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

Abstract

To provide therapy vectors such as AAV vectors expressing β-sarcoglycan, and a method of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy.SOLUTION: A recombinant AAV (rAAV) comprises a polynucleotide sequence comprising a specific nucleotide sequence. The polynucleotide sequence comprises i) two complementary nucleotide sequences each encoding a gene of interest, where the two complementary nucleotide sequences encoding the gene of interest flank a 5' ITR sequence, and ii) two complementary polyadenylation sequences, where the polynucleotide sequence is flanked by two 3' ITR sequences, where the two 3' ITR sequences are complementary.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 256368, filed on October 15, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] <亮 Incorporation by reference of electronically submitted materials This application includes a Sequence Listing in computer-readable form, identified as follows and incorporated herein by reference in its entirety as a separate part of this disclosure: File name: 56758_Seqlisting.txt, size: 28,732 bytes, created: September 28, 2022.

[0003] Therapeutic vectors such as AAV vectors expressing β-sarcoglycan, and methods of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy are described herein.

Background Art

[0004] LGMD is a rare condition, and symptoms vary from person to person with respect to age of onset, area of muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or later. Both genders are equally affected. LGMD causes weakness in the shoulders and pelvic girdle, and the muscles near the upper arms and wrists may also weaken over time. Weakness in the legs often appears before weakness in the arms. Facial muscles are usually not affected. As the condition progresses, people may have trouble walking and may need to use a wheelchair over time. When the muscles of the shoulders and arms are involved, it can be difficult to raise the arms above the head or lift objects. Depending on the type of LGMD, the heart and respiratory muscles may be involved.

[0005] There are at least 19 forms of LGMD, which are classified by the associated genetic defects.

Table 1

[0006] Limb-girdle muscular dystrophy (LGMD) type 2E (LGMD2E) is an autosomal recessive disorder caused by mutations in the gene encoding β-sarcoglycan (SGCB), resulting in the loss of a functional protein. LGMD2E represents a relatively common and severe form of LGMD in the United States, with an incidence reported worldwide of 1 in 200,000 to 1 in 350,000 (Moore et al. J Neuropathol Exp Neurol 2006;65:995-1003). The absence of β-sarcoglycan causes a progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis, all of which lead to deterioration of muscle strength and function. (Araishi et al., Hum Mol Genet 1999;8:1589-1598, Durbee et al., Mol Cell 2000;5:141-151) As a complex, sarcoglycans (α-, β, γ-, δ-), which range in size from 35 to 50 kD, are transmembrane proteins that confer stability to the muscle sheath, providing protection from mechanical stress during muscle activity. (Araishi et al., Hum Mol Genet 1999;8:1589-1598) Loss of β-sarcoglycan in LGMD2E usually causes an accompanying loss of other sarcoglycan proteins to varying degrees, contributing to the fragility of the sarcolemma that leads to muscle fiber loss. 1 The clinical phenotype of LGMD2E varies, but diagnosis typically occurs by age 10, and loss of ambulation occurs from mid- to late teens. Patients present with elevated serum creatine kinase (CK), proximal muscle weakness, difficulty rising from the floor, and progressive loss of ambulation. Cardiac involvement occurs in 50% of cases.

[0007] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the AAV serotype genomes are known. For example, the complete genome of AAV-1 is available under GenBank access number NC_002077, the complete genome of AAV-2 is available under GenBank access number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is available under GenBank access number NC_1829, the complete genome of AAV-4 is available under GenBank access number NC_001829, the genome of AAV-5 is available under GenBank access number AF085716, the complete genome of AAV-6 is available under GenBank access number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are available under GenBank access numbers AX753246 and AX753249, respectively, and the genome of AAV-9 is available under Gao et al. The AAV-10 genome is available in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is available in U.S. Patent No. 9,434,928, incorporated herein by reference. The Cis action sequence, which directs viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration, is contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) promote the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19), linked to differential splicing of a single AAV intron (at nucleotides 2107 and 2227), produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene.The Rep protein possesses 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 are involved in the production of the 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).

[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, since 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] 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 Sci 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 leads to the appearance of the transgene product in the systemic circulation after 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 cellular factors necessary for the correct glycosylation, folding, and secretion of antibodies, showing that muscles can stably express secreted protein therapeutics.

[0010] A newly emerging form of therapy for LGMD2E is virus-mediated gene delivery to restore wild-type protein to affected muscle, leading to recovery of muscle function. Considering that a subset of patients may develop cardiomyopathy (Fannin et al., Neuromusc Disord 2003;13:303-309, Sveen et al., Arch Neurol 2008;65:1196-1201, Melacini et al., Muscle Nerve 1999;22:473-479, Barresi et al., J Med Genet 2000;37:102-107), this should be considered in the long-term care of these patients. In previous reports, Sgcb null mice were well-characterized. Araishi et al. developed a β-sarcoglycan-deficient mouse that reproduced the clinical picture seen in LGMD2E, with loss of all sarcoglycans, not just sarcospan, while preserving at least small amounts of merosin, dystroglycan, and dystrophin. The histological changes in this animal model also served as a prototype for clinical responses, including overt skeletal muscular fibrosis. (Gibertini et al., Cell Tissue Res 2014;356:427-443) Dressman et al. (Dressman et al., Hum Gene Ther 2002;13:1631-1646) injected rAAV2.CMV.SGCB into the transversus abdominis muscle. Expression persisted for 21 months, and muscle fibers were protected from recurrent necrosis. The use of self-complementary AAVs to enhance transgene expression (McCarty et al., Gene Ther 2001;8:1248-1254), muscle-specific promoters for better targeting of skeletal muscle (Wang et al., Gene Ther 2008;15:1489-1499, Rodino-Klapac et al., Neurology 2008;71:240-247), and optimization of the human β-sarcoglycan gene (hSGCB) are also described. 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. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Moore et al. J Neuropathol Exp Neurol 2006;65:995-1003 [Non-Patent Document 2] Araishi et al., Hum Mol Genet 1999;8:1589-1598 [Non-Patent Document 3] Durbee et al., Mol Cell 2000;5:141-151 [Overview of the Initiative] [Means for solving the problem]

