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BR-122025011514-A2 - USE OF A COMPOSITION COMPRISING RECOMBINANT ADENOASSOCIATED VIRUS (rAAV) IN THE TREATMENT OF MUSCULAR DYSTROPHY

BR122025011514A2BR 122025011514 A2BR122025011514 A2BR 122025011514A2BR-122025011514-A2

Abstract

The present disclosure provides gene therapy vectors, for example, recombinant adeno-associated virus (rAAV), produced in adherent mammalian cells cultured under suspension conditions, for the expression of a human microdystrophin gene. The present disclosure also provides compositions and methods of utilizing these rAAVs to treat muscular dystrophy such as, for example, Duchenne muscular dystrophy.

Inventors

  • Maroof ALAM
  • Louise Rodino-Klapac

Assignees

  • SAREPTA THERAPEUTICS, INC.

Dates

Publication Date
20260317
Application Date
20220513
Priority Date
20210517

Claims (18)

  1. 1. Use of a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin characterized in that it is used in the manufacture of a medicament for the treatment of muscular dystrophy, wherein the medicament is for administration to a genotyped patient, wherein the patient is genotyped for at least one mutation in exons 18-79 of the human dystrophin (DMD) gene.
  2. 2. Use according to claim 1, characterized in that the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ. ID. NO: 9; b) rAAV particles comprising nucleotides 55-5021 of SEQ. ID. NO: 3; and/or c) rAAV particles comprising nucleotides 1-4977 of SEQ. ID. NO: 8.
  3. 3. Use according to claim 1 or 2, characterized in that the medicinal product comprising rAAV is for administration by injection, infusion or implantation.
  4. 4. Use according to claim 3, characterized in that the medicinal product comprising rAAV is for administration by infusion over approximately one hour; optionally in that the rAAV is for administration intravenously via a peripheral vein of the limb.
  5. 5. Use, according to any one of claims 1 to 4, characterized in that the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.
  6. 6. Use, according to claim 5, characterized in that the muscular dystrophy is Duchenne muscular dystrophy.
  7. 7. Use, according to any one of claims 1 to 6, characterized in that the level of microdystrophin gene expression in a cell of the individual is increased after administration of rAAV, compared with the level of microdystrophin gene expression before administration of rAAV.
  8. 8. Use, according to claim 7, characterized in that the expression of the microdystrophin gene in the cell is detected by measuring the level of microdystrophin protein by WESTERN BLOT in muscle biopsied before and after administration of the medicinal product comprising rAAV.
  9. 9. Use according to claim 8, characterized in that expression is at least 55.4% after administration of rAAV, compared previously.
  10. 10. Use, according to any one of claims 1 to 9, characterized in that the average percentage of microdystrophin-positive fibers in the individual's muscle tissue is increased after administration of the drug comprising rAAV, compared with the number of microdystrophin-positive fibers before administration of the drug comprising rAAV.
  11. 11. Use, according to claim 10, characterized in that the average percentage of microdystrophin-positive fibers is at least 70.5% and the average intensity is at least 116.9% as detected by immunofluorescence (IF) in muscle biopsies before and after administration of rAAV.
  12. 12. Use, according to any one of claims 1 to 11, characterized in that microdystrophin transduction by vector genome count is at least 3.87 average vector genome copies per nucleus.
  13. 13. Use, according to any one of claims 1 to 12, further characterized by comprising genotyping of the DMD gene of the human individual prior to administration of the composition to said human individual.
  14. 14. Use, according to claim 13, characterized in that genotyping detects at least one mutation in exons 18 to 79 of the DMD gene.
  15. 15. Use, according to claim 14, characterized in that at least one mutation is a frameshift deletion, a frameshift duplication, a premature stop, or other pathogenic variant that results in the absence of expression of the human dystrophin protein.
  16. 16. Use, according to any one of claims 1 to 15, characterized in that the medicinal product comprising rAAV is for administration using a systemic route of administration and at a dose of about 5.0 x 1012 vg/kg to about 1.0 x 1015 vg/kg.
  17. 17. Use, according to any one of claims 1 to 16, characterized in that the systemic route of administration is an intravenous route and the dose of the medicinal product comprising rAAV is for administration of approximately 2 x 1014 vg/kg.
  18. 18. Use, according to any one of claims 1 to 17, characterized in that the dose of rAAV is for administration at a concentration of approximately 10 ml/kg.

