 or 10. In other embodiments, the two ITRs comprise the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 12, and one of the ITRs comprises the nucleotide sequence of SEQ ID NO: 9.

[0029] AAV can be any serotype of, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74, or a synthetic AAV serotype. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0030] The Disclosure also provides compositions comprising any of the disclosed rAAVs or any of the disclosed polynucleotides. In some embodiments, the compositions further comprise pharmaceutically acceptable carriers, diluents, and / or auxiliaries. For example, the compositions comprise any of the rAAVs, buffers, ionic strengtheners, and surfactants of the Disclosure.

[0031] This disclosure provides a method for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in one of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0032] This disclosure also provides a method for doing so in subjects requiring an increase in muscle strength and / or muscle mass, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in one of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0033] This disclosure provides a method for doing so in a subject requiring fibrosis relief, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in one of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0034] This disclosure provides a method for reducing contraction-induced injury in subjects requiring such reduction, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in one of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0035] The present disclosure provides a method of doing so in a subject that requires treatment of alpha-sarcoglycanopathy, the method comprising the step of administering any one of the rAAVs disclosed herein, wherein the rAAV is administered by a systemic route. In particular, in any one of the disclosed methods, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic administration route.

[0036] In any one of the disclosed methods, the rAAV is based on supercoiled DNA or plasmid as a quantitative standard, and is about 1.0×10 12 vg / kg to about 5.0×10 15 vg / kg. For example, the rAAV is based on supercoiled DNA or plasmid as a quantitative standard, and 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 13 vg / kg to about 2×10 14 vg / kg, or the 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.

[0037] In another embodiment, in any of the disclosed methods, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, resulting in approximately 1.85 × 10⁻⁶ units. 13 vg / kg or 7.41 × 10 13 It is administered at a dose of vg / kg. For example, rAAV is measured at approximately 1.0 × 10⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 It is administered at a dose of vg / kg.

[0038] In addition, in any of the disclosed methods, the systemic route of administration is an intravenous route. For example, in any of the disclosed methods, rAAV is administered by injection, infusion, or implantation. In some embodiments, rAAV is administered by an intravenous route via peripheral limb veins.

[0039] 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).

[0040] In an exemplary embodiment, a method for treating muscular dystrophy includes administering rAAV to a subject suffering from limb-girdle muscular dystrophy, wherein rAAV is measured approximately 5 × 10⁻¹⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14 Administered by intravenous infusion at a dose of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1.

[0041] In exemplary embodiments, the present disclosure provides a method for treating muscular dystrophy in a subject requiring treatment, the method comprising the step of administering rAAV to a subject, the subject having limb-girdle muscular dystrophy, and the rAAV being approximately 5 × 10⁻¹⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14 Administered by intravenous infusion at a dose of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1. For example, in these methods, the level of alpha-sarcoglycan gene expression in the target cells increases after rAAV administration compared to the level of alpha-sarcoglycan gene expression before rAAV administration.

[0042] In any of the disclosed methods, the level of alpha-sarcoglycan gene expression in the cells of interest increases after administration of rAAV compared to the level of alpha-sarcoglycan gene expression before administration of rAAV, and / or the serum CK level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV, and / or spontaneous movement and specific force generation increase, fibrosis is reduced, resistance to contraction-induced injury of the tibialis anterior muscle increases, and / or the muscle tissue of the subject The number of alpha-sarcoglycan-positive fibers in the tissue increased after rAAV administration compared to the number of alpha-sarcoglycan-positive fibers before rAAV administration, or fibrosis was reduced in subjects after rAAV administration compared to before rAAV administration, and / or fibrosis was reduced in subjects after rAAV administration compared to before rAAV administration, and / or specific force, fiber diameter size, and / or eccentric contraction in the subject's muscle increased after rAAV administration compared to before rAAV administration.

[0043] In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0044] In another embodiment, the Disclosure provides a method for expressing an alpha-sarcoglycan gene in cells, comprising administering one of the disclosed rAAVs to a target. For example, the Disclosure provides a method for expressing an alpha-sarcoglycan gene in cells, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1 to a target. In addition, in one of the methods, the expression of an alpha-sarcoglycan gene in cells is detected by measuring alpha-sarcoglycan protein levels by Western blotting in a muscle biopsy. Alternatively, in one of the methods, the expression of an alpha-sarcoglycan gene in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of an alpha-sarcoglycan gene is measured in a target by detecting the number of vector genomes per microgram of genomic DNA.

[0045] This disclosure provides a method for reducing serum CK levels in subjects requiring such reduction, the method comprising administering one of the disclosed rAAVs to a subject. For example, this disclosure provides a method for reducing serum CK levels in subjects requiring such reduction, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1 to a subject.

[0046] In another aspect, the Disclosure provides a method for increasing alpha-sarcoglycan-positive fibers in target muscle tissue, comprising administering one of the disclosed rAAVs to target. For example, the Disclosure provides a method for doing so in target muscle tissue that requires an increase in alpha-sarcoglycan-positive fibers, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1 to target.

[0047] This disclosure also provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, including administering one of the disclosed rAAVs to the subject. For example, this disclosure provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, the method comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1 to the subject. In addition, in one of the disclosed methods, alpha-sarcoglycan gene expression in the cells of the subject is detected by measuring alpha-sarcoglycan protein levels by Western blotting in a muscle biopsy. Alternatively, in one of the methods, alpha-sarcoglycan gene expression in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, alpha-sarcoglycan gene expression is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0048] This disclosure provides compositions for treating muscular dystrophy in subjects requiring such treatment, comprising one of the rAAVs disclosed herein, and formulated for systemic administration. In particular, in one of the compositions, the rAAV is AAVrh74.tMCK.hSCGA.

[0049] This disclosure also provides compositions for doing so in subjects requiring an increase in muscle strength and / or muscle mass, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in any of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0050] This disclosure provides compositions for doing so in subjects requiring fibrosis relief, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in any of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0051] This disclosure provides compositions for reducing contraction-induced injury in subjects requiring such reduction, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in any of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0052] This disclosure provides compositions for treating alpha-sarcoglycan disorders in subjects requiring treatment, comprising the step of administering one of the rAAVs disclosed herein, wherein the rAAV is administered via a systemic route. In particular, in any of the methods disclosed, the rAAV is AAVrh74.tMCK.hSCGA, and the rAAV is administered using a systemic route.

[0053] Any of the disclosed compositions, based on superhelical DNA or plasmids as a quantitative standard, yields approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 15 Contains rAAV in doses of vg / kg. For example, rAAV is approximately 1.0 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 1013 vg / kg ~ approx. 2×10 14 The dose of vg / kg, or rAAV, is approximately 5 × 10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, or approximately 5 × 10 14 The dosage is in 1g / kg.

[0054] In another embodiment, in any of the disclosed compositions, rAAV is measured based on linearized DNA or plasmid as a quantitative standard, resulting in approximately 1.85 × 10⁻¹⁶ units. 13 vg / kg or approximately 7.41 × 10 13 It is administered at a dose of vg / kg. For example, rAAV is measured at approximately 1.0 × 10⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 It is administered at a dose of vg / kg.

[0055] In addition, any of the disclosed compositions is formulated for administration by an intravenous route, such as a composition formulated for administration by injection, infusion, or implantation. In some embodiments, the disclosed compositions are formulated for administration by an intravenous route through a peripheral limb vein.

[0056] Any of the disclosed compositions is for the treatment of limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0057] In an exemplary embodiment, the present disclosure provides a composition for treating a subject suffering from limb-girdle muscular dystrophy, the composition being based on supercoiled DNA or plasmid as a quantitative standard, about 5×10 13 vg / kg to about 2×10 14 vg / kg and containing a dose of rAAV, the composition being formulated for administration by intravenous infusion, and the rAAV containing the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 1.

[0058] In addition, the present disclosure provides a composition for performing it in a subject that requires treating limb-girdle muscular dystrophy, the composition being based on supercoiled DNA or plasmid as a quantitative standard, about 5×10 13 vg / kg to about 2×10 14 vg / kg and containing a dose of rAAV, the composition being formulated for administration by intravenous infusion, and the rAAV containing the nucleotide sequence of the scAAVrh74.tMCK.hSGCA construct of SEQ ID NO: 1. For example, administration of the composition increases the level of alpha-sarcoglycan gene expression in the cells of the subject as compared to the level of alpha-sarcoglycan gene expression prior to administration of the composition.

[0059] In addition, administration of any of the disclosed compositions increases the level of alpha-sarcoglycan gene expression in the target cells compared to the level of alpha-sarcoglycan gene expression before administration of the composition, and / or administration of the disclosed compositions decreases the serum CK level in the subject compared to the serum CK level before administration of the composition, and / or increases spontaneous movement and specific force generation, reduces fibrosis, increases resistance to contraction-induced injury of the tibialis anterior muscle, and / or administration of the compositions increases the number of alpha-sarcoglycan-positive fibers in the target muscle tissue compared to the number of alpha-sarcoglycan-positive fibers before administration of the composition, and / or administration of the compositions reduces fibrosis in the subject compared to before administration of rAAV, and / or the compositions reduce fibrosis compared to before administration of the composition, or administration of the compositions increases specific force, fiber diameter size, and / or eccentric contraction in the target muscle compared to before administration of the composition. In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0060] In another embodiment, the Disclosure provides compositions for expressing alpha-sarcoglycan genes in cells, the compositions comprising any of the disclosed rAAVs. For example, the Disclosure provides a composition for expressing alpha-sarcoglycan genes in cells comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1. In addition, in any of the compositions, the expression of alpha-sarcoglycan genes in the cells of interest is detected by measuring alpha-sarcoglycan protein levels by Western blotting in a muscle biopsy. Alternatively, in any of the methods, the expression of alpha-sarcoglycan genes in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of alpha-sarcoglycan genes is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0061] This disclosure provides compositions for achieving a reduction in serum CK levels in subjects requiring such reduction, the compositions comprising one of the disclosed rAAVs. For example, this disclosure provides compositions for achieving a reduction in serum CK levels in subjects requiring such reduction, the compositions comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1.

[0062] In another embodiment, the Disclosure provides a composition for increasing alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the composition comprising one of the disclosed rAAVs. For example, the Disclosure provides a composition for increasing alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the composition comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1.

[0063] This disclosure also provides compositions for increasing alpha-sarcoglycan expression in subjects requiring it, the compositions comprising one of the disclosed rAAVs. For example, this disclosure provides compositions for increasing alpha-sarcoglycan expression in subjects requiring it, the compositions comprising the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1. In addition, after administration of one of the disclosed compositions, alpha-sarcoglycan gene expression in cells of a subject is detected by measuring alpha-sarcoglycan protein levels by Western blotting in muscle biopsy. Alternatively, after administration of one of the disclosed compositions, alpha-sarcoglycan gene expression in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in muscle biopsy. In other embodiments, after administration of one of the disclosed compositions, alpha-sarcoglycan gene expression is measured in a subject by detecting the number of vector genomes per microgram of genomic DNA.

[0064] This disclosure provides the use of any of the disclosed rAAVs for the preparation of a medicament for treating muscular dystrophy in subjects requiring such treatment, the medicament being formulated for systemic administration. In particular, this disclosure provides the use of AAVrh74.tMCK.hSCGA for the preparation of a medicament for treating muscular dystrophy, the medicament being formulated for systemic administration.