[0012] This specification describes gene therapy vectors expressing the β-sarcoglycan gene (e.g., AAV), as well as methods for delivering β-sarcoglycan to muscle to alleviate and / or prevent fibrosis, and / or increase muscle strength, and / or treat mammalian subjects suffering from muscular dystrophy. Methods for treating muscular dystrophy, including administration of a self-complementary recombinant AAV (rAAV)scAAVrh74.MHCK7.hSGCB vector, methods for expressing the beta-sarcoglycan gene in a patient, pharmaceutical compositions containing rAAV, and methods for producing rAAV are described herein. In embodiments of the present invention, 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 polynucleotide sequence described in item 1, wherein the nucleotide sequence includes the nucleotide sequence of sequence number 1. (Item 3) Recombinant AAV (rAAV) comprising a polynucleotide sequence, wherein the polynucleotide sequence comprises i) two complementary nucleotide sequences each encoding a target gene, wherein the two complementary nucleotide sequences encoding the target gene are adjacent to a 5'ITR sequence, and ii) two complementary polyadenylated sequences. A recombinant AAV wherein the polynucleotide sequence is flanked by two 3'ITR sequences, and the two 3'ITR sequences are complementary. (Item 4) Recombinant AAV as described in item 3, wherein the target gene includes the human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, or human ANO5.calpain-3 (Cap 3) gene. (Item 5) 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 6) The recombinant AAV according to items 3-5, 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 7) The recombinant AAV described in item 6, 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, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C5-12, 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 8) The recombinant AAV described in item 7, wherein the muscle-specific regulatory element is a shortened MCK (tMCK). (Item 9) The recombinant AAV described in item 7, wherein the muscle-specific regulatory element is MHCK7. (Item 10) A recombinant AAV as described in any one of items 3 to 9, further comprising two complementary chimerintrons. (Item 11) A recombinant AAV according to any one of items 3 to 10, further comprising three reverse terminal repeats (ITRs), one of which is adjacent to the two complementary muscle-specific control elements. (Item 12) Recombinant AAV as described in any one of items 3 to 11, comprising the nucleotide sequence of SEQ ID NO: 1. (Item 13) The recombinant AAV described in any one of items 3 to 12, wherein the vector is serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV rh.74. (Item 14) A composition comprising a recombinant AAV as described in any one of items 3 to 13. (Item 15) A method for treating muscular dystrophy in a subject requiring treatment, comprising administering to the subject a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14. (Item 16) 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 items 3 to 13 or a composition described in item 14. (Item 17) 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 items 3 to 13 or a composition described in item 14. (Item 18) 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 items 3 to 13 or a composition described in item 14. (Item 19) A method for treating β-sarcoglycan disorder in a subject, comprising administering to the subject a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14. (Item 20) A method for increasing beta-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 items 3 to 13 or a composition described in item 14. (Item 21) The method according to item 20, wherein the expression of the beta-sarcoglycan gene or the number of positive beta-sarcoglycan-positive fibers is detected by measuring the beta-sarcoglycan protein level by Western blotting in muscle biopsies before and after rAAV administration. (Item 22) The method according to item 20, wherein the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan-positive muscle fibers is detected by measuring the beta-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of rAAV. (Item 23) The method described in any one of items 15 to 22, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 24) The method according to any one of items 15 to 23, wherein the recombinant AAV or the composition is administered by intramuscular or intravenous injection. (Item 25) The method according to any one of items 15 to 23, wherein the recombinant AAV or the composition is administered systemically. (Item 26) The method according to item 25, wherein the recombinant AAV or the composition is administered parenterally by injection, infusion, or implantation. (Item 27) The recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 7.41 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, resulting in 2 × 10⁶ doses. 14 The method described in any one of items 15-26, administered at an equivalent dose of vg / kg. (Item 28) The recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 1.85 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 The method described in any one of items 15-26, administered at an equivalent dose of vg / kg. (Item 29) The method according to item 27 or 28, wherein the recombinant AAV is administered systemically. (Item 30) The method according to item 27 or 28, wherein the recombinant AAV is administered intravenously. (Item 31) A composition comprising a recombinant AAV as described in any one of items 3 to 13 or a composition as described in item 14. (Item 32) A composition comprising a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14, for increasing muscle strength and / or muscle mass in mammals suffering from muscular dystrophy. (Item 33) A composition comprising a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14, for the purpose of alleviating fibrosis in mammals suffering from muscular dystrophy. (Item 34) A composition comprising a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14, for reducing contraction-induced injury in mammals suffering from muscular dystrophy. (Item 35) A composition comprising a recombinant AAV described in any one of items 3 to 13 or a composition described in item 14, for the treatment of β-sarcoglycan disorders in mammals requiring treatment. (Item 36) A composition comprising a recombinant AAV according to any one of items 3 to 13 or a composition according to item 14 for increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in target muscle tissue. (Item 37) The composition according to item 36, wherein the expression of the beta-sarcoglycan gene or the number of positive beta-sarcoglycan-positive fibers is detected by measuring the beta-sarcoglycan protein level by Western blotting in muscle biopsies before and after administration of the rAAV. (Item 38) The composition according to item 36, wherein the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan-positive muscle fibers is detected by measuring the beta-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 39) The composition according to any one of items 32 to 38, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 40) A composition according to any one of items 32 to 39, formulated for intramuscular or intravenous injection. (Item 41) A composition described in any one of items 32 to 39, formulated for systemic administration. (Item 42) The composition according to item 41, wherein the systemic administration is parenteral administration by injection, infusion, or transplantation. (Item 43) The recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 7.41 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, resulting in 2 × 10⁶ doses. 14A composition according to any one of items 32 to 42, in an equivalent dose of vg / kg. (Item 44) The recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 1.85 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 A composition according to any one of items 32 to 42, administered in an equivalent dose of vg / kg. (Item 45) The composition according to item 43 or 44, wherein the composition is formulated for systemic administration. (Item 46) The composition according to item 43 or 44, wherein the composition is formulated for intravenous administration. (Item 47) Use of recombinant AAV as described in any one of items 3 to 13 or the composition described in item 14 for the preparation of drugs for the treatment of muscular dystrophy. (Item 48) Use of a recombinant AAV vector described in any one of items 3 to 13 or a composition described in item 14 for the preparation of a drug for increasing muscle strength and / or muscle mass in mammals suffering from muscular dystrophy. (Item 49) Use of a recombinant AAV vector described in any one of items 3 to 13 or a composition described in item 14 for the preparation of a drug to alleviate fibrosis in mammals suffering from muscular dystrophy. (Item 50) Use of a recombinant AAV vector described in any one of items 3 to 13 or a composition described in item 14 for the preparation of a drug to reduce contraction-induced injury in subjects suffering from muscular dystrophy. (Item 51) Use of recombinant AAVs described in any one of items 3 to 13 or compositions described in item 14 for the preparation of agents for treating β-sarcoglycan disorders in mammals requiring treatment. (Item 52) Use of the recombinant AAV according to any one of items 3 to 13 or the composition according to item 14 for the preparation of an agent for increasing beta-sarcoglycan positive fibers and / or decreasing CK levels in a target muscle tissue. (Item 53) The use according to item 52, wherein the expression of the beta-sarcoglycan gene or the number of positive beta-sarcoglycan positive fibers is detected by measuring the beta-sarcoglycan protein level by Western blot in muscle biopsies before and after administration of the rAAV. (Item 54) The use according to item 52, wherein the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan positive muscle fibers is detected by measuring the beta-sarcoglycan protein level by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 55) The use according to any one of items 47 to 54, wherein the subject is suffering from limb-girdle muscular dystrophy. (Item 56) The use according to any one of items 47 to 55, wherein the agent is formulated for intramuscular injection or intravenous injection. (Item 57) The use according to any one of items 47 to 55, wherein the agent is formulated for systemic administration. (Item 58) The use according to item 57, wherein the systemic administration is parenteral administration by injection, infusion, or transplantation. (Item 59) The use according to any one of items 47 to 58, wherein the recombinant AAV is at a dosage of 7.41×10 13 vg / kg measured by qPCR using a linear reference plasmid, or an equivalent dosage of 2×10 14 vg / kg measured by qPCR using a supercoiled reference plasmid. (Item 60) The recombinant AAV is 1.85×10 measured by qPCR using a linear reference plasmid.13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 Use as described in any one of items 47-58, administered at an equivalent dose of vg / kg. (Item 61) The method according to item 59 or 60, wherein the recombinant AAV is formulated for systemic administration. (Item 62) The method according to item 59 or 60, wherein the recombinant AAV is formulated for intravenous administration. (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) The rAAV vector described in item 63, wherein the target gene comprises human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, and human ANO5.calpain-3 (Cap 3) genes. (Item 65) The rAAV vector according to item 63, wherein the promoter is a muscle-specific regulatory element, and the muscle-specific regulatory element comprises a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C5-12, 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).

[0013] A self-complementary AAV (scAAV) expressing a β-sarcoglycan gene is provided herein. For example, the provided scAAV comprises a polynucleotide sequence comprising: i) two nucleotide sequences encoding a self-complementary β-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).

[0014] Furthermore, recombinant AAV (rAAV) vectors containing a polynucleotide sequence are also provided, 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. In one embodiment, the gene of interest includes the human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, human ANO5, or calpain-3 (Cap 3) 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 factors (MEFs), muscle creatine kinase (MCK) promoters, MCK enhancers, 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 response elements (GREs).

[0015] 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.

[0016] This specification provides a polynucleotide comprising two self-complementary nucleotide sequences (also referred to as expression cassettes), each nucleotide sequence comprising an MHCK7 promoter, a chimeric intron, an hSGCB 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.