Description

CROSS-REFERENCE WITH RELATED ORDERS [001] This application claims the benefit of U.S. Provisional Application No. 63/253,998, filed October 8, 2021, U.S. Provisional Application No. 63/243,944, filed September 14, 2021, U.S. Provisional Application No. 63/209,733, filed June 11, 2021, and U.S. Provisional Application No. 63/189,676, filed May 17, 2021, which are hereby incorporated by reference in their entirety. REFERENCE TO THE LIST OF SEQUENCES SUBMITTED ELECTRONICALLY [002] The content of the sequence listing submitted electronically in an ASCII text file (Name: 4140_052PC04_Seqlisting_ST25.txt; size: 60,194 bytes and Creation Date: May 13, 2022), filed with the application, is incorporated in this descriptive report by reference in its entirety. FIELD [003] The present disclosure is in the field of gene therapy. More particularly, the present disclosure provides gene therapy vectors, for example, adeno-associated virus (AAV) vectors, for the expression of a miniaturized human microdystrophin gene, wherein the AAVs are produced by adherent cells cultured under suspension conditions. The present disclosure also provides methods of utilizing these vectors to express microdystrophin in skeletal muscles, including the diaphragm and cardiac muscle, and to protect muscle fibers from injury, increase muscle strength, and reduce and/or prevent fibrosis in individuals suffering from muscular dystrophy. BACKGROUND [004] The importance of muscle mass and strength for daily activities, such as locomotion and respiration, and for whole-body metabolism is unequivocal. Deficits in muscle function produce muscular dystrophies (MDs), which are characterized by weakness and loss of muscle mass and have serious impacts on quality of life. The best-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with loss of anchoring of the sarcolemmal cytoskeleton by DAPC. Duchenne Muscular Dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn males. [005] DMD is caused by mutations in the DMD gene that lead to reductions in mRNA and the absence of dystrophin, a 427 kDa sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51: 919 28, 1987). The DAPC is composed of multiple proteins in the muscle sarcolemma that form the structural link between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, an actin-binding protein, and alpha-dystroglycan, a laminin-binding protein. These structural links act to stabilize the muscle cell membrane during contraction and protect against contraction-induced damage. With the loss of dystrophin, membrane fragility results in sarcolemmal tears and an influx of calcium, triggering calcium-activated proteases and segmental necrosis of the fiber (Straub et al., Curr Opin. Neurol. 10: 168-75 (1997)). This uncontrolled cycle of muscle degeneration and regeneration eventually depletes the population of muscle stem cells (Sacco et al., Cell 143: 1059-1071 (2010); Wallace et al., Annu. Rev. Physiol. 71: 37-57 (2009)), resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scarring. [006] Without membrane stabilization by dystrophin or a microdystrophin, DMD will manifest as uncontrolled cycles of tissue injury and repair, ultimately replacing lost muscle fibers with fibrotic scar tissue through connective tissue proliferation. Fibrosis is characterized by excessive deposits of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced by cytokines such as TGFβ, which are released by activated fibroblasts responding to stress and inflammation. Although the primary pathological feature of DMD is myofiber degeneration and necrosis, fibrosis as a pathological consequence has equal repercussions. The overproduction of fibrotic tissue restricts muscle regeneration and contributes to progressive muscle weakness in the patient with DMD. In one study, the presence of fibrosis in muscle biopsies of early DMD was highly correlated with poor motor outcome in a 10-year follow-up (Desguerre et al., J. Neuropathol. Exp. Neurol. 68: 762-767 (2009)). These results point to fibrosis as an important contributor to muscle dysfunction in DMD and highlight the need for early intervention before fibrosis becomes evident. [007] International Publication No. WO 2019/245973 A1, incorporated by reference in this descriptive report in its entirety, describes the AAV vector delivery of the microdystrophin gene to treat muscular dystrophy (e.g., DMD) in a human individual. There is a continued need in the art, however, for improved production methods of these AAV vectors, especially methods suitable for the large-scale fabrication of these gene therapy vectors. SUMMARY [008] The present disclosure is directed to gene therapy vectors, for example, AAV vectors, pr