[0065] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a medicament to achieve an increase in muscle strength and / or muscle mass in subjects requiring such an increase. In particular, this disclosure provides a use in which the rAAV is AAVrh74.tMCK.hSCGA and the rAAV is administered via a systemic route of administration.

[0066] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a medicament to do so in subjects requiring fibrosis relief. In particular, this disclosure provides a use in which the rAAV is AAVrh74.tMCK.hSCGA and the rAAV is administered via a systemic route.

[0067] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a pharmacopoeia to do so in subjects requiring mitigation of contraction-induced injury. In particular, this disclosure provides a use in which the rAAV is AAVrh74.tMCK.hSCGA and the rAAV is administered via a systemic route of administration.

[0068] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a medicament to treat alpha-sarcoglycan disorders in subjects requiring such treatment. In particular, this disclosure provides a use in which the rAAV is AAVrh74.tMCK.hSCGA and the rAAV is administered via a systemic route of administration.

[0069] In any of the disclosed uses, the pharmaceutical product is measured based on superhelical DNA or plasmids as a quantitative standard, approximately 1.0 × 10⁶ 12 vg / kg ~ approx. 5.0×1015 It contains rAAV at a dose of vg / kg. For example, rAAV is based on supercoiled DNA or plasmid as a quantification standard, about 1.0×10 12 vg / kg to about 2.0×10 15 vg / kg, about 5×10 12 vg / kg to about 1.0×10 15 vg / kg, about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, about 2.0×10 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 dose, or 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 dose.

[0070] In another embodiment, in any of the disclosed uses, the medicament contains rAAV at a dose of about 1.85×10 13 vg / kg or 7.41×10 13 vg / kg. For example, the medicament is based on linearized DNA or plasmid as a quantification standard, 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, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 Includes rAAV at a dose of vg / kg.

[0071] In addition, in any of the disclosed uses, the medicament is formulated for administration via an intravenous route. For example, in any of the disclosed uses, the medicament is formulated for administration by injection, infusion, or implantation. In some embodiments, the medicament is formulated for administration via an intravenous route through peripheral limb veins.

[0072] In any of the disclosed uses, the medicament is for the treatment of limb-girdle muscular dystrophy, such as limb-girdle muscular dystrophy type 2D (LGMD2D).

[0073] In exemplary embodiments, the present disclosure provides the use of rAAV for the preparation of a pharmacopoeia for the treatment of limb-girdle muscular dystrophy, the pharmacopoeia being formulated for administration by intravenous infusion, and the rAAV being approximately 5 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg ~ approx. 2×10 14 In a dose of vg / kg, rAAV contains the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1. For example, administration of the drug to a subject requiring drug administration results in an increase in alpha-sarcoglycan gene expression in the subject's cells compared to the level of alpha-sarcoglycan gene expression before rAAV administration.

[0074] In any of the disclosed uses, administration of the pharmacoglycan to a subject requiring the administration of the pharmacoglycan results in an increase in the level of alpha-sarcoglycan gene expression in the subject's cells compared to the level of alpha-sarcoglycan gene expression before administration of the pharmacoglycan, and / or administration of the pharmacoglycan to the subject results in a decrease in serum CK levels in the subject compared to serum CK levels before administration of the pharmacoglycan, and / or increased spontaneous movement and specific force generation, reduced fibrosis, increased resistance to contraction-induced injury of the tibialis anterior muscle, and / or administration of the pharmacoglycan to the subject results in an increase in the number of alpha-sarcoglycan-positive fibers in the subject's muscle tissue compared to the number of alpha-sarcoglycan-positive fibers before administration of the pharmacoglycan, and / or administration of the pharmacoglycan to a subject requiring the administration of the pharmacoglycan results in reduced fibrosis in the subject compared to before administration of the pharmacoglycan, and / or increased specific force, fiber diameter size, and / or eccentric contraction in the subject's muscles compared to before administration of the pharmacoglycan. In some embodiments, alpha-sarcoglycan gene expression is detected by measuring alpha-sarcoglycan protein levels by Western blotting and / or immunohistochemistry.

[0075] In another embodiment, the Disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmaceutical for doing so in a subject requiring the expression of an alpha-sarcoglycan gene in cells. For example, the Disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical for doing so in a subject requiring the expression of an alpha-sarcoglycan gene in cells, the scAAVrh74.tMCK.hSGCA construct comprising the nucleotide sequence of SEQ ID NO: 1. In addition, in any of the uses, the expression of the alpha-sarcoglycan gene in the cells of interest is detected by measuring the alpha-sarcoglycan protein level by Western blotting in a muscle biopsy. Alternatively, in any of the uses, the expression of the alpha-sarcoglycan gene in cells is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in a muscle biopsy. In other embodiments, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0076] This disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmacopoeia to achieve a reduction in serum CK levels in subjects requiring such reduction. For example, this disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmacopoeia to achieve a reduction in serum CK levels in subjects requiring such reduction, the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 1.

[0077] In another aspect, the Disclosure provides the use of any of the disclosed rAAVs for the preparation of a pharmacopoeia to increase alpha-sarcoglycan-positive fibers in a muscle tissue of interest. For example, the Disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmacopoeia to increase alpha-sarcoglycan-positive fibers in a muscle tissue of interest, the scAAVrh74.tMCK.hSGCA construct comprising the nucleotide sequence of SEQ ID NO: 1.

[0078] This disclosure also provides the use of any of the disclosed rAAVs for the preparation of a pharmaceutical to do so in subjects requiring increased alpha-sarcoglycan expression. For example, this disclosure provides the use of the scAAVrh74.tMCK.hSGCA construct for the preparation of a pharmaceutical to do so in subjects requiring increased alpha-sarcoglycan expression, the scAAVrh74.tMCK.hSGCA construct comprises the nucleotide sequence of SEQ ID NO: 1. In addition, in any of the disclosed uses, alpha-sarcoglycan gene expression in cells of interest is detected by measuring alpha-sarcoglycan protein levels by Western blotting in muscle biopsy. Alternatively, in any of the disclosed uses, alpha-sarcoglycan gene expression in cells is detected by measuring alpha-sarcoglycan protein levels by immunohistochemistry in muscle biopsy. In other embodiments, alpha-sarcoglycan gene expression is measured in subjects by detecting the number of vector genomes per microgram of genomic DNA.

[0079] In any of the disclosed methods, compositions, or uses, the subjects are human subjects aged 4 to 15 years, or human subjects aged 25 to 55 years, or human subjects over 50 years.

[0080] In any of the disclosed methods, compositions, or uses, the subjects are children, adolescents, or young adults. Alternatively, the subjects are middle-aged adults or elderly.

[0081] For example, in any of the disclosed methods, compositions, or uses, the subjects are human subjects aged 4 to 15 years, having alpha-sarcoglycan (SGCA) mutations confirmed in both alleles, being negative for AAVrh74 antibody, and / or having undergone a 100-meter walking test of more than 40% or the usual rate.

[0082] In another embodiment, a method for generating rAAV as disclosed herein is provided, comprising transferring a plasmid into a cell, wherein the plasmid comprises a nucleotide sequence that is at least 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1. In particular, the plasmid comprises the nucleotide sequence of SEQ ID NO: 1.

[0083] In any of the disclosed methods for generating rAAV, the method further comprises transferring a packaging plasmid and / or a helper virus into a host cell. In addition, in any of the disclosed methods for generating rAAV, the packaging cell comprises a stably integrated AAV cap gene and / or the packaging cell comprises a stably integrated AAV rep gene.

[0084] In another embodiment, the disclosure provides a host cell comprising an AAV vector plasmid containing a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the host cell comprises an AAV vector plasmid containing the nucleotide sequence of SEQ ID NO: 1.

[0085] A method for producing recombinant AAV vector particles is also provided, comprising culturing cells transfected with a plasmid described herein and recovering recombinant AAV particles from the supernatant of the transfected cells. Viral particles comprising any of the recombinant AAV vectors described herein are also contemplated. In one embodiment, a method for producing rAAV comprises transferring an AAV vector plasmid to a host cell. In another embodiment, the plasmid comprises a nucleotide sequence that is at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1. In another aspect, the disclosure provides cells comprising an AAV vector plasmid comprising the nucleotide sequence of SEQ ID NO: 1. The cells described herein may be insect cells, e.g., Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells), or mammalian cells (preferably human cells, e.g., primary human cells or established cell lines). In one embodiment, the mammalian cells are 293 cells, COS cells, HeLa cells, or KB cells.

[0086] In another embodiment, the plasmid contains a nucleotide sequence that is at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1. In some embodiments, the vector plasmid contains any one of the nucleotide sequences in SEQ ID NO: 1. In some embodiments, the AAV vector plasmid is stably expressed in host cells. Host cells stably harboring the AAV vector plasmid can be used to generate rAAV.

[0087] A method for producing recombinant AAV vector particles provided herein may further include the step of transferring a packaging plasmid and / or a helper virus into a host cell. For example, the method further includes the step of the packaging cell containing a stably incorporated AAVcap gene, and / or the packaging cell containing a stably incorporated AAVrep gene. The present invention also provides cells containing a plasmid containing a nucleotide sequence that is at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1, or a plasmid containing the nucleotide sequence of SEQ ID NO: 1. Cells containing the nucleotide sequence of SEQ ID NO: 1 are also provided.

[0088] Methods for alleviating fibrosis in subjects requiring it are also provided. In this regard, the method comprises administering a therapeutically effective dose of the AAV vector described herein (or a composition comprising the rAAV vector described herein) to a mammalian subject. In some embodiments, the subject suffers from muscular dystrophy. In some embodiments, administration of the rAAV vector described herein (or a composition comprising the rAAV vector described herein) alleviates fibrosis in the skeletal or cardiac muscle of the subject.

[0089] As used herein, the term “muscular dystrophy” refers to a disorder characterized by a gradual decline in strength and muscle mass. Non-exclusive examples of muscular dystrophy include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, sarcoglycan disorders, congenital muscular dystrophy such as congenital muscular dystrophy due to partial LAMA2 deficiency, merosin-deficient congenital muscular dystrophy, type 1D congenital muscular dystrophy, Fukuyama congenital muscular dystrophy, limb-girdle type 1A muscular dystrophy, limb-girdle type 2A muscular dystrophy, limb-girdle type 2B muscular dystrophy, and limb-girdle type 2C muscular dystrophy. Examples of muscular dystrophy include dystrophy, limb-girdle type 2D muscular dystrophy, limb-girdle type 2E muscular dystrophy, limb-girdle type 2F muscular dystrophy, limb-girdle type 2G muscular dystrophy, limb-girdle type 2H muscular dystrophy, limb-girdle type 2I muscular dystrophy, limb-girdle type 2I muscular dystrophy, limb-girdle type 2J muscular dystrophy, limb-girdle type 2K muscular dystrophy, limb-girdle type IC muscular dystrophy, ankylosing vertebral muscular dystrophy with simple epidermolysis bullosa, oculopharyngeal muscular dystrophy, Ulrich type congenital muscular dystrophy, and Ulrich type scleroatnik muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy. In some embodiments, the subject suffers from limb-girdle muscular dystrophy type 2D (LGMD2D).