[0017] 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 4511 nucleotide sequence comprising two hSGCB cDNA sequences (SEQ ID NO: 2 and / or SEQ ID NO: 11) that encode the amino acid sequence of SEQ ID NO: 3 and hybridize with each other, two chimeric intron sequences (SEQ ID NO: 4 and / or SEQ ID NO: 11) that hybridize with each other, and two MHCK7 promoters (SEQ ID NO: 5 and / or SEQ ID NO: 13) and two polyadenylated sequences (SEQ ID NO: 6 and / or SEQ ID NO: 14) 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: 7. Additional ITR sequences are shown as SEQ ID NOs: 8 and 15.

[0018] 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.

[0019] 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.

[0020] 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-globulin These include the following genes: Gag, MG1MA3, L523S, METRAP, GDNF, AQP1, PG9DP, HBB, ADA, TCR, CAR, filgrastim, IL-12, GM-CSF, ICP34.5, PENK, RB94, SST2, DCK.P53, HSC, human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, human ANO5, and calpain-3 (Cap 3) 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.

[0021] The recombinant AAV according to claim 3, wherein the target gene is the human sarcoglycan-β (hSCGB), human sarcoglycan-γ (hSCGG), human dysferlin, human ANO5, or calpain-3 (Cap 3) gene.

[0022] This disclosure also provides a polynucleotide-containing rAAV, the polynucleotide sequence comprising i) two self-complementary nucleotide sequences, each encoding a human β-sarcoglycan (hSGCB) 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 hSGCB 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 hSGCB protein comprises the nucleotide sequence of SEQ ID NO: 2. For example, the polyadenylated sequence comprises the nucleotide sequence of SEQ ID NO: 6.

[0023] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding β-sarcoglycan are described herein. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical sequences to the nucleotide sequence described in SEQ ID NO: 5 or SEQ ID NO: 2, and encodes a protein that retains β-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises the nucleotide sequence described in SEQ ID NO: 2. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan consists of the nucleotide sequence described in SEQ ID NO: 5 or SEQ ID NO: 2.

[0024] In another embodiment, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding a β-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 β-sarcoglycan activity.

[0025] In another embodiment, recombinant AAV vectors comprising a polynucleotide sequence encoding a functional β-sarcoglycan, or its complement, which includes a nucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2 under stringent conditions, are described herein.

[0026] 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).

[0027] 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, between 1% and 15% of the stated numerical value or numerical range.

[0028] 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.

[0029] 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 factors (MEFs), muscle creatine kinase (MCK) promoters, MCK elements, truncated MCK (tMCK) promoters, tMCK elements, myosin heavy chain (MHC) promoters, MHCK7 promoters (hybrid versions of MHC and MCK), C5-12 promoters (synthetic 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, steroid-induced elements, or glucocorticoid-responsive elements (GREs).

[0030] In some embodiments, the disclosed rAAV comprises a polynucleotide sequence, each of which two complementary nucleotide sequences is operably coupled to a muscle-specific regulatory element MCK (tMCK) promoter containing the nucleotide sequence of SEQ ID NO: 9. In other embodiments, the disclosed rAAV comprises a polynucleotide sequence, each of which two complementary nucleotide sequences is operably coupled to a muscle-specific regulatory element MHCK7 promoter containing the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 5.

[0031] In further embodiments, the disclosed rAAV comprises a polynucleotide sequence containing two complementary chimeric introns. For example, the chimeric introns comprise the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 4.

[0032] 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 comprise SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 15. In a particular example, the ITR flanked by two self-complementary muscle-specific regulatory elements comprises the nucleotide sequence of SEQ ID NO: 7. In other embodiments, the two ITRs comprise the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 15, with one of the ITRs comprising the nucleotide sequence of SEQ ID NO: 7.

[0033] 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, and AAVrh.74. The production of pseudotype 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).

[0034] The Disclosure also provides compositions comprising either the disclosed rAAV or the disclosed polynucleotide. In some embodiments, the compositions further comprise pharmaceutically acceptable carriers, diluents, and / or auxiliaries. For example, the compositions comprise either the rAAV of the Disclosure, buffers, ionic strengtheners, and surfactants.

[0035] In one embodiment, a method for treating muscular dystrophy in a subject requiring treatment is described herein, comprising the step of administering to the subject any of the disclosed rAAVs or any of the disclosed compositions.

[0036] In another aspect, the Disclosure provides a method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering to the subject any of the disclosed rAAVs or any of the disclosed compositions.

[0037] The disclosure also provides a method for reducing contraction-induced injury in a subject suffering from muscular dystrophy, comprising administering to the subject any of the disclosed rAAVs or any of the disclosed compositions.

[0038] In addition, the disclosure provides a method for treating β-sarcoglycan disorders in a subject, comprising administering to the subject any of the disclosed rAAVs or any of the disclosed compositions.

[0039] This disclosure also provides a method for increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, comprising administering to the subject any of the disclosed rAAVs or any of the disclosed compositions. For example, in any of the disclosed methods, the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan-positive fibers is detected by measuring beta-sarcoglycan protein levels by Western blotting in muscle biopsies before and after rAAV administration. Alternatively, in any of the disclosed methods, the expression of the beta-sarcoglycan gene or the number of beta-sarcoglycan-positive muscle fibers is detected by measuring beta-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after rAAV administration.

[0040] In all of the disclosed methods, the subjects suffer from limb-girdle muscular dystrophy.

[0041] In any of the disclosed methods, recombinant AAV or composition is administered by intramuscular or intravenous injection. In other embodiments, rAAV or composition is administered systemically, for example, by intravenous injection.

[0042] In any of the disclosed methods, recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 7.41 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, resulting in 2 × 10⁶ doses. 14 It is administered at an equivalent dose of vg / kg.

[0043] In another embodiment, in any of the disclosed methods, recombinant AAV is measured by qPCR using a linear reference plasmid and yields 1.85 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 It is administered at an equivalent dose of vg / kg.

[0044] In one embodiment, the disclosure provides a composition for treating muscular dystrophy in a subject requiring treatment, the composition comprising any of the disclosed rAAVs or any of the disclosed compositions.

[0045] In another embodiment, the Disclosure provides compositions for increasing muscle strength and / or muscle mass in subjects suffering from muscular dystrophy, the compositions comprising any of the disclosed rAAVs or any of the disclosed compositions.

[0046] This disclosure also provides compositions for reducing contraction-induced injury in subjects suffering from muscular dystrophy, the compositions comprising any of the disclosed rAAVs or any of the disclosed compositions.

[0047] In addition, the Disclosure provides compositions for treating β-sarcoglycan disorders in subjects, the compositions comprising any of the disclosed rAAVs or any of the disclosed compositions.

[0048] The disclosure also provides compositions for increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of interest, the compositions comprising any of the disclosed rAAVs or any of the disclosed compositions. For example, administration of the disclosed compositions results in increased expression of the beta-sarcoglycan gene or an increase in the number of positive beta-sarcoglycan-positive fibers, as detected by measuring beta-sarcoglycan protein levels by Western blotting in muscle biopsies before and after rAAV administration. Alternatively, administration of the disclosed compositions results in increased expression of the beta-sarcoglycan gene or an increase in the number of beta-sarcoglycan-positive muscle fibers, as detected by measuring beta-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after rAAV administration.

[0049] In all of the disclosed compositions, the subjects suffer from limb-girdle muscular dystrophy.

[0050] In any of the disclosed compositions, recombinant AAV or the composition is formulated for administration by intramuscular or intravenous injection. In other embodiments, rAAV or the composition is formulated for systemic administration, such as intravenous administration.

[0051] In any of the disclosed compositions, recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 7.41 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, resulting in 2 × 10⁶ doses. 14 This is an equivalent dosage in vg / kg.

[0052] In another embodiment, in any of the disclosed compositions, recombinant AAV is measured by qPCR using a linear reference plasmid and yields 1.85 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 This is an equivalent dosage in vg / kg.