[0090] As used herein, the term “fibrosis” refers to the excessive or uncontrolled deposition of extracellular matrix (ECM) components and abnormal repair processes in post-injury tissues, including skeletal muscle, cardiac muscle, liver, lungs, kidneys, and pancreas. The deposited ECM components include collagen (e.g., collagen 1, collagen 2, or collagen 3) and fibronectin.

[0091] In another embodiment, a method for increasing muscle strength and / or muscle mass in a mammalian subject is described herein, comprising administering a therapeutically effective amount of the AAV vector described herein (or a composition comprising the AAV vector described herein) to the mammalian subject. In one embodiment, the subject is a human.

[0092] The provided formulation or composition contains one or more buffering agents from among Tris, Trisine, Bis-Trisine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. For example, the buffering agent may contain Tris at a concentration of about 5 mM to about 40 mM and a pH of 8.0, or the buffering agent may contain Tris at about 20 mM and a pH of 8.0.

[0093] In any of the provided formulations or compositions, the ionic strengthening agent comprises one or more of the following: potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. For example, the ionic strengthening agent may contain MgCl2 at a concentration of about 0.2 mM to about 4 mM, or the ionic strengthening agent may contain NaCl at a concentration of about 50 mM to about 500 mM, or the ionic strengthening agent may contain MgCl2 at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM, or the ionic strengthening agent may contain MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM.

[0094] In any of the provided formulations or compositions, the surfactant comprises one or more of the following: sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. For example, the poloxamer comprises one or more of the following: poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. The poloxamer may be present in a concentration of about 0.00001% to about 1%. An example surfactant is poloxamer 188 at a concentration of about 0.001%.

[0095] The preceding paragraphs are not intended to define all aspects of the Invention, and additional aspects are described in other sections, such as the detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein should be considered even if no combination of features is found together in the same sentence, paragraph, or section of this document. The Invention includes, as additional aspects, all embodiments of the Invention that are somewhat narrower in scope than the variations defined in the particular paragraphs above. For example, if a particular aspect of the Invention is described as a genus, each member of the genus should be understood to be an aspect of the Invention individually. In certain embodiments, for example, the following items are provided: (Item 1) A polynucleotide sequence containing a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1. (Item 2) The nucleotide sequence is a polynucleotide sequence according to any one of the preceding items, wherein the nucleotide sequence includes a nucleotide sequence or Sequence ID No. 1. (Item 3) Recombinant AAV (rAAV) comprising a polynucleotide sequence, wherein the polynucleotide sequence comprises i) two complementary nucleotide sequences each encoding the amino acid sequence of SEQ ID NO: 3, and ii) two complementary polyadenylated sequences. (Item 4) The recombinant AAV according to any one of the preceding items, wherein each of the two complementary nucleotide sequences is operably linked to a muscle-specific regulatory element, and the two muscle-specific regulatory elements are complementary to each other. (Item 5) The recombinant AAV described in any one of the preceding items, wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor (MEF) element, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, an MHCK7 promoter, a C5-12 promoter, a mouse creatine kinase enhancer element, a skeletal fast-twitch muscle troponin C gene element, a slow-twitch muscle cardiac troponin C gene element, a slow-twitch muscle troponin I gene element, a hypoxia-induced nuclear factor-binding element, a steroid-induced element, or a glucocorticoid response element (GRE). (Item 6) The recombinant AAV described in any one of the preceding items, wherein the muscle-specific regulatory element is a shortened MCK (tMCK). (Item 7) Recombinant AAV as described in any one of the preceding items, further comprising two complementary chimerintrons. (Item 8) A recombinant AAV according to any one of the preceding items, further comprising three reverse terminal repeats (ITRs), one of which is adjacent to the two complementary muscle-specific control elements. (Item 9) A recombinant AAV as described in any one of the preceding items, comprising the nucleotide sequence of SEQ ID NO: 1. (Item 10) The recombinant AAV described in any one of the preceding items, wherein the vector is serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh.74, or a synthetic AAV serotype. (Item 11) A composition comprising recombinant AAV as described in any one of the preceding items. (Item 12) A method for treating muscular dystrophy in a subject requiring treatment for muscular dystrophy, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 13) A method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 14) A method for alleviating fibrosis in a subject suffering from muscular dystrophy, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 15) A method for reducing contraction-induced injury in a subject suffering from muscular dystrophy, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 16) A method for treating alpha-sarcoglycan disorder in a subject, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 17) A method for increasing alpha-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, comprising administering to the subject a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items. (Item 18) The method according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of positive alpha-sarcoglycan-positive fibers is detected by measuring the alpha-sarcoglycan protein level by Western blotting in muscle biopsies before and after rAAV administration. (Item 19) The method according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of alpha-sarcoglycan-positive muscle fibers is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of rAAV. (Item 20) The method described in any one of the preceding items, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 21) The method according to any one of the preceding items, wherein the recombinant AAV or the composition is administered by intramuscular injection or intravenous injection. (Item 22) The method according to any one of the preceding items, wherein the recombinant AAV or the composition is administered systemically. (Item 23) The method according to any one of the preceding items, wherein the recombinant AAV or the composition is administered parenterally by injection, infusion, or transplantation. (Item 24) The recombinant AAV, based on superhelical DNA or linear plasmid as a quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 5.0×10 15 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 25) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 26) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 27) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 28) The rAAV is administered via a systemic route, and based on the linearized DNA or plasmid used as the quantitative standard, approximately 1.85 × 10⁻⁶ units are obtained. 13 vg / kg ~ approx. 7.41×10 13 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 29) A composition for treating muscular dystrophy in mammals, comprising recombinant AAV as described in any one of the preceding items or a composition as described in any one of the preceding items. (Item 30) A composition comprising a recombinant AAV or a composition described in any one of the preceding items, for increasing muscle strength and / or muscle mass in mammals suffering from muscular dystrophy. (Item 31) A composition comprising a recombinant AAV described in any one of the preceding items or a composition described in any one of the preceding items, for the purpose of alleviating fibrosis in mammals suffering from muscular dystrophy. (Item 32) A composition comprising a recombinant AAV or a composition described in any one of the preceding items, for reducing contraction-induced injury in subjects suffering from muscular dystrophy. (Item 33) A composition comprising recombinant AAV as described in any one of the preceding items or a composition as described in any one of the preceding items, for the treatment of β-sarcoglycan disorders in mammals requiring treatment. (Item 34) A composition comprising recombinant AAV as described in any one of the preceding items or a composition as described in any one of the preceding items for increasing alpha-sarcoglycan-positive fibers and / or decreasing CK levels in target muscle tissue. (Item 35) The composition according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of positive alpha-sarcoglycan-positive fibers is detected by measuring the alpha-sarcoglycan protein level by Western blotting in muscle biopsies before and after administration of the rAAV. (Item 36) The composition according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of alpha-sarcoglycan-positive muscle fibers is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 37) The composition according to any one of the preceding items, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 38) A composition according to any one of the preceding items, formulated for intramuscular or intravenous injection. (Item 39) A composition described in any one of the preceding items, formulated for systemic administration. (Item 40) The composition according to any one of the preceding items, wherein the systemic administration is parenteral administration by injection, infusion, or transplantation. (Item 41) The recombinant AAV, based on superhelical DNA or linear plasmid as a quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 5.0×10 15A composition according to any one of the preceding items, administered at a dosage of vg / kg. (Item 42) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 A composition according to any one of the preceding items, administered in a dose of vg / kg. (Item 43) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 A composition according to any one of the preceding items, administered in a dose of vg / kg. (Item 44) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 A composition according to any one of the preceding items, administered in a dose of vg / kg. (Item 45) The rAAV is administered via a systemic route, and based on the linearized DNA or plasmid used as the quantitative standard, approximately 1.85 × 10⁻⁶ units are obtained. 13 vg / kg ~ approx. 7.41×10 13 A composition according to any one of the preceding items, administered in a dose of vg / kg. (Item 46) Use of recombinant AAV as described in any one of the preceding items or a composition as described in any one of the preceding items for the preparation of a medicament for the treatment of muscular dystrophy. (Item 47) Use of a recombinant AAV vector or a composition described in any one of the preceding items for the preparation of a pharmacopoeia for increasing muscle strength and / or muscle mass in mammals suffering from muscular dystrophy. (Item 48) Use of a recombinant AAV vector or a composition described in any one of the preceding items for the preparation of a pharmaceutical product for reducing fibrosis in mammals suffering from muscular dystrophy. (Item 49) Use of a recombinant AAV vector or a composition described in any one of the preceding items for the preparation of a pharmacopoeia for reducing contraction-induced injury in subjects suffering from muscular dystrophy. (Item 50) Use of recombinant AAV or a composition described in any one of the preceding items for the preparation of a pharmaceutical product for treating β-sarcoglycan disorders in mammals requiring treatment for β-sarcoglycan disorders. (Item 51) Use of recombinant AAV or a composition described in any one of the preceding items for the preparation of a pharmacopoeia to increase alpha-sarcoglycan-positive fibers and / or decrease CK levels in target muscle tissue. (Item 52) The use according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of positive alpha-sarcoglycan-positive fibers is detected by measuring the alpha-sarcoglycan protein level by Western blotting in muscle biopsies before and after administration of rAAV. (Item 53) The use according to any one of the preceding items, wherein the expression of the alpha-sarcoglycan gene or the number of alpha-sarcoglycan-positive muscle fibers is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of rAAV. (Item 54) The use described in any one of the preceding items, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 55) The use of the pharmaceutical product as described in any one of the preceding items, wherein the pharmaceutical product is formulated for intramuscular or intravenous injection. (Item 56) The use of the aforementioned pharmaceutical product as described in any one of the preceding items, wherein the pharmaceutical product is formulated for systemic administration. (Item 57) The use described in any one of the preceding items, wherein the systemic administration is parenteral administration by injection, infusion, or transplantation. (Item 58) The recombinant AAV, based on the superhelical DNA or linear plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 5.0×10 15 Use as described in any one of the preceding items, administered at a dose of vg / kg. (Item 59) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, yields approximately 1.0 × 10⁻⁶ units. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 2.0×10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5 × 10 13 vg / kg ~ approx. 2×10 14 Use as described in any one of the preceding items, administered at a dose of vg / kg. (Item 60) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg ~ approx. 2×10 14 Use as described in any one of the preceding items, administered at a dose of vg / kg. (Item 61) The rAAV, based on the superhelical DNA or plasmid used as the quantitative standard, is approximately 5 × 10⁻⁶. 13 vg / kg, approximately 1×10 14 vg / kg, or approximately 2 × 10⁻⁶ 14 Use as described in any one of the preceding items, administered at a dose of vg / kg. (Item 62) The rAAV is administered via a systemic route, and based on the linearized DNA or plasmid used as the quantitative standard, approximately 1.85 × 10⁻⁶ units are obtained. 13 vg / kg ~ approx. 7.41×10 13 The method described in any one of the preceding items, administered at a dose of vg / kg. (Item 63) A recombinant AAV (rAAV) vector comprising a polynucleotide sequence, wherein the polynucleotide sequence is arranged in the 5' to 3' direction, (1) Complementary sequences of polyadenylated sequences, (2) The complementary sequence of the target gene, (3) Complementary arrangement of introns, (4) Complementary sequence of promoters, (5) 5'ITR sequence and, (6) The promoter and, (7) The intron and, (8) The target gene and, (9) The polyadenylated sequence and, A recombinant AAV vector wherein the polynucleotide sequence is flanked by two 3'ITR sequences, and the two 3'ITR sequences are complementary to each other. (Item 64) An rAAV vector according to any one of the preceding items, wherein the target gene includes the human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, human ANO5, and calpain-3 (Cap3) genes. (Item 65) The rAAV vector according to any one of the preceding items, wherein the promoter is a muscle-specific regulatory element, and the muscle-specific regulatory element includes a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor (MEF) element, a muscle creatine kinase (MCK) element, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, an MHCK7 promoter, a C5-12 promoter, a mouse creatine kinase enhancer element, a skeletal fast-twitch muscle troponin C gene element, a slow-twitch muscle cardiac troponin C gene element, a slow-twitch muscle troponin I gene element, a hypoxia-induced nuclear factor-binding element, or a steroid-induced element, or a glucocorticoid response element (GRE). (Summary) Methods for treating muscular dystrophy, including administration of a self-complementary recombinant AAV (rAAV) scAAVrh74.tMCK.hSGCA vector, methods for expressing the alpha-sarcoglycan gene in a patient, pharmaceutical compositions containing rAAV, and methods for producing rAAV are described herein. [Brief explanation of the drawing]