[0053] In one embodiment, the use of any of the disclosed rAAVs or any of the disclosed compositions for the preparation of a drug for the treatment of muscular dystrophy in a subject requiring such treatment is described herein.

[0054] In another aspect, the disclosure provides the use of any of the disclosed rAAVs or any of the disclosed compositions for the preparation of agents for increasing muscle strength and / or muscle mass in mammals suffering from muscular dystrophy.

[0055] This disclosure also provides the use of any of the disclosed rAAVs or any of the disclosed compositions for the preparation of agents to reduce contraction-induced injury in subjects suffering from muscular dystrophy.

[0056] In addition, this disclosure provides the use of any of the disclosed rAAVs or any of the disclosed compositions for the preparation of agents for treating β-sarcoglycan disorders in subjects.

[0057] This disclosure also provides the use of any of the disclosed rAAVs or any of the disclosed compositions for the preparation of agents to increase beta-sarcoglycan-positive fibers and / or decrease CK levels in muscle tissue of interest. For example, in either use, administration of rAAV or any of the compositions resulted in an increase in beta-sarcoglycan gene expression or the number of positive beta-sarcoglycan-positive fibers, as detected by measuring beta-sarcoglycan protein levels by Western blotting in muscle biopsies before and after rAAV administration. Alternatively, in either use of the disclosed compositions, administration of rAAV or any of the disclosed compositions resulted in an increase in beta-sarcoglycan gene expression or the number of beta-sarcoglycan-positive muscle fibers, as detected by measuring beta-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after rAAV administration.

[0058] In all of the disclosed uses, the subjects suffer from limb-girdle muscular dystrophy.

[0059] In any of the disclosed uses, the recombinant AAV vector or composition is formulated for administration by intramuscular or intravenous injection. In other embodiments, the rAAV or composition is formulated for systemic administration, such as intravenous administration.

[0060] In any of the disclosed uses, recombinant AAV was measured by qPCR using a linear reference plasmid, yielding 7.41 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, resulting in 2 × 10⁶ doses. 14 This is an equivalent dosage in vg / kg.

[0061] In another embodiment, in any of the disclosed uses, recombinant AAV is measured by qPCR using a linear reference plasmid and yields 1.85 × 10⁶ 13 The dose was measured by vg / kg, or by qPCR using a supercoiled reference plasmid, with a result of 5 × 10⁶. 13 This is an equivalent dosage in vg / kg.

[0062] In any of the disclosed methods, compositions, or uses, serum creatine kinase (CK) levels in the subject decrease after administration of rAAV or the composition compared to serum CK levels before administration of rAAV or the composition.

[0063] In another embodiment, in any of the disclosed methods, compositions, or uses, the level of beta-sarcoglycan gene expression in the cells of interest increases after administration of rAAV or the composition compared to the level of beta-sarcoglycan gene expression before administration of rAAV or the composition; the number of beta-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of rAAV; or motor function is improved in the subject compared to the motor function of the subject before administration of rAAV, and motor function is determined by a 100-meter timed walking test.

[0064] In one embodiment, in any of the disclosed methods, compositions, or uses, the level of alpha-sarcoglycan gene expression increases in subjects requiring it after administration of rAAV or the composition, compared to the level of alpha-sarcoglycan gene expression before administration of rAAV or the composition. In another embodiment, in any of the disclosed methods, compositions, or uses, administration of rAAV or the composition results in increased localization of alpha-sarcoglycan to the cell membrane in subjects requiring it. In yet another embodiment, in any of the disclosed methods, compositions, or uses, the level of alpha-sarcoglycan expression increases in muscle tissue, or muscle function improves in subjects requiring it after administration of rAAV, compared to the level of alpha-sarcoglycan expression or muscle function before administration of rAAV or the composition.

[0065] In another embodiment, the disclosure provides a method for increasing sarcoglycan expression in muscle tissue of a subject, comprising administering the subject one of the disclosed rAAVs encoding an hSGCB and detecting an increase in the expression of at least a second sarcoglycan in the cell membrane of cells expressing the hSGCB. In some embodiments, the second sarcoglycan is α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD).

[0066] 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.

[0067] In any of the methods, compositions, and uses provided, the level of beta-sarcoglycan gene expression in the cells of interest increases after administration of rAAV or the composition compared to the level of beta-sarcoglycan gene expression before administration of rAAV or the composition; the serum creatine kinase (CK) level in the subject decreases after administration of rAAV or the composition compared to the serum CK level before administration of rAAV or the composition; and / or the number of beta-sarcoglycan-positive fibers in the muscle tissue of the subject increases after administration of rAAV or the composition compared to the number of beta-sarcoglycan-positive fibers before administration of rAAV.

[0068] In another embodiment, in any of the methods, compositions, and uses provided, motor function is improved in the subject compared to the subject's motor function before administration of rAAV or the composition, and motor function is determined by a 100-meter timed walking test. For example, motor function improves by at least 5% at 1 month or 30 days after gene transfer, at least 10% at 2 months or 60 days after gene transfer, or at least 15% at 3 months or 90 days after gene transfer. In some embodiments, motor function improves by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50%.

[0069] For example, in any of the methods, compositions, and uses provided, the systemic route of administration is an intravenous route. For example, rAAV is administered using an intravenous route, and the dose of rAAV administered is approximately 1.85 × 10⁻⁶ based on a linearized plasmid as a quantitative standard. 13 vg / kg or approximately 7.41 × 10 13 The dose of rAAV administered is approximately 5 × 10⁶, based on the supercoiled plasmid as a quantitative standard, whether in vg / kg. 13 vg / kg or approximately 2 x 10 14 It is vg / kg.

[0070] In some embodiments, the dose of rAAV is administered via an intravenous route, and the dose is approximately 1.0 × 10⁶ based on a supercoiled plasmid as a quantitative standard.13 vg / kg ~ approx. 5×10 14 Based on linearized plasmids as a quantitative standard, approximately 1.0 × 10⁶ 13 vg / kg ~ approx. 1.0×10 14 It is vg / kg.

[0071] In addition, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 13 vg~approx. 2×10 16 vg, or 1.5 × 10 13 vg~1×10 16 vg, or approximately 1.5 × 10 13 vg~approx. 2×10 15 vg, or approximately 1.5 × 10 13 vg~approx. 1×10 15 It is vg. In addition, in any of the methods, provided methods, compositions and uses, the dose of rAAV is administered at a concentration of approximately 10 mL / kg. In any of the methods, provided methods, compositions and uses, the muscular dystrophy is limb-girdle muscular dystrophy.

[0072] In any of the methods, uses, and compositions for treating muscular dystrophy provided, the subjects are 4 to 15 years old, have confirmed beta-sarcoglycan (SGCB) mutations in both alleles, are negative for AAVrh74 antibody, and / or have a 100-meter walk test score greater than 40% or normal. In any of the methods, uses, and compositions for treating muscular dystrophy provided, the subjects are pediatric subjects. In some embodiments, the subjects are pediatric subjects, for example, in the range of 1 to 10 years old. In some embodiments, the subjects are 4 to 15 years old. In one embodiment, the subjects are adolescent subjects, for example, in the range of 10 to 19 years old. In addition, in one embodiment, the subjects are young adult subjects, for example, in the range of late teens or early twenties, for example, the subjects may be in the range of 15 to 29 years old. In some embodiments, the subjects are middle-aged adults or elderly subjects, as a result, middle-aged adults may be in the range of 25 to 55 years old, and elderly subjects may be in the range of over 50 years old.

[0073] In some embodiments, rAAV is administered by injection, infusion, or implantation. For example, rAAV is administered by infusion over approximately 1-2 hours. In addition, rAAV is administered via an intravenous route through peripheral limb veins.

[0074] In any of the methods, uses, or compositions provided, the subject has a gene mutation in a gene encoding a sarcoglycan or muscular dystrophy. In some embodiments, the sarcoglycan is β-sarcoglycan (SGCB), α-sarcoglycan (SGCA), γ-sarcoglycan (SGCG), or δ-sarcoglycan (SGCD). In some embodiments, the sarcoglycan is β-sarcoglycan or α-sarcoglycan.