[0096] [Figure 1] This provides a schematic diagram of the scAAVrh74.tMCK.hSGCA therapeutic alpha-sarcoglycan transgene cassette. It is a self-complementary AAV vector containing the codon-optimized human alpha-sarcoglycan gene (hSGCA). A muscle-specific tMCK promoter drives expression. The cassette also includes a chimeric intron to enhance processing and polyadenylation signals for stability.

[0097] [Figure 2-1] This provides the annotated nucleotide sequence of scAAVrh74.tMCK.hSGCA. [Figure 2-2] This provides the annotated nucleotide sequence of scAAVrh74.tMCK.hSGCA. [Figure 2-3] This provides the annotated nucleotide sequence of scAAVrh74.tMCK.hSGCA. [Figure 2-4] This provides the annotated nucleotide sequence of scAAVrh74.tMCK.hSGCA. [Modes for carrying out the invention]

[0098] The implementation of this invention will, unless otherwise indicated, utilize conventional methods of virology, microbiology, molecular biology, and recombinant DNA technology, within the scope of the art of those skilled in the art. Such techniques are fully described in the literature. For example, Sambrook et al.Molecular Cloning: A Laboratory Manual (Current Edition), DNA Cloning: A Practical Approach, Vol. I&II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., Current Edition), Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition), Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition), CRC Handbook of Parvoviruses, vol. I&II (P. Tijssen, ed.), Fundamental Virology, 2nd Edition, vol. I & II (BNFields and DMKnipe, eds.), Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley-Liss, Third Edition), and Ausubel et al. al. (1991) Current Protocols in Molecular Biology (Wiley Interscience, NY).

[0099] definition The singular forms "a," "an," and "the" include plural referents unless otherwise specified in the context. For example, a reference to "cells" includes multiple such cells, and a reference to "culture" includes one or more cultures and their equivalents as known to those skilled in the art. A reference to "recombinant AAV" includes a mixture of two or more rAAV virions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0100] The use of the term “or” in the claims is used to mean “and / or” unless expressly indicated to mean only the alternatives, or unless the alternatives are mutually exclusive; however, this disclosure supports the definition that refers only to the alternatives and “and / or.”

[0101] Throughout this application, the term “approximately” is used to indicate that the value includes the statistical experimental error (standard deviation of the error) to the device or method used to determine the value.

[0102] The term "vector" refers to any genetic element that can replicate when associated with appropriate regulatory elements and can transfer gene sequences between cells, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions. In one embodiment, the vector is a viral vector.

[0103] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells where certain functions are provided by co-infecting helper viruses. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165–174 of Parvoviruses and Human Disease, JRPattison, ed., and Rose, Comprehensive Virology 3: 1–61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all possessing three related capsid proteins, including one expressed in AAV2. The degree of relevance is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the terminals corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0104] As used herein, “AAV vector” refers to one or more target polynucleotides (or transgenes) adjacent to an AAV terminal repeat sequence (ITR). Such an AAV vector can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing rep and cap gene products. In one embodiment, the AAV vector is a vector derived from adeno-associated virus serotypes, 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 AAVrh.74. The AAV vector is preferably a rep and / or cap gene, in which one or more of the AAV wild-type genes are deleted in whole or in part, but which can retain a functional adjacent ITR sequence. Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Therefore, AAV vectors are defined herein to contain at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). ITRs do not need to be wild-type nucleotide sequences and can be modified, for example, by nucleotide insertions, deletions, or substitutions, as long as the sequence provides functional rescue, replication, and packaging.

[0105] The term "AAV helper function" refers to AAV-derived coding sequences that can be expressed to provide the AAV gene product that then functions in trans for productive AAV replication. Thus, AAV helper functions include the major AAV open reading frame (ORF), rep, and cap. Rep expression products have been shown to possess many functions, including, among others, recognition, binding, and nicking of AAV origins in DNA replication, DNA helicase activity, and regulation of transcription from AAV (or other heterologous) promoters. Cap expression products provide the necessary packaging functions. AAV helper functions are used herein to complement the trans AAV functions lost from AAV vectors.

[0106] A "recombinant virus" refers to a virus that has been genetically modified, for example, by adding or inserting a different nucleic acid sequence into the viral particle.

[0107] "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a virus particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed in the capsid. In one embodiment, the AAV virion contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell). In some embodiments, the production of an AAV virus particle involves the production of an AAV vector, for example, the vector being contained within the AAV vector particle.

[0108] AAV genomes, such as transgenes delivered to mammalian cells, are typically referred to as "AAV vector particles" or simply "AAV vectors." Therefore, since such vectors are contained within AAV vector particles, the production of AAV vector particles inevitably involves the production of AAV vectors.

[0109] For example, wild-type (wt) AAV virus particles contain a linear single-stranded AAV nucleic acid genome associated with the AAV capsid protein coat. AAV virions can be either single-stranded (ss) AAV or self-complementary (SC) AAV. In one embodiment, a single-stranded AAV nucleic acid molecule, either a complementary sense, e.g., a "sense" or "antisense" strand, may be packaged in an AAV virion, and both strands are equally infectious.

[0110] The terms “recombinant AAV” or “rAAV” are defined herein as infectious replication-deficient viruses comprising an AAV protein shell in which an AAV ITR encapsulates a heterologous nucleotide sequence of interest flanked on both sides. In one embodiment, rAAV is produced in a suitable host cell, which has an AAV vector introduced therein, AAV helper function, and accessory function. Thus, the host cell can encode an AAV polypeptide required to package the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0111] The term "transfection" refers to the uptake of foreign DNA by cells, and cells are "transfected" when exogenous DNA is introduced into the cell membrane. Many transfection techniques are commonly known in this field. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous DNA portions, such as nucleotide insertion vectors and other nucleic acid molecules, can be introduced into suitable host cells.

[0112] The term “host cell” means, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of AAV helper constructs, AAV vector plasmids, accessory functional vectors, or other transfer DNA. This term includes offspring of the transfected original cell. Thus, as used herein, “host cell” generally refers to a cell transfected with an exogenous DNA sequence. It is understood that offspring of a single parent cell may not necessarily be completely identical in morphology or genomic or whole DNA complement due to natural, accidental, or intentional mutations.

[0113] The term "transduction" is used to refer to the administration / delivery of a target polynucleotide (e.g., a polynucleotide sequence encoding β-sarcoglycan) to recipient cells either in vivo or in vitro via the described replication-deficient rAAV, resulting in the expression of β-sarcoglycan by the recipient cells.

[0114] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived from the digestive tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, including myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac muscle cells.

[0115] The term "heterogeneous," when relating to nucleic acid sequences such as coding and regulatory sequences, refers to sequences that are not normally bound together and / or are not normally associated with a particular cell. Therefore, the "heterogeneous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in nature in association with other molecules. For example, the heterogeneous region of a nucleic acid construct may include a coding sequence adjacent to a coding sequence that is not found in association with a naturally occurring coding sequence. Another example of a heterogeneous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from those in a native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterogeneous for the purposes of this invention. As used herein, allelic mutations or naturally occurring mutational events do not produce heterogeneous DNA.

[0116] A "coding sequence," or sequence that "codes" a particular protein, is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into a polypeptide (in the case of DNA) and translated (in the case of mRNA) in vitro or in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxy) end. Coding sequences may include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence will typically be located on the 3' side of the coding sequence.

[0117] Nucleic acid sequences refer to DNA or RNA sequences. Nucleic acids include 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseuduracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, and 5-methylaminomethyluracil. DNA and RNA base analogs include, but are not limited to, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosin, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosin, 2-thiocytosine, and 2,6-diaminopurines.

[0118] The term DNA “regulatory sequences” collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRESs"), enhancers, etc., which collectively provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these regulatory sequences are always necessary, as long as the selected coding sequence can be replicated, transcribed, and translated in a suitable host cell.

[0119] The term “promoter” is used herein in its usual sense to refer to a nucleotide region containing a DNA regulatory sequence, the regulatory sequence being derived from a gene that can bind to RNA polymerase and initiate transcription of a downstream (3'-direction) coding sequence. Transcription promoters may include “inducible promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.” In one embodiment, the promoter is a muscle-specific promoter, which includes, but is not limited to, 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 α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch muscle troponin c gene element, slow-twitch muscle cardiac troponin c gene element, slow-twitch muscle 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.

[0120] The term "operably linked" refers to the arrangement of elements configured so that the components described in this way perform their normal functions. Thus, control sequences operably linked to a coding sequence can influence the expression of the coding sequence. Control sequences do not need to be adjacent to the coding sequence as long as they function to direct its expression. For example, an intervening untranslated but transcribed sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0121] When RNA polymerase binds to a promoter sequence, transcribes the coding sequence into mRNA, and then translates it into the polypeptide encoded by the coding sequence, the promoter "directs the transcription" of the coding sequence within the cell.

[0122] An “expression cassette” or “expression construct” refers to an assembly that can direct the expression of a sequence or gene of interest. An expression cassette, as described above, includes regulatory elements such as a promoter that is operablely linked to the sequence or gene of interest (to direct transcription), and often also includes a polyadenylated sequence. In certain embodiments of the present invention, the expression cassettes described herein may be contained within a plasmid construct. In addition to the components of an expression cassette, a plasmid construct may also include one or more selectable markers, signals that enable the plasmid construct to exist as single-stranded DNA, at least one multicloning site, and a “mammalian” origin of replication (e.g., SV40 or an adenovirus origin of replication).

[0123] When referring to a nucleotide sequence, "isolated" means that the indicated molecule exists in the substantial absence of other nucleotide sequences, chromatin material, or other biological macromolecules. Therefore, an "isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule that substantially contains no other nucleic acid molecules that do not encode the polypeptide in question, however, the molecule may contain some additional bases or parts that do not adversely affect the fundamental properties of the composition.