[0075] In any of the methods, uses, or compositions provided, the level of beta-sarcoglycan protein is at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44% after rAAV administration. or increase by at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 63%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. For example, when the level of beta-sarcoglycan protein is detected by measuring the level of beta-sarcoglycan protein by Western blotting in muscle biopsies before and after rAAV administration, it increases by at least 33%, or when the level of beta-sarcoglycan protein is detected by measuring the level of beta-sarcoglycan protein by immunohistochemistry in muscle biopsies before and after rAAV administration, it increases by at least 38% or at least 39%.

[0076] In any of the methods, uses, or compositions provided herein, the serum CK level in the subject decreases after administration of rAAV compared to the serum CK level before administration of rAAV. For example, serum CK levels in subjects decrease by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 63%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, or at least 98% compared to serum CK levels before rAAV administration, 60–90 days, 60 days, or 90 days after rAAV administration.

[0077] In any of the methods, uses, or compositions provided herein, the number of beta-sarcoglycan-positive fibers in the muscle tissue of interest increases after administration of rAAV compared to the number of beta-sarcoglycan-positive fibers before administration of rAAV. For example, the number of beta-sarcoglycan-positive fibers is detected by measuring the beta-sarcoglycan protein level by Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration. For example, the number of beta-sarcoglycan-positive fibers in the muscle tissue of interest increases by at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%, or at least 36%, or at least 37%, or at least 38%, or at least 39%, or at least 40%, or at least 41%, or at least 42%, or at least 43%, or at least 44%, or at least 4% after administration of rAAV. It increases by 5% or at least 46%, or at least 47%, or at least 48%, or at least 49%, or at least 50%, or at least 51%, or at least 52%, or at least 53%, or at least 54%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59%, or at least 60%, or at least 63%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%.

[0078] In any of the methods, compositions, and uses provided herein, the level of alpha-sarcoglycan in a subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of rAAV. 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 rAAV administration.

[0079] In any of the methods, uses, or compositions provided for expressing the beta-sarcoglycan gene in cells, the expression of the beta-sarcoglycan gene in cells is detected by measuring the beta-sarcoglycan protein level by Western blotting or immunohistochemistry in muscle biopsies before and after administration of any of the rAAV or compositions disclosed herein. For example, the cells have more than 1 AAV virus copy number. In addition, the beta-sarcoglycan gene is measured in the subject by detecting more than 1 rAAV vector genome copy per nucleus.

[0080] In any of these methods, uses, and compositions, serum CK levels in the subject are reduced by at least 82% by 60 days after administration of any of the disclosed rAAVs or compositions, compared to serum CK levels before administration of rAAV.

[0081] In any of these methods, uses, and compositions, the number of beta-sarcoglycan-positive fibers is detected by measuring beta-sarcoglycan protein levels by Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration. In addition, in any of these methods, uses, and compositions, the number of beta-sarcoglycan-positive fibers is measured by detecting more than one rAAV vector genome copy per nucleus.

[0082] In any of these methods, uses, and compositions, 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 rAAV administration. In addition, in any of the methods, uses, and compositions provided, alpha-sarcoglycan colocalizes to the membrane of cells expressing beta-sarcoglycan encoded by scAAVrh74.MHCK7.hSGCB.

[0083] 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 include, and may include, insect cells, e.g., Drosophila cells (e.g., S2 cells or Kc cells), silkworm cells (e.g., Bme21 cells), or mosquito cells (e.g., C6 / 36 cells), or mammalian cells (preferably human cells, e.g., human primary cells or established cell lines). In one embodiment, the mammalian cell is 293 cells, COS cells, HeLa cells, or KB cells.

[0084] 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. rAAV can be generated using host cells that stably possess the AAV vector plasmid. In one embodiment, the AAV vector plasmid is the pAAV.MHCK7.hSGCB.KAN plasmid.

[0085] 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.

[0086] Methods for alleviating fibrosis in subjects requiring it are also provided. In this regard, the method comprises administering a therapeutically effective amount of the rAAV 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.

[0087] 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 2E (LGMD2E).

[0088] As used herein, the term “fibrosis” refers to the excessive or uncontrolled deposition of extracellular matrix (ECM) components and the abnormal repair processes in post-injury tissues, including skeletal muscle, cardiac muscle, liver, lungs, kidneys, and pancreas. The deposited ECM components include collagen (e.g., collagen 1, collagen 2, or collagen 3) and fibronectin.

[0089] 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 human.

[0090] 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.

[0091] 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.

[0092] 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%.

[0093] 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. [Brief explanation of the drawing]

[0094] [Figure 1] This provides a schematic diagram of the rAAVrh74.MHCK7.SGCB therapeutic β-sarcoglycan transgene cassette. It is a self-complementary AAV vector containing the codon-optimized human β-sarcoglycan gene (hSGCB). A muscle-specific MHCK7 promoter drives expression. The cassette also includes a chimeric intron to enhance processing and polyadenylation signals for stability. [Figure 2A-1] rAAVrh74.MHCK7.SGCB is a nucleotide to which the annotation has been added. [Figure 2A-2] rAAVrh74.MHCK7.SGCB is a nucleotide to which the annotation has been added. [Figure 2B] Nucleotides annotated with rAAVrh74.MHCK7.SGCB [Figure 2C] This provides a nucleotide sequence annotated with rAAVrh74.MHCK7.SGCB. [Figure 3] This shows robust expression and myocyte membrane localization of SGCB 60 days after injection of rAAVrh74.MHCK7.SGCB. [Figure 4] This shows increased expression of SGCD and SGCG proteins in the muscle cell membrane 60 days after injection of rAAVrh74.MHCK7.SGCB. [Figure 5] This shows the reduction in creatine kinase (CK) after treatment with rAAVrh74.MHCK7.SGCB (SRP-9003). LLN = lower limit of normal, ULN = upper limit of normal. [Figure 6] This study demonstrates that rAAVrh74.MHCK7.SGCB(SRP-9003) treatment leads to sustained improvement in the total NSD score. [Figure 7] This study demonstrates that patients treated with rAAVrh74.MHCK7.SGCB(SRP-9003) show improvement in total NSAD scores compared to natural progression data. [Modes for carrying out the invention]

[0095] 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).

[0096] 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.

[0097] 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.”

[0098] 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.

[0099] 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.

[0100] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells, provided with certain functions by co-infecting helper viruses. Currently, there are 13 serotypes of AAV that have been characterized. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all of which possess three related capsid proteins, including the one expressed in AAV2. The degree of relatedness 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 ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] "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.

[0105] 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.

[0106] 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.

[0107] 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 desired heterogeneous nucleotide sequence 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 desired recombinant nucleotide sequence) into infectious recombinant virion particles for subsequent gene delivery.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] "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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] The term “promoter” is used herein in its usual sense to refer to a nucleotide region containing a DNA regulatory sequence, the regulatory sequence being derived from a gene that can bind to RNA polymerase and initiate transcription of a downstream (3'-direction) coding sequence. Transcription promoters may include “inducible promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.” In one embodiment, the promoter is a muscle-specific promoter, which includes, but is not limited to, human skeletal actin gene elements, cardiac actin gene elements, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer-binding factor mef-binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer elements, skeletal fast-twitch muscle troponin C gene elements, slow-twitch muscle cardiac troponin C gene elements, slow-twitch muscle troponin i gene elements, hypoxia-inducible nuclear factor (HIF) response elements (HRE), steroid-inducible elements, and glucocorticoid response elements (GRE). In another embodiment, the promoter is the MCK promoter, tMCK promoter, or MHCK7 promoter.

[0117] 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.

[0118] 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.

[0119] 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).

[0120] 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.

[0121] 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.

[0122] 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.