[0124] When a particular nucleotide sequence is described as being located "upstream," "downstream," "3'," or "5'" relative to another sequence, for the purpose of describing the relative position of a nucleotide sequence within a particular nucleic acid molecule throughout this application, it should be understood that this refers to the position of the sequence in the "sense" or "coding" strand of the DNA molecule, as is customary in the art.

[0125] In the context of nucleic acid sequences or amino acid sequences, the terms “sequence identity,” “percentage of sequence identity,” or “percentage of identity” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison can be the full length of the genome, the full length of the gene coding sequence, or preferably a fragment of at least about 500–5000 nucleotides. However, identity between smaller fragments, such as at least about 9 nucleotides, usually at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 or more nucleotides, may also be desired. The percentage of sequence identity can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information of two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as an alignment tool, the following default parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; prediction = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swissprotein+Spupdate+PIR.

[0126] The term “subject” refers to any member of the animal kingdom, including, but not limited to, humans and non-human primates such as chimpanzees and other apes and monkey species, domesticated animals such as cattle, sheep, pigs, goats and horses, domesticated mammals such as dogs and cats, and experimental animals such as rodents such as mice, rats and guinea pigs. In some embodiments, the subject is a human being in the range of birth to 2 years, 1 to 10 years, or 4 to 15 years, or 10 to 19 years, or 20 to 40 years, or 15 to 29 years or 25 to 55 years, or 40 to 60 years, or 50 years or older, or 60 years or older, or 65 years or older, or 70 years or older.

[0127] AAV Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains a 145-nucleotide terminal inversion (ITR). Multiple serotypes of AAV exist. The nucleotide sequences of the AAV serotype genomes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), modified by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is available under GenBank acceptance number NC_002077, the complete genome of AAV-3 is available under GenBank acceptance number NC_1829, the complete genome of AAV-4 is available under GenBank acceptance number NC_001829, the genome of AAV-5 is available under GenBank acceptance number AF085716, the complete genome of AAV-6 is available under GenBank acceptance number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are available under GenBank acceptance numbers AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 for AAV-8), and the genome of AAV-9 is available under GenBank acceptance number AX753246 and AX753249, respectively. The AAV-10 genome is available in al., J. Virol., 78:6381-6388 (2004), presented in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is available in Virology, 330(2):375-383 (2004). Cloning of AAVrh.74 serotypes is described in Rodino-Klapac., et al. Journal of Translational Medicine 5, 45 (2007). Cis action sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 of AAV2), two rep promoters (p5 and p19) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites contribute to the production of these three related capsid proteins. A single-consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0128] AAV possesses unique characteristics that make it attractive, for example, as a vector for delivering foreign DNA to cells in gene therapy. AAV infection of cells in culture is non-cellular, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells and allows for the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genomic capsid formation, and integration are contained within the ITR of the AAV genome, some or all of the approximately 4.3 kb inside the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing the promoter, the DNA of interest, and polyadenylation signals. The rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. This means it readily withstands the conditions used to inactivate adenoviruses (56°C–65°C for several hours), reducing the importance of chilling AAV. AAV can be freeze-dried. Finally, AAV-infected cells do not show resistance to co-infection.

[0129] Multiple studies have demonstrated long-term (over 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997), Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). Also see Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscles are highly angiogenic, recombinant AAV transduction resulted in the emergence of the transgene product in systemic circulation following intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, showing that muscles are capable of stable expression of secreted protein therapeutics.

[0130] The recombinant AAV genomes of this disclosure include the nucleic acid molecule and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA of the rAAV genome may be derived from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV serotypes AAVrh.74, 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, AAVrh.10, and AAVrh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background information section above, the nucleotide sequences of various AAV serotype genomes are known in the art. AAVrh.74 can be used to promote muscle-specific expression.

[0131] The DNA plasmid of this disclosure comprises the rAAV genome of this disclosure. The DNA plasmid is transferred to a cell tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function are provided to the cell to be packaged, are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAV rep and cap genes isolated from (i.e., not present in) the rAAV genome, and the helper virus function be present in a single cell (referred to herein as the packaging cell). The rep and cap genes of AAV may originate from any AAV serotype from which the recombinant virus may originate, and may originate from AAV serotypes different from the rAAV genome ITR, such as AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAVrh.10, AAVrh.74, and AAV-13, among others. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated in its entirety herein by reference.

[0132] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the rAAV genome, and selectable markers such as the neomycin resistance gene, is incorporated into the cell genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a preferred method is to use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0133] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Patent No. 5,173,414, WO95 / 13365, and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul et al. This is described in al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production.

[0134] Accordingly, this disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0135] The recombinant AAVs of this disclosure (i.e., infectious capsidized rAAV particles) comprise an rAAV genome. In exemplary embodiments, the genomes of both rAAVs lack the rep and cap DNA of AAV, i.e., there is no rep or cap DNA of AAV between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise the nucleic acid molecules of this disclosure are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), which is incorporated herein by reference in its entirety.

[0136] In exemplary embodiments, the recombinant AAV vector of this disclosure is produced by triple transfection using the AAV vector plasmid scAAV.tMCK.hSCGA, pNLRep2-Caprh74, and pHelp (Xiao et al., J Virol 72, 2224-2232 (1998)), where rAAV contains an hSCGA gene expression cassette flanked by an AAV2 reverse terminal repeat (ITR). This sequence is capsidated to AAVrh.74 virions. The plasmid contains the hSCGA sequence, as well as core promoter elements of the MHCK7 enhancer and muscle-specific promoter that drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) that promotes high levels of gene expression, and a bovine growth hormone polyadenylation signal is used for efficient transcription termination.

[0137] pNLREP2-Caprh74 is an AAV helper plasmid encoding four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins derived from serotype rh74.

[0138] The pHELP adenovirus helper plasmid is 11,635 bp long and was obtained from Applied Viromics. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid represent only about 40% of the adenovirus genome and do not include cis elements important for replication, such as the terminal repeat sequences of the adenovirus. Therefore, it is not expected that infectious adenoviruses will be produced from such a production system.

[0139] rAAV can be purified by methods standard in the art, such as by column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0140] In another embodiment, the Disclosure envisions a composition comprising rAAV of the Disclosure. The composition of the Disclosure comprises rAAV and a pharmaceutically acceptable carrier. The composition may also include other components such as diluents and adjuvants. The acceptable carrier, diluent, and adjuvant are nontoxic to the recipient, preferably inert at the adopted dosage and concentration, and include buffers and surfactants such as Pluronic®.

[0141] The titer of rAAV administered by the methods of the present disclosure can vary, for example, depending on the particular rAAV, mode of administration, treatment goal, targeted individual, and cell type, and can be determined by standard methods in the art. The titer of rAAV can range from about 1×106, about 1×107, about 1×108, about 1×109, about 1×1010, about 1×1011, about 1×1012, about 1×1013 to about 1×1014 or more DNase-resistant particles (DRP) per mL. The dosage may be expressed in units of viral genome (vg). One exemplary method for determining the titer of the encapsulated vector genome is to use quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25(4):855-869, 2017). Unless otherwise specified, the dosages described herein correspond to dosages determined by the supercoiled DNA standard.

[0142] Methods of transducing target cells with rAAV, either in vivo or in vitro, are contemplated by the present disclosure. The in vivo method includes administering a composition comprising an effective dose or effective multiple doses of the rAAV of the present disclosure to an animal (including humans) that needs it. When the dose is administered before the onset of the disorder / disease, the administration is prophylactic. When the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, the effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the severity of the disease, results in remission (partial or complete) of the disease, and / or extends survival. An example of a disease contemplated for prevention or treatment by the methods of the present disclosure is muscular dystrophy, such as limb-girdle muscular dystrophy or Duchenne muscular dystrophy.

[0143] Combination therapies are also contemplated by the present disclosure. The combinations used herein include both concurrent and sequential therapies. Combinations of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids), such as combinations with novel therapies, are particularly contemplated.

[0144] Administration of the compositions, combination therapies or formulations at effective dosages can be by standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV component of the rAAV of the present disclosure (specifically, AAV ITR and capsid protein) can be selected and / or adapted by those skilled in the art considering the infectious disease and / or disease state being treated, and the target cell / tissue expressing the hSCGA protein.

[0145] The present disclosure provides for local and systemic administration of effective dosages of the rAAV, formulations, and compositions of the present disclosure. For example, systemic administration is administration to the circulatory system such that the whole body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract, and parenteral administration through injection, infusion, or transplantation.

[0146] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method of transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, injection into muscle and injection into the bloodstream. It has been demonstrated that simply resuspending rAAV in phosphate buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). The capsid protein of rAAV may be modified such that the rAAV is targeted to a specific target tissue such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been developed previously and can be used in practicing the present disclosure. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0147] For intramuscular injection, adjuvant solutions such as sesame oil or peanut oil, aqueous propylene glycol solutions, and sterile aqueous solutions can be used. Such aqueous solutions can be buffered as needed, and the liquid diluent is first isotonicized with physiological saline or glucose. Solutions of rAAV as free acid (DNA contains acidic phosphate groups) or pharmacokinetically acceptable salts 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 mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0148] Pharmaceutical carriers, diluents, or excipients suitable for injection applications include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow for easy syringe use. The form must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. The prolonged absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0149] Sterile injectable solutions are prepared by combining the required amount of rAAV in a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by mixing the sterilized active ingredient with a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired components from those solutions that have been previously sterilized and filtered.

[0150] Transduction with rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are isolated from the target, transduced with rAAV, and reintroduced into the target. Alternatively, syngeneic or heterologous muscle cells may be used if those cells do not produce an inappropriate immune response in the target.

[0151] Suitable methods for transduction into a target and reintroduction of transduced cells are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in a suitable culture medium and screening cells with the desired DNA using conventional techniques such as Southern blotting and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into a target by various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle using a catheter, for example.

[0152] Transduction of cells with rAAVs of this disclosure results in sustained expression of the hSCGA protein. Therefore, this disclosure provides methods for administering / delivering rAAVs expressing the hSCGA protein to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of this disclosure. Transduction may be carried out with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present disclosure, though not limited to these, may be derived from the actin and myosin gene families, e.g., the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)), muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], human skeletal muscle actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], cardiac muscle actin gene, muscle creatine kinase sequence elements (Johnson et al. The present invention provides a method for transducing muscle cells and muscle tissue induced by muscle-specific regulatory elements, including regulatory elements derived from mouse creatine kinase enhancer (mCK) elements, regulatory elements derived from skeletal fast-twitch muscle troponin C gene, slow-twitch muscle cardiac troponin C gene, and slow-twitch muscle troponin I gene: hypoxia-induced nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), steroid-induced elements, and promoters containing glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), as well as other regulatory elements.

[0153] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. This disclosure aims to achieve sustained expression of hSCGA derived from transduced myofibrils.

[0154] Therefore, this disclosure provides a method for administering an effective dose (or doses administered essentially simultaneously or at intervals) of rAAV encoding hSCGA to a subject in need thereof.