[0123] The term "subject" refers to any member of the animal kingdom, including, but not limited to, humans and non-human primates such as chimpanzees and other apes and monkey species, domesticated animals such as cattle, sheep, pigs, goats and horses, domesticated mammals such as dogs and cats, and experimental animals including rodents such as mice, rats and guinea pigs. In some embodiments, the subject is a human being in the range of birth to 2 years, 1 to 10 years, or 4 to 15 years, or 10 to 19 years, or 20 to 40 years, or 15 to 29 years or 25 to 55 years, or 40 to 60 years, or 50 years or older, or 60 years or older, or 65 years or older, or 70 years or older.

[0124] 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 publicly 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 presented under GenBank access number NC_002077, the complete genome of AAV-3 is presented under GenBank access number NC_1829, the complete genome of AAV-4 is presented under GenBank access number NC_001829, the genome of AAV-5 is presented under GenBank access number AF085716, the complete genome of AAV-6 is presented under GenBank access number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are presented under GenBank access numbers AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8), and the genome of AAV-9 is presented by Gao et al. The AAV-10 genome was presented in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome 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). The Cis action sequence that directs viral DNA replication (rep), capsid formation / packaging, and integration into host cell chromosomes is 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).

[0125] 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. In addition, AAV can transduce slow-dividing and non-dividing cells and persist essentially for 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, since the 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.

[0126] 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). See also 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 leads to the appearance of the transgene product in the systemic circulation after 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 cellular factors necessary for the correct glycosylation, folding, and secretion of antibodies, showing that muscles can stably express secreted protein therapeutics.

[0127] The recombinant AAV genome of this disclosure comprises the nucleic acid molecule of this disclosure 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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).

[0132] 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.

[0133] In exemplary embodiments, the recombinant AAV vector of this disclosure is produced by a triple transfection method using the AAV vector plasmid scAAV.MHCK7.hSCGB, pNLRep2-Caprh74, and pHelp (Xiao et al., J Virol 72, 2224-2232 (1998)), where rAAV contains an hSCGB gene expression cassette flanked by an AAV2 reverse terminal repeat (ITR), which is the sequence to be capsidated to AAVrh.74 virions. The plasmid contains the hSCGB sequence as well as the MHCK7 enhancer and core promoter elements of a muscle-specific promoter that drives 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.

[0134] 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. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 3.

[0135] 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, infectious adenovirus is not expected to be produced from such a production system. A schematic diagram of the pHELP plasmid is shown in Figure 4.

[0136] 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.

[0137] 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®.

[0138] The titer of rAAV administered by the method of this disclosure will vary depending, for example, on the specific rAAV, mode of administration, therapeutic target, targeted individual, and cell type, and may be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 The range may be from or above the DNase-resistant particle (DRP) range. Dosage may be expressed in units of viral genome (vg). One exemplary method for determining the encapsulated vector genome titer 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 the dose determined by a supercoiled DNA standard.

[0139] A method for transducing target cells with rAAV in vivo or in vitro is envisioned by this disclosure. The in vivo method comprises the step of administering a composition comprising an effective dose or multiple effective doses of the rAAV of this disclosure to an animal (including humans) in need thereof. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In embodiments of this disclosure, the effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition being treated, delays or prevents progression to the disorder / disease condition, reduces the severity of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease envisioned for prevention or treatment by the method of this disclosure is muscular dystrophy, e.g., limb-girdle muscular dystrophy or Duchenne muscular dystrophy.

[0140] Combination therapies are also contemplated in this disclosure. The combinations used herein include both concurrent and sequential therapies. In particular, combinations of the methods disclosed herein with standard medical treatments (e.g., corticosteroids), such as in combination with novel therapies, are contemplated.

[0141] The effective dose of the composition, combination therapy, or formulation may be administered via a route standard 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 this disclosure (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 the hSCGB protein.

[0142] This disclosure provides topical and systemic administration of effective doses of the rAAV, formulations, and compositions of this disclosure. 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 through injection, infusion, or transplantation.

[0143] In particular, the practical administration of rAAV according to this disclosure can be achieved by using any physical method for transporting the rAAV recombinant vector to the target tissue of an animal. Administration according to this disclosure includes, but is not limited to, intramuscular injection 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 may be administered co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV is targeted to a specific target tissue of interest, 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 in advance and may be used when carrying out this disclosure. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] Transduction of cells with rAAVs of this disclosure results in sustained expression of the hSCGB protein. Therefore, this disclosure provides methods for administering / delivering rAAVs expressing the hSCGB 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, includes the actin and myosin gene families, e.g., those derived from the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)), muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)), regulatory elements derived from the human skeletal muscle actin gene (Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)), regulatory elements derived from the cardiac actin gene, and muscle creatine kinase sequence elements (Johnson et al. The present invention provides a method for transducing muscle cells and muscle tissue directed by muscle-specific regulatory elements, including a mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the skeletal fast-twitch muscle troponin C gene, the slow-twitch muscle cardiac troponin C gene, and the slow-twitch muscle troponin I gene, a hypoxia-induced nuclear factor-binding element (Semenza et al., Proc. Natl. Acad. Sci. USA 88:5680-5684 (1991)), a glucocorticoid response element (GRE), steroid-induced elements and promoters (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), and other regulatory elements.

[0150] 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 hSCGB derived from transduced myofibrils.

[0151] Therefore, this disclosure provides a method for administering an effective dose (or doses administered essentially simultaneously or at intervals) of rAAV encoding hSCGB to a subject in need of it.

[0152] The titer of rAAV administered by the method of the present invention varies depending, for example, on the specific rAAV, the method of administration, the treatment target, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per mL. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 The above range may apply to DNase-resistant particles (DRPs). The dosage may be expressed in units of viral genome (vg). The titer of rAAV may be determined by a supercoiled plasmid quantitative standard or a linearized plasmid quantitative standard.

[0153] The present invention envisions a method for transducing target cells with rAAV in vivo or in vitro. The in vivo method comprises the step of administering an effective dose or multiple effective doses of a composition comprising rAAV of the present invention to an animal (including humans) in need. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition to be treated, a dose that slows or prevents progression to the disorder / disease condition, a dose that reduces the 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 for which the method of the present invention is envisioned for prevention or treatment is muscular dystrophy, e.g., limb-girdle muscular dystrophy. Thus, provided is a method for transducing target cells with rAAV scAAVrh74.MHCK7.hSGCB, comprising the nucleotide sequence of SEQ ID NO: 1.

[0154] 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.

[0155] 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.

[0156] 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 assay and liver function monitoring by GGT.

[0157] 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.

[0158] For example, therapeutically effective doses of rAAV vector are approximately 1e13vg / kg, 2e13vg / kg, 3e13vg / kg, 4e13vg / kg, 5e13vg / kg, 6e13vg / kg, 7e13vg / kg, 7.4e13vg / kg, 8e13vg / kg, 9e13vg / kg, 1e14vg / kg, 2e14vg / kg, 3e14vg / kg, 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 supercoiled standard qPCR titration or linear standard qPCR titration. In one embodiment, the therapeutically effective dose of rAAV is a dose of 5 e13 vg / kg based on a supercoiled 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, the therapeutically effective dose of rAAV is a dose of 2 e14 vg / kg based on a supercoiled 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.MHCK7.hSGCB 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, based on the supercoil plasmid as a quantitative standard. e13vg / 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 approximately 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, and It is 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 g to approximately 1e14vg / kg, or approximately 5e13vg / kg to approximately 2e14vg / kg, or 5e13vg / kg to approximately 3e14vg / kg, or approximately 5e13vg / kg to approximately 4e14vg / kg, or approximately 5e13vg / kg to approximately 5e14vg / kg, or approximately 1e14vg / kg to approximately 2e14vg / kg, or 1e14vg / kg to approximately 3e14vg / kg, or approximately 1e14vg / kg 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 these doses of rAAV vectors.

[0159] 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.

[0160] 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.

[0161] 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 (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference.

[0162] 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 × 10⁻⁶ 12 vg / ml ~ approx. 5.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 5.0 × 10⁶ based on a supercoiled plasmid as a quantitative standard. 12 vg / ml ~ approx. 1.0×10 14 The concentration is vg / ml. In another embodiment, rAAV is approximately 2.0 × 10⁶ based on a supercoiled plasmid as a quantitative standard. 13 The concentration is vg / ml. In one embodiment, rAAV is the scAAVrh74.MHCK7.hSGCB vector. In one embodiment, the concentration of rAAV in the composition or formulation is 1 × 10⁻¹⁶ based on a supercoiled plasmid as a quantitative standard. 13 vg / ml ~ 2 × 10 14 The concentration is vg / ml. In another embodiment, the concentration is 2 × 10 based on a supercoiled plasmid as a quantitative standard. 13 vg / ml, 4 x 10 13 vg / ml, or 5×10 13The concentration is vg / ml. In one embodiment, the buffer comprises one or more of Tris, Trisine, 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, 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.

[0163] Accordingly, this specification also describes a method for administering an effective dose (or a dose essentially administered simultaneously or at intervals) of rAAV encoding β-sarcoglycan to a mammalian subject in need thereof.

[0164] 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.

[0165] 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]

[0166] Preclinical studies using scAAVrh74.MHCK7.hSGCB are described in International Patent Publication No. 2017 / 180976, which is incorporated herein by reference in its entirety.

[0167] Example 1 Construction of scAAVrh.74.MHCK7.hSGCB A transgene cassette containing codon-optimized full-length human SCGB cDNA, as shown in Figure 1, was constructed. The cassette contains a consensus Kozak sequence (CCACC), an SV40 chimeric intron, a synthetic polyadenylation site, and muscle-specific MHCK7, which is used to drive cassette expression. This is an MCK-based promoter that utilizes a 206 bp enhancer harvested from approximately 1.2 kb at 5' of the transcription start site within an endogenous muscle creatine kinase gene (enh358MCK, 584 bp) with a proximal promoter. The cassette was packaged in a self-complementary (sc)AAVrh.74 vector that is 93% homologous to AAV8. AAVrh.74 has been shown to be safe and effective in mice and non-human primates, particularly when crossing the blood-barrier upon delivery to muscle via circulation. (Chicoine et al.,Mol Ther 2014;22:713-724.,Rodino-Klapac et al.,Mol Ther 2010;18:109-117,Chicoine et al.,Mol Ther 2014;22:338-347)

[0168] 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 2]

[0169] Example 2 LGMD2E Open-Label Trial Six patients treated with rAAVrh74.MHCK7.SGCB demonstrated clinical efficacy through robust target tissue transduction and transgene expression (biceps brachii, TA) (Figure 3), as well as improvements in multiple outpatient clinical outcome measures. This clinical dose-escalation study evaluated two dose cohorts (each containing n=3 patients), in which patients received rAAVrh74.MHCK7.SGCB (referred to herein as "SRP-9003") at 1.85 × 10⁶ doses. 13 vg / kg and 7.41 × 10 13 The drug was administered via intravenous infusion at a dose of vg / kg. 1.85 × 10⁶ 13 The dosage is 5 × 10⁻⁶ g / kg. 13 The results were measured by qPCR using linear reference plasmid DNA qPCR with its supercoiled reference DNA equivalent at vg / kg. 7.41 × 10⁻⁶ 13 vg / kg is 2 × 10 14 The measurement was performed by qPCR using linear reference plasmid DNA qPCR with its supercoiled reference DNA equivalent at vg / kg.

[0170] As shown in Table 1, dose-dependent increases were observed in both target tissue transduction (measured as vector genome copies per nucleus) and β-SG protein percentage (measured as immunofluorescence of mean β-sarcoglycan-positive fiber percentage (PβSGPF), immunofluorescence of mean fluorescence expression percentage (PFE), and total protein expression by Western blotting). Furthermore, 1.84 x 10⁻¹⁰ 13 In Cohort 1 patients treated with vg / kg and whose 2-year biopsies were recently obtained, β-SG expression was maintained at a high level from day 60 to year 2. [Table 3]

[0171] Deficiency of β-sarcoglycan (SGCB) protein leads to a reduction or complete loss of other sarcoglycan subunits from the membrane. Following administration of SRP-9003, reconstitution of SGCB expression resulted in increased expression of δ-sarcoglycan (SGCD) and γ-sarcoglycan (SGCG) (Figure 4). The simultaneous increase in the expression of other sarcoglycans and the localization of β-SG to the myocyte membrane (measured by SGCB-positive fiber percentage and fiber strength) indicates that SGCB expression induced by rAAVrh74.MHCK7.SGCB was functional and correctly localized within myocytes, as expected from the native SCGB protein.

[0172] Elevated CK levels were statistically significantly inversely correlated with disease duration (Semplicini 2015), indicating that significant increases in CK, such as those observed at baseline in patients in Cohort 1, signify an active process of muscle deterioration (i.e., active disease before permanent deterioration). Systemic gene transfection in SRP-9003 resulted in an early and substantial reduction in mean CK levels, which was maintained at the 750-day assessment (74%) (Figure 5).

[0173] Patients treated with rAAVrh74.MHCK7.SGCB showed clinically meaningful improvements in multiple functional motor assessments and time-dependent functional tests, including the North Star Assessment of Dysferlinopathy (NSAD), between years 1 and 2 (Figure 6 and Table 2). Functional improvements in both cohorts were most impressively demonstrated by an overall 5-point improvement in the total NSAD score across the two dose cohorts. Importantly, the improvements observed in treated patients persisted to the end of year 2. [Table 4]

[0174] Example 3 Natural progression clinical trial In the rAAVrh74.MHCK7.SGCB trial, the following criteria, the same as those used in the open-label clinical trial, were applied to the "natural course" dataset to select patients for the "natural course" control cohort for comparison with treated patients: ●Limited to LGMD2E / R4 outpatients only. For this cohort, it was defined as having no missing 10MWR values. ● Baseline age: 4 to 15 years old ● 100MWR trial results: More than 40% of the predicted results for age, height, sex, and weight were consistent with healthy controls.

[0175] To derive a natural course comparator for patients treated with rAAVrh74.MHCK7.SGCB(SRP-9003), five control patients meeting the criteria applied to the entire “natural course” database were identified from the overall dataset of 35 patients. Baseline comparisons between the treatment cohort and the natural course cohort are shown in Table 3. Age and sex are well-balanced between the rAAVrh74.MHCK7.SGCB treatment population and the “natural course” control cohort. In particular, baseline functional endpoint scores are higher in the NCH control cohort. [Table 5]

[0176] Following administration of rAAVrh74.MHCK7.SGCB, overall improvement was observed in subjects treated with gene therapy compared to the declining trend in the “natural course” control cohort (Figure 7). Changes from baseline in NSAD scores for individual subjects are provided as NH = natural course, NSAD = Northstar assessment of dysferlinopathy, and SD = standard deviation. Patients treated with SRP-9003 showed clinically meaningful improvement in functional outcomes in an exploratory comparison with the LGMD2E / R4 natural course cohort, as measured by NSAD.