[0155] The titer of rAAV administered by the method of the present invention varies depending, for example, on the specific rAAV, the method of administration, the treatment target, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per mL. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 The DNase-resistant particle (DRP) range may be from or above. The dosage may be expressed in units of viral genome (vg). The titer of rAAV may be determined by a superhelical plasmid quantification standard or a linearized plasmid quantification standard.

[0156] The present invention envisions a method for transducing target cells with rAAV in vivo or in vitro. The in vivo method comprises the step of administering an effective dose or multiple effective doses of a composition containing rAAV of the present invention to an animal (including humans) in need. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition to be treated, a dose that slows or prevents progression to the disorder / disease condition, a dose that reduces the scope of the disease, a dose that results in remission (partial or complete) of the disease, and / or a dose that prolongs survival. An example of a disease envisioned for prevention or treatment by the method of the present invention is muscular dystrophy, e.g., limb-girdle muscular dystrophy. Thus, a method for transducing target cells with rAAV scAAVrh74.tMCK.hSGCA, comprising the nucleotide sequence of SEQ ID NO: 1, is provided.

[0157] Combination therapies are also envisioned by the present invention. Combination therapies as used herein include concurrent or sequential treatments. Combinations of the methods of the present invention with standard medical treatments (e.g., steroids, corticosteroids, and / or glucocorticoids comprising one or more of prednisone, prednisolone, and deflazacort, but not limited to these) are specifically envisioned, as are combinations with novel therapies. In this view, these combinations include administering one or more steroids, corticosteroids, and / or glucocorticoids comprising one or more of prednisone, prednisolone, and deflazacort, to the target before administering the methods of the present invention to the target, simultaneously with or after administering the rAAV to the target.

[0158] In related embodiments of the combination therapy envisioned by the present invention, glucocorticoids include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, or triamcinolone.

[0159] Antigen-specific T cell responses are recognized as possible in subjects administered with rAAV vectors. This is a response expected 2–4 weeks after gene transfer. One possible consequence of such an antigen-specific T cell response is the clearance of transduced cells and loss of transgene expression. To attenuate the host immune response to rAAV-based therapy, subjects may be initiated with oral prophylactic prezonisone or an equivalent glucocorticoid at approximately 1 mg / kg / day, pre-treatment, for example, 24 hours before the treatment procedure, up to a maximum dose of 60 mg / day. If necessary, an equivalent glucocorticoid may be administered intravenously at a dose of approximately 1 mg / kg / day. Treatment lasts for approximately one month. Protocols for tapering the dose of prezonisone or an equivalent glucocorticoid can be implemented based on the individual subject's immune response to gene transfer and evaluated by ELISpot assays and liver function monitoring by GGT.

[0160] Provided is a method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, where rAAV is approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 15 It is administered using a dose of vg / kg. For example, in any of the methods provided, the dose of rAAV administered is approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×1013 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The value is vg / kg. In another embodiment, the dose is approximately 5.0 × 10 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10 14 The dosage is vg / kg. In one embodiment, rAAV is administered via a systemic route, including an intravenous route. In another embodiment, rAAV is administered at approximately 5.0 × 10⁻⁶ 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10 14 It is administered intravenously at a dose of vg / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy.

[0161] In addition, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 13 vg~approx.3.5×10 16 vg, or approximately 3 × 10 13 vg~approx. 1.0×10 16 vg, or approximately 1.5 × 10 13 vg ~ approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 15 The dose is in vg. In addition, in any of the methods, the dose of rAAV is administered at a concentration of approximately 10 mL / kg. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy. In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy type 2D. The doses in this disclosure are expressed in either vg or vg / kg and are based on titration methods by quantitative PCR (qPCR). qPCR-based titration methods are known in the art.

[0162] In addition, provided is a method for treating muscular dystrophy in subjects requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, where rAAV is administered via a systemic route at a dose of approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 2.0×1015 When administered at a dose of vg / kg, the level of alpha-sarcoglycan gene expression in the target cells increases after rAAV administration compared to the level of alpha-sarcoglycan gene expression before rAAV administration, the serum CK level in the target decreases after rAAV administration compared to the serum CK level before rAAV administration, and / or spontaneous movement and specific force generation increase, fibrosis is reduced, resistance to contraction-induced injury of the tibialis anterior muscle increases, and / or the number of alpha-sarcoglycan-positive fibers in the target muscle tissue increases after rAAV administration compared to the number of alpha-sarcoglycan-positive fibers before rAAV administration, the fiber diameter size in the target muscle tissue increases after rAAV administration compared to the number of fiber diameters before rAAV administration, or centronucleation in the target muscle tissue is reduced after rAAV administration compared to centronucleation before rAAV administration. Muscle tissue includes, but is not limited to, the triceps brachii, tibialis anterior, soleus, gastrocnemius, biceps brachii, trapezius, gluteus, psoas major, deltoid, quadriceps femoris, and diaphragm. In one embodiment, muscle tissue includes the tibialis anterior, gastrocnemius, gluteus, psoas major, and triceps brachii. Alpha-sarcoglycan expression is determined by methods known to those skilled in the art. In one embodiment, expression is determined by Western blotting, immunochemistry in muscle biopsy, and / or by detecting the number of vector genomes per microgram of genomic DNA.

[0163] In some embodiments, the disclosure includes a method for treating muscular dystrophy in a subject requiring treatment, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein motor function is significantly improved in the subject compared to the motor function of the subject before administration of rAAV.

[0164] A method is provided for increasing alpha-sarcoglycans in patients requiring such an increase, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 1 to the patient.

[0165] In any of the methods, uses, and compositions for treating muscular dystrophy provided, the subject is between 4 and 15 years old, has an alpha-sarcoglycan (SGCA) mutation identified in both alleles, is negative for the AAVrh74 antibody, and / or has a walking test of more than 40% or normal for 100 meters. In any of the methods, uses, and compositions for treating muscular dystrophy provided, the subject is a pediatric subject. In some embodiments, the subject is a pediatric subject, for example, a subject in the range of 1 to 21 years old. In some embodiments, the subject is 1 to 10 years old, or 2 to 12 years old, 4 to 15 years old, or 10 to 19 years old. In one embodiment, the subject is a young adult subject, for example, a subject in the range of 12 to 21 years old. Additionally, the subject is, in one embodiment, a young adult subject such as a subject in the age range of 15 to 29 years old or 18 to 39 years old. In some embodiments, the subject is a middle-aged adult or an elderly subject. As a result, the middle-aged adult may be in the range of 25 to 55 years old, adult subjects of older age may be in the range of over 50 years old, and elderly subjects may be in the range of over 65 years old. In some embodiments, rAAV is administered by injection, infusion, or transplantation. For example, rAAV is administered by infusion over approximately 1 to 2 hours. Additionally, rAAV is administered by an intravenous route via a peripheral limb vein.

[0166] A method of doing so in a subject that requires treatment for muscular dystrophy, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK.hSGCA, wherein rAAV is administered at a dose of about 1.0×10 12 vg / kg to about 5.0×10 14 vg / kg using a systemic administration route, and 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, rAAV comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0167] In one embodiment, 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, rAAV comprises a nucleotide sequence encoding a protein comprising the polypeptide sequence shown in SEQ ID NO: 2. In addition, any of the disclosed rAAVs further comprises a promoter such as the tMCK promoter sequence of SEQ ID NO: 7 and / or SEQ ID NO: 9. In some embodiments, rAAV is rAAV of serotype AAVrh.74. In one embodiment, rAAV comprises the 5' inverted terminal repeat sequence of SEQ ID NO: 8. In another embodiment, rAAV comprises the 3' inverted terminal repeat sequence of SEQ ID NO: 4 and / or SEQ ID NO: 11. In another embodiment, rAAV includes the polyA sequence of sequence number 5 and / or sequence number 10.

[0168] AAV dosage can be determined by several methods, including but not limited to LISA, reverse transcriptase activity assessment, FACS, transduction assays (Northern blotting, e.g., semi-quantitative Northern), dot blot analysis, or PCR (e.g., qPCR). It is well known that AAV dosage can be determined by measuring the AAV vector genome with quantitative real-time PCR (qPCR). Such qPCR methods overcome the inconsistent or arbitrary results from conventional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. Plasmid morphology may affect the dosage results by qPCR. In one embodiment, circular or superhelical DNA or plasmid is used as a quantitative standard. In another embodiment, linearized DNA or plasmid is used as a quantitative standard.

[0169] The terms “superhelical DNA” or “superhelical plasmid” refer to DNA or plasmids that do not contain free ends. The terms “linearized DNA” or “linearized plasmid” refer to DNA or plasmids that contain free 5' ends and free 3' ends that are not linked to each other. In one embodiment, linearized DNA or plasmids are obtained by restriction digestion of circular DNA (e.g., plasmid DNA) or by restriction digestion of dbDNA. In another embodiment, restriction digestion is performed using an enzyme that produces at least one blunt end.

[0170] In an exemplary embodiment, a method for doing so in a subject requiring treatment for muscular dystrophy includes the step of administering recombinant adeno-associated virus (rAAV) scAAVrh74.tMCK7.hSGCA, where rAAV is administered via a systemic route in a dose of approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 5.0×10 14 The dose was administered at vg / kg, and the human subjects suffered from limb-girdle muscular dystrophy. In one embodiment, rAAV was measured based on superhelical DNA or plasmid as a quantitative standard, approximately 5.0 × 10⁶ 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10 14 The dose is administered by intravenous infusion over approximately 1-2 hours at a dose of vg / kg, and rAAV contains the scAAVrh74.tMCK7.hSGCA construct nucleotide sequence of SEQ ID NO: 1. In another embodiment, the dose is approximately 1.85 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 10 13 It is vg / kg.

[0171] The disclosure also provides a method for increasing sarcoglycan expression in target muscle tissue, the method comprising administering to the target a scAAVrh74.tMCK.hSGCA construct containing a nucleotide sequence at least 90%, at least 95%, or 99% identical to SEQ ID NO: 1.

[0172] The disclosure also further provides a method for improving muscle function in a subject, the method comprising administering a construct to a subject that comprises a nucleotide sequence that is at least 90% identical, at least 95% identical, or 99% identical to SEQ ID NO: 1.

[0173] In some embodiments, the subjects suffer from a gene mutation in the gene encoding the sarcoglycan protein, or from muscular dystrophy. In some embodiments, the subjects suffer from a gene mutation in the gene encoding the alpha-sarcoglycan protein.

[0174] In any of the provided methods, the level of alpha-sarcoglycan gene expression in the target cells increases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the level of alpha-sarcoglycan gene expression before administration of the scAAVrh74.tMCK.hSGCA construct.

[0175] In addition, in any of the provided methods, the expression of the alpha-sarcoglycan gene in cells is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0176] In any of the provided methods, the level of alpha-sarcoglycan protein increases after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of alpha-sarcoglycan protein increases by at least 33% when detected by measuring the alpha-sarcoglycan protein level in Western blots of muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct, or the level of alpha-sarcoglycan protein is measured by detecting immunohistochemistry and / or vector genome count per microgram of genomic DNA in muscle biopsies taken before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0177] In any of the methods provided herein, serum CK levels in the subject decrease after administration of the scAAVrh74.tMCK.hSGCA construct compared to serum CK levels before administration of the scAAVrh74.tMCK.hSGCA construct.