[0177] The safety and tolerability of rAAVrh74.MHCK7.SGCB were determined by evaluating adverse events, physical examination, vital signs, serum and urinalysis results, and immunogenicity at each visit up to 18 months for Cohort 1 and up to 6 months for Cohort 2. No other laboratory abnormalities indicating safety concerns were observed in either cohort. No abnormal platelet counts outside the normal range were observed. There were no clinical sequelae associated with complement activation. The results did not indicate any new safety signals, and all treatment-related adverse events occurred early, were transient, and manageable. References: 1 Bonnemann CG, Modi R, Noguchi S, Mizuno Y, Yoshida M, Gussoni E et al. Beta-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995;11:266-273. 2 Moore SA, Shilling CJ, Westra S, Wall C, Wicklund MP, Stolle C et al. Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 2006;65:995-1003. 3 Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E et al.Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in beta-sarcoglycan-deficient mice.Hum Mol Genet 1999;8:1589-1598. 4 Durbeej M,Cohn RD,Hrstka RF,Moore SA,Allamand V,Davidson BL et al.Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell 2000;5:141-151. 5 Bonnemann CG,Passos-Bueno MR,McNally EM,Vainzof M,de Sa Moreira E,Marie SK et al.Genomic screening for beta-sarcoglycan gene mutations:missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E).Hum Mol Genet 1996;5:1953-1961. 6 Angelini C,Fanin M,Freda MP,Duggan DJ,Siciliano G,Hoffman EP.The clinical spectrum of sarcoglycanopathies.Neurology 1999;52:176-179. 7 Sandona D,Betto R.Sarcoglycanopathies:molecular pathogenesis and therapeutic prospects.Exp Rev Mol Med 2009;11:e28. 8 Fanin M,Melacini P,Boito C,Pegoraro E,Angelini C.LGMD2E patients risk developing dilated cardiomyopathy.Neuromusc Disord 2003;13:303-309. 9 Sveen ML,Thune JJ,Kober L,Vissing J.Cardiac involvement in patients with limb-girdle muscular dystrophy type 2 and Becker muscular dystrophy.Arch Neurol 2008;65:1196-1201. 10 Melacini P,Fanin M,Duggan DJ,Freda MP,Berardinelli A,Danieli GA et al.Heart involvement in muscular dystrophies due to sarcoglycan gene mutations.Muscle Nerve 1999;22:473-479. 11 Narayanaswami P,Weiss M,Selcen D,David W,Raynor E,Carter G et al.Evidence-based guideline summary:diagnosis and treatment of limb-girdle and distal dystrophies:report of the guideline development subcommittee of the American Academy of Neurology and the practice issues review panel of the American Association of Neuromuscular & Electrodiagnostic Medicine.Neurology 2014;83:1453-1463. 12 Wong-Kisiel LC,Kuntz NL.Two siblings with limb-girdle muscular dystrophy type 2E responsive to deflazacort.Neuromusc Disord 2010;20:122-124. 13 Barresi R,Di Blasi C,Negri T,Brugnoni R,Vitali A,Felisari G et al.Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.J Med Genet 2000;37:102-107. 14 Gibertini S,Zanotti S,Savadori P,Curcio M,Saredi S,Salerno F et al.Fibrosis and inflammation are greater in muscles of beta-sarcoglycan-null mouse than mdx mouse.Cell Tissue Res 2014;356:427-443. 15 McCarty DM,Fu H,Monahan PE,Toulson CE,Naik P,Samulski RJ.Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther 2003;10:2112-2118. 16 McCarty DM,Monahan PE,Samulski RJ.Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.Gene Ther 2001;8:1248-1254. 17 Chicoine LG,Rodino-Klapac LR,Shao G,Xu R,Bremer WG,Camboni M et al.Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin alpha2 surrogates.Mol Ther 2014;22:713-724. 18 Rodino-Klapac LR,Montgomery CL,Bremer WG,Shontz KM,Malik V,Davis N et al.Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.Mol Ther 2010;18:109-117. 19 Rodino-Klapac LR,Janssen PM,Montgomery CL,Coley BD,Chicoine LG,Clark KR et al.A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy.J Transl Med 2007;5:45. 20 Wang B,Li J,Fu FH,Chen C,Zhu X,Zhou L et al.Construction and analysis of compact muscle-specific promoters for AAV vectors.Gene Ther 2008;15:1489-1499. 21 Chicoine LG,Montgomery CL,Bremer WG,Shontz KM,Griffin DA,Heller KN et al.Plasmapheresis eliminates the negative impact of AAV antibodies on micro-dystrophin gene expression following vascular delivery.Mol Ther 2014;22:338-347. 22 Matsuda R,Nishikawa A,Tanaka H.Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue:evidence of apoptosis in dystrophin-deficient muscle.J Biochem 1995;118:959-964. 23 Straub V,Rafael JA,Chamberlain JS,Campbell KP.Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.J Cell Biol 1997;139:375-385. 24 Mendell JR,Sahenk Z,Malik V,Gomez AM,Flanigan KM,Lowes LP et al.A phase 1 / 2a follistatin gene therapy trial for becker muscular dystrophy.Mol Ther 2015;23:192-201. 25 Dressman D,Araishi K,Imamura M,Sasaoka T,Liu LA,Engvall E et al.Delivery of alpha- and beta-sarcoglycan by recombinant adeno-associated virus:efficient rescue of muscle,but differential toxicity.Hum Gene Ther 2002;13:1631-1646. 26 Rodino-Klapac LR,Lee JS,Mulligan RC,Clark KR,Mendell JR.Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D.Neurology 2008;71:240-247. 27 Shield MA,Haugen HS,Clegg CH,Hauschka SD.E-box sites and a proximal reg-ulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol Cell Biol 1996;16:5058-5068. 28 Rabinowitz JE,Rolling F,Li C,Conrath H,Xiao W,Xiao X et al.Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.J Virol 2002;76:791-801. 29 Grieger JC,Choi VW,Samulski RJ.Production and characterization of adeno-associated viral vectors.Nat Protoc 2006;1:1412-1428. 30 Clark KR,Liu X,McGrath JP,Johnson PR.Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.Hum Gene Ther 1999;10:1031-1039. 31 Liu M,Yue Y,Harper SQ,Grange RW,Chamberlain JS,Duan D.Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury.Mol Ther 2005;11:245-256. 32 Hakim CH,Grange RW,Duan D.The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to 20-mo-old mdx mice.J Appl Physiol 2011;110:1656-1663. 33 Wein N,Vulin A,Falzarano MS,Szigyarto CA,Maiti B,Findlay A et al.Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.Nat Med 2014;20:992-1000.

Claims

1. A polynucleotide sequence comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:1, wherein said nucleotide sequence is self-complementary and encodes a protein that retains beta-sarcoglycan activity.

2. The polynucleotide sequence of claim 1, wherein at least 90% are determined by BLAST as an alignment tool using default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; explanation=50 sequences; permutation=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss Protein+Spupdate+PIR.

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

1.

4. A recombinant AAV (rAAV) comprising a polynucleotide sequence, the polynucleotide sequence comprising, in the 5' to 3' direction, 1) a complementary sequence of a polyadenylation sequence, (2) a sequence complementary to a cDNA sequence encoding the amino acid sequence of SEQ ID NO: 3, (3) a complementary sequence of an intron, (4) a complementary sequence of a muscle-specific promoter, (5) a 5' ITR sequence, (6) the muscle-specific promoter sequence, (7) the intron, (8) a cDNA sequence encoding the amino acid sequence of SEQ ID NO: 3, and (9) the polyadenylation sequence; A recombinant AAV, wherein the polynucleotide sequence is flanked by two 3' ITR sequences, and the two 3' ITR sequences are complementary.

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 muscle cell-specific enhancer-binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C5-12, 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 described in claim 5, wherein the muscle-specific control element is truncated MCK (tMCK).

7. The recombinant AAV described in claim 5, wherein the muscle-specific regulatory element is MHCK7.

8. A recombinant AAV as described in claim 4, comprising the nucleotide sequence of SEQ ID NO:

1.

9. The recombinant AAV of claim 4, wherein the vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAV rh.

74.

10. A composition comprising the recombinant AAV described in claim 4.

11. A composition for use in the treatment of muscular dystrophy, comprising the recombinant AAV of claim 4.

12. A composition for increasing muscle strength and / or muscle mass in a mammalian subject suffering from muscular dystrophy, comprising the recombinant AAV of claim 4.

13. A composition for use according to claim 11 or claim 12, wherein the systemic administration is parenteral administration by injection, infusion, or implantation.

14. A composition for use as described in claim 11 or claim 12, wherein the composition is formulated for systemic administration.

15. A composition for use according to claim 11 or claim 12, wherein the composition is formulated for intravenous administration.

Images