[0178] In any of the methods provided herein, the number of alpha-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of the scAAVrh74.tMCK.hSGCA construct compared to the number of alpha-sarcoglycan-positive fibers before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of alpha-sarcoglycan-positive fibers is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. For example, the number of alpha-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of the scAAVrh74.tMCK.hSGCA construct.

[0179] In any of the methods provided herein, the level of alpha-sarcoglycan in the subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of the scAAVrh74.tMCK.hSGCA construct. For example, the level of alpha-sarcoglycan is detected by measuring the alpha-sarcoglycan protein level by immunohistochemistry or Western blotting in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct.

[0180] Another embodiment provides a method for expressing the alpha-sarcoglycan gene in a patient's cells, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 1 to the patient. In any of the provided methods for expressing the alpha-sarcoglycan gene in a patient's cells, the expression of the alpha-sarcoglycan gene in the patient's cells is detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the scAAVrh74.tMCK.hSGCA construct. In one embodiment, the alpha-sarcoglycan gene is measured in the patient by detecting more than one rAAV vector genome copy per nucleus. In another embodiment, the expression of the alpha-sarcoglycan gene is measured in the subject by detecting the number of vector genomes per microgram of genomic DNA.

[0181] A method is also provided for doing so in patients who require a reduction in serum CK levels, which involves administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of Sequence ID No. 1.

[0182] A method is provided for increasing alpha-sarcoglycan-positive fibers in a patient's muscle tissue, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1. In any of these methods, the number of alpha-sarcoglycan-positive fibers is detected by measuring alpha-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0183] Another embodiment provides a method for increasing alpha-sarcoglycan expression in subjects requiring it, comprising administering the scAAVrh74.tMCK.hSGCA construct nucleotide sequence of SEQ ID NO: 1 to the subject. In either of these methods, the level of alpha-sarcoglycan is detected by measuring the alpha-sarcoglycan protein level by Western blotting or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0184] The therapeutically effective dose of rAAV vector is approximately 1.0 × 10⁻⁶. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×10 13 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The dose of rAAV is in the range of vg / kg. In another embodiment, the dose is approximately 5.0 × 10 13 vg / kg, approximately 1.0×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 14 The value is vg / kg. In another embodiment, the dose is 5.0 × 10⁻⁶ 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10 14 The value is vg / kg. The present invention is also intended to include compositions comprising rAAV vectors within these ranges.

[0185] The dosage may be expressed in units of viral genome (vg). The titer of rAAV may be determined by a superhelical DNA or plasmid quantitative standard or a linearized DNA or plasmid quantitative standard. The titer or dosage of the AAV vector may vary based on the physical form of the plasmid or DNA as the quantitative standard. For example, the titer or dosage value may vary based on a superhelical standard qPCR titration method or a linearized standard qPCR titration method. In one embodiment, the dosage in this disclosure is based on superhelical DNA or plasmid as the quantitative standard. In another embodiment, the dosage in this disclosure is based on linearized DNA or plasmid as the quantitative standard. Thus, in one embodiment, the therapeutically effective dose of rAAV vector is about 1.0 × 10⁻¹⁶ based on superhelical DNA or plasmid as the quantitative standard. 12 vg / kg ~ approx. 2.0×10 15 vg / kg, approx. 5×10 12 vg / kg ~ approx. 1.0×10 15 vg / kg, approximately 1.0×10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, approx. 5×10 13 vg / kg ~ approx. 2×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 13 vg / kg ~ approx. 3.0×10 14 The dose of rAAV is in the range of vg / kg. In another embodiment, the dose is approximately 5.0 × 10⁶ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg, approximately 1.0×10 14 vg / kg, or approximately 2.0 × 10⁻⁶ 14 The dose is vg / kg. In another embodiment, the dose is 5.0 × 10⁻¹⁰ based on superhelical DNA or plasmid as a quantitative standard. 13 vg / kg, 1.0 × 10 14 vg / kg, or 2.0 × 10 14 It is vg / kg.

[0186] In another embodiment, the therapeutically effective dose of the rAAV vector is approximately 1.0 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.6×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.8×10 13 vg / kg ~ approx. 8.0×10 13 vg / kg, approximately 1.2×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, approximately 1.4×10 13 vg / kg ~ approx. 7.4×10 13 vg / kg, approximately 1.9×10 13 vg / kg ~ approx. 7.5×10 13 vg / kg, or approximately 1.8 × 10⁻⁶ 13 vg / kg ~ approx. 8.0×10 13 The dose of rAAV is in the range of vg / kg. For example, the therapeutically effective dose of rAAV vector is approximately 1.85 × 10⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 10 13 The dosage is in 1g / kg.

[0187] In one embodiment, 5.0 × 10⁶ units based on superhelical DNA or plasmid as a quantitative standard are used. 13 The dose of vg / kg is 1.85 × 10⁴ based on linearized DNA or plasmid as a quantitative standard. 13 This is equivalent to a dose of vg / kg. In another embodiment, 2.0 × 10 based on superhelical DNA or plasmid 14 The dose of vg / kg is 7.41 × 10⁴ based on linearized DNA or plasmid as a quantitative standard. 13 This is equivalent to vg / kg. Therefore, in another embodiment, approximately 1.85 × 10⁻¹⁶ based on linearized DNA or plasmid as a quantitative standard. 13 vg / kg or 7.41 × 10 13 It is vg / kg.

[0188] The effective dose of the composition may be administered via a standard route in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV component of the rAAV of the present invention (specifically, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state being treated, as well as the target cells / tissues expressing alpha-sarcoglycans.

[0189] The present invention provides topical and systemic administration of effective doses of the rAAV and composition of the present invention. For example, systemic administration means administration to the circulatory system so that the whole body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration by injection, infusion, or transplantation.

[0190] In particular, the actual administration of rAAV according to the present invention can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (although DNA-degrading compositions should be avoided in the usual manner with rAAV). The rAAV capsid protein may be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference.

[0191] The therapeutically effective dose of rAAV vector is approximately 1 e13 vg / kg to approximately 5 e14 vg / kg, or approximately 1 e13 vg / kg to approximately 2 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 3 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 4 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 5 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 6 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 7 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 8 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 9 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 1 e14 vg / kg, or approximately 1 e13 vg / kg to approximately 2 e14 vg / kg, or 1 e13 vg / kg to approximately 3 e14 vg / kg, or approximately 1 e13 to approximately 4 e14 vg / kg, or approximately 3 e13 vg / kg to approximately 4 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 5 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 6 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 7 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 8 e13 vg / kg, or approximately 3 e13 vg / kg to approximately 9 e13 vg / kg, or approximately 3e13vg / kg to approximately 1e14vg / kg, or approximately 3e13vg / kg to approximately 2e14vg / kg, or approximately 3e13vg / kg to approximately 3e14vg / kg, or approximately 3e13 to approximately 4e14vg / kg, or approximately 3e13vg / kg to approximately 5e14vg / kg, or approximately 5e13vg / kg to approximately 6e13vg / kg, or approximately 5e13vg / kg to approximately 7e13vg / kg, or approximately 5e13vg / kg to approximately 8e13vg / kg, or approximately 5e13vg / kg to approximately 9e13vg / kg, or approximately 5e13vg / kg to approximately 1e14vg / kg, The dosages of rAAVs are in the range of approximately 5e13vg / kg to approximately 2e14vg / kg, or 5e13vg / kg to approximately 3e14vg / kg, or approximately 5e13 to approximately 4e14vg / kg, or approximately 5e13vg / kg to approximately 5e14vg / kg, or approximately 1e14vg / kg to approximately 2e14vg / kg, or 1e14vg / kg to approximately 3e14vg / kg, or approximately 1e14 to approximately 4e14vg / kg, or approximately 1e14vg / kg to approximately 5e14vg / kg, 6e14vg / kg, 7e14vg / kg, 8e14vg / kg, or 9e14vg / kg. The present invention also includes compositions comprising rAAV vectors in these ranges.

[0192] For example, therapeutically effective doses of rAAV vector are 1e13vg / kg, approximately 2e13vg / kg, approximately 3e13vg / kg, approximately 4e13vg / kg, approximately 5e13vg / kg, approximately 6e13vg / kg, approximately 7e13vg / kg, approximately 7.4e13vg / kg, approximately 8e13vg / kg, approximately 9e13vg / kg, approximately 1e14vg / kg, approximately 2e14vg / kg, approximately 3e14vg / kg, approximately 4e14vg / kg, and 5e14vg / kg. The titer or dosage of AAV vector may vary based on the physical morphology of plasmid DNA as a quantitative standard. For example, the titer or dosage value may vary based on superhelical standard qPCR titration or linear standard qPCR titration. In one embodiment, a therapeutically effective dose of rAAV is a dose of 5 e13 vg / kg based on a superhelical plasmid as a quantitative standard, or a dose of 1.85 e13 vg / kg based on a linearized plasmid as a quantitative standard. In another embodiment, a therapeutically effective dose of rAAV is a dose of 2 e14 vg / kg based on a superhelical plasmid as a quantitative standard, or a dose of 7.41 e13 vg / kg based on a linearized plasmid as a quantitative standard.In another embodiment, a therapeutically effective amount of scAAVrh74.tMCK.hSGCA is approximately 1 e13 vg / kg to approximately 5 e14 vg / kg, or approximately 1 e13 vg / kg to approximately 2 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 3 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 4 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 5 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 6 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 7 e13 vg / kg, or approximately 1 e13 vg / kg to approximately 8 e13 vg / kg, or approximately 1e13vg / kg to approximately 9e13vg / kg, or approximately 1e13vg / kg to approximately 1e14vg / kg, or approximately 1e13vg / kg to approximately 2e14vg / kg, or 1e13vg / kg to approximately 3e14vg / kg, or approximately 1e13 to approximately 4e14vg / kg, or approximately 3e13vg / kg to approximately 4e13vg / kg, or approximately 3e13vg / kg to approximately 5e13vg / kg, or approximately 3e13vg / kg to approximately 6e13vg / kg, or approximately 3e13vg / kg to approximately 7e13vg / kg, or approximately 3e13vg / kg to approximately 8e13vg / kg, or approximately 3e13vg / kg to approximately 9e13vg / kg, or approximately 3e13vg / kg to approximately 1e14vg / kg, or approximately 3e13vg / kg to approximately 2e14vg / kg, or approximately 3e13vg / kg to approximately 3e14vg / kg, or approximately 3e13 to approximately 4e14vg / kg, or approximately 3e13vg / kg to approximately 5e14vg / kg, or approximately 5e13vg / kg to approximately 6e13vg / kg, or approximately 5e13vg / kg to approximately 7e13vg / kg, or approximately 5e13vg / kg to approximately 8e13vg / kg, or approximately 5e13vg / kg to approximately 9e13vg / kg, or approximately 5e13vg / kg The dosage ranges from approximately 1 e14 vg / kg, or approximately 5 e13 vg / kg to approximately 2 e14 vg / kg, or 5 e13 vg / kg to approximately 3 e14 vg / kg, or approximately 5 e13 to approximately 4 e14 vg / kg, or approximately 5 e13 vg / kg to approximately 5 e14 vg / kg, or approximately 1 e14 vg / kg to approximately 2 e14 vg / kg, or 1 e14 vg / kg to approximately 3 e14 vg / kg, or approximately 1 e14 to approximately 4 e14 vg / kg, or approximately 1 e14 vg / kg to approximately 5 e14 vg / kg, 6 e14 vg / kg, 7 e14 vg / kg, 8 e14 vg / kg, or 9 e14 vg / kg.The present invention also includes compositions comprising these doses of rAAV vectors.

[0193] The effective dose of the composition may be administered via a standard route in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV component of the rAAV of the present invention (specifically, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state being treated, as well as the target cells / tissues expressing β-sarcoglycans.

[0194] The present invention provides topical and systemic administration of effective doses of the rAAV and composition of the present invention. For example, systemic administration means administration to the circulatory system so that the whole body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration by injection, infusion, or transplantation.

[0195] In particular, the actual administration of rAAV according to the present invention can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (although DNA-degrading compositions should be avoided in the usual manner with rAAV). The rAAV capsid protein may be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference.

[0196] The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation delivered to the muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been developed to date and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. Therefore, in another embodiment, this application relates to a formulation comprising rAAV containing a capsid derived from AAVrh74, a buffer, an ionic strengthener, and a surfactant. In one embodiment, the rAAV is approximately 1.0 x 10⁻¹⁶ 12 vg / ml ~ approx. 5.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 5.0 x 10⁻¹⁶ based on a superhelical plasmid as a quantitative standard. 12 vg / ml ~ approx. 1.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 2.0 × 10⁶ based on a superhelical plasmid as a quantitative standard. 13 The concentration is vg / ml. In one embodiment, rAAV is the scAAVrh74.tMCK.hSGCA vector. In one embodiment, the concentration of rAAV in the composition or formulation is 1 × 10⁻¹⁶ based on a superhelical plasmid as a quantitative standard. 13 vg / ml ~ 2 × 10 14 The concentration is vg / ml. In another embodiment, the concentration is 2 × 10⁻¹⁶ based on a superhelical plasmid as a quantitative standard. 13 vg / ml, 4 x 10 13 vg / ml, or 5×10 13The concentration is vg / ml. In one embodiment, the buffer comprises one or more of Tris, Trisine, Bis-Trisine, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In another embodiment, the buffer comprises Tris at a concentration of about 5 mM to about 40 mM and a pH of 8.0. In one embodiment, the buffer comprises Tris at a concentration of about 20 mM and a pH of 8.0. In one embodiment, the ionic strengthener comprises one or more of potassium chloride (KCl), potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride (NH4Cl), ammonium acetate, magnesium chloride (MgCl2), magnesium acetate, magnesium sulfate, manganese chloride (MnCl2), manganese acetate, manganese sulfate, sodium chloride (NaCl), sodium acetate, lithium chloride (LiCl), and lithium acetate. In one embodiment, the ionic strengthener comprises MgCl2 at a concentration of about 0.2 mM to about 4 mM. In another embodiment, the ionic strengthener comprises NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strengthening agent contains MgCl2 at a concentration of about 0.2 mM to about 4 mM and NaCl at a concentration of about 50 mM to about 500 mM. In another embodiment, the ionic strengthening agent contains MgCl2 at a concentration of about 1 mM and NaCl at a concentration of about 200 mM. In one embodiment, the surfactant contains one or more of the following: sulfonates, sulfates, phosphonates, phosphates, poloxamers, and cationic surfactants. In one embodiment, the poloxamer contains one or more of the following: poloxamer 124, poloxamer 181, poloxamer 184, poloxamer 188, poloxamer 237, poloxamer 331, poloxamer 338, and poloxamer 407. In one embodiment, the surfactant contains poloxamer at a concentration of about 0.00001% to about 1%. In another embodiment, the surfactant contains poloxamer 188 at a concentration of about 0.001%. For intramuscular injection, adjuvant solutions such as sesame oil or peanut oil, or aqueous propylene glycol solutions and sterile aqueous solutions can be used. Such aqueous solutions can be buffered as needed, and the liquid diluent is first isotonicized with physiological saline or glucose.Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacokinetically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0197] Therefore, this specification also describes a method for administering an effective dose (or a dose essentially administered simultaneously or at intervals) of rAAV encoding alpha-sarcoglycans to a mammalian subject in need thereof.

[0198] All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the event of any conflict, this application shall prevail, including any definitions herein.

[0199] The present invention is further illustrated in the following embodiments, which do not limit the scope of the invention as described in the claims. [Examples]

[0200] Preclinical studies using AAVrh74.tMCK.hSCGA are described in International Patent Publication No. 2013 / 078316 and U.S. Patents No. 9,434,928 and No. 10,105,453, which are incorporated herein by reference in their entirety.

[0201] Example 1 Construction of scAAVrh74.tMCK.hSGCA The scAAVrh74.tMCK.hSGCA transgene cassette was constructed by inserting a tMCK expression cassette driving a codon-optimized human alpha-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 sequence contained in this vector is the AAV2 inverted end repeat, which is necessary for both viral DNA replication and packaging of the rAAV vector genome. One of the inverted end repeats (ITRs) has a targeted deletion of a terminal degradation site (TRS) to restrict replication from this ITR and promote the generation of a dimeric replicate for self-complementary vector packaging. The AAVrh74 virus has been proven safe and highly efficient for transduction into muscle across the blood-vascular barrier in mice, non-human primates (NHPs), and humans.

[0202] Recombinant AAV, (sc)rAAVrh74.tMCK.hSGCA, was constructed by triple transfection. The encapsulated vg titer was determined using a qPCR-based titration method and a Prism 7500 Fast Taqman detector system (PE Applied Biosystems). The construct contains a chimeric intron to promote high levels of expression. The chimeric intron consists of the 5' donor region from the first intron and branch point of the human β-globin gene, and the 3' splice acceptor region from the intron between the leader and body of the immunoglobulin gene's heavy chain variable region. rAAV also contains a synthetic SV40 polyadenylation signal, which is used for efficient transcription termination. A schematic diagram of the expression cassette is shown in Figure 1 below. The vector was produced using a human alpha-sacroglycan (alpha-SG) gene, capsid-formed on the AAVrh74 virion, adjacent to the AAV2 ITR sequence. The construct contains a pre-initial promoter / enhancer for tMCK (GenBank accession number M21390) and uses a β-globin intron for high levels of expression.

[0203] Single-stranded AAV vectors (ssAAVs), upon entering the nucleus, require cell-mediated synthesis of a second strand before they are ready for replication and transcription. Exemplary self-complementary AAV vectors (scAAVs) have the structure shown in Figure 1 and the annotated nucleotide sequence provided in Figure 2, as listed in the table below. Because scAAVs bypass the rate-limiting step of cell synthesis of the second strand required in ssAAVs, they are superior to ssAAVs in gene therapy. [Table 1]

Claims

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

1.

2. The polynucleotide sequence of claim 1, wherein the nucleotide sequence comprises the nucleotide sequence or SEQ ID NO:

1.

3. A recombinant AAV (rAAV) comprising a polynucleotide sequence, the polynucleotide sequence comprising: i) two complementary nucleotide sequences, each encoding the amino acid sequence of SEQ ID NO:3; and ii) two complementary polyadenylation sequences.

4. The recombinant AAV of claim 3, wherein each of the two complementary nucleotide sequences is operably linked to a muscle-specific regulatory element, and the two muscle-specific regulatory elements are complementary to each other.

5. 5. The recombinant AAV of claim 4, wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF) element, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, an MHCK7 promoter, a C5-12 promoter, a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor-binding element, a steroid-inducible element, or a glucocorticoid response element (GRE).

6. The recombinant AAV of any one of claims 3 to 5, further comprising two complementary chimeric introns.

7. The recombinant AAV of any one of claims 3 to 5, further comprising three inverted terminal repeats (ITRs), one ITR flanked by the two complementary muscle-specific regulatory elements.

8. The recombinant AAV of any one of claims 3 to 5, comprising the nucleotide sequence of SEQ ID NO:

1.

9. The recombinant AAV according to any one of claims 3 to 5, wherein the vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh.74, or a variant thereof.

10. A composition comprising the recombinant AAV of any one of claims 3 to 5.

11. A recombinant AAV described in any one of claims 3 to 5 for use in a method for treating alpha-sarcoglycanopathy muscular dystrophy in a subject in need of treatment for alpha-sarcoglycanopathy muscular dystrophy, the method comprising administering the recombinant AAV to the subject.

12. 6. The recombinant AAV of any one of claims 3 to 5 for use in a method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, said method comprising administering said recombinant AAV to said subject.

13. 6. The recombinant AAV of any one of claims 3 to 5 for use in a method for reducing fibrosis in a subject suffering from muscular dystrophy, said method comprising administering said recombinant AAV to said subject.

14. 6. The recombinant AAV of any one of claims 3 to 5 for use in a method for reducing contraction-induced damage in a subject suffering from muscular dystrophy, said method comprising administering said recombinant AAV to said subject.

15. A recombinant AAV according to any one of claims 3 to 5 for use in a method for increasing alpha-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, the method comprising administering the recombinant AAV to the subject.

16. The recombinant AAV for use according to claim 11, wherein the subject suffers from limb-girdle muscular dystrophy.

17. The recombinant AAV for use according to claim 11, characterized in that the recombinant AAV or the composition is administered systemically.

18. The rAAV is: i) approximately 5 x 10 based on supercoiled DNA or plasmid as a quantification standard; １３ vg / kg ~ approx. 2×10 １４ 12. The recombinant AAV for use according to claim 11, wherein the recombinant AAV is administered at a dose of: ii) about 5x1013 vg / kg based on supercoiled DNA or plasmid as a quantification standard; iii) about 1x1014 vg / kg based on supercoiled DNA or plasmid as a quantification standard; iv) 2x1014 vg / kg based on supercoiled DNA or plasmid as a quantification standard; v) about 1.85x1013 vg / kg based on linearized DNA or plasmid as a quantification standard; or vi) 7.41x1013 vg / kg based on linearized DNA or plasmid as a quantification standard.

19. 1. A recombinant AAV (rAAV) vector comprising a polynucleotide sequence, the polynucleotide sequence comprising, in 5' to 3' direction: (1) a complementary sequence of the polyadenylation sequence; (2) a complementary sequence of a gene of interest, wherein the gene of interest encodes the amino acid sequence of SEQ ID NO: 3; and (3) a complementary sequence of the intron; and (4) a complementary sequence of the promoter; and (5) a 5' ITR sequence; (6) the promoter; (7) the intron; (8) the gene of interest; (9) The polyadenylation sequence, A recombinant AAV vector, wherein the polynucleotide sequence is flanked by two 3' ITR sequences, and the two 3' ITR sequences are complementary to each other.

20. A host cell comprising the polynucleotide described in claim 1.

21. The cell of claim 20, which is a eukaryotic cell, a bacterial cell, an insect cell or a yeast cell.

22. The cell according to claim 20, which is a 293 cell, a HeLa cell, an MRC-5 cell, a WI-38 cell, a Vero cell, or an FrhL-2 cell.

23. A method for producing recombinant AAV (rAAV) particles, comprising culturing the host cell described in claim 20.

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