阅读说明：本技术 用于治疗杜兴肌营养不良的组合物和方法 (Compositions and methods for treating duchenne muscular dystrophy ) 是由 H·斯特德曼 于 2019-04-16 设计创作，主要内容包括：本文描述了肌养蛋白或肌养蛋白相关蛋白的三重剪接突变体及其用于治疗杜兴肌营养不良的方法。还提供了病毒载体,其包含编码在指导其表达的调节元件控制下的三重剪接突变肌养蛋白或肌养蛋白相关蛋白的核酸。还提供了组合物,其含有配制用于递送给人患者的这种病毒载体。(Triple splice mutants of dystrophin or dystrophin-related proteins and methods of their use for treating duchenne muscular dystrophy are described herein. Also provided are viral vectors comprising a nucleic acid encoding a triple-spliced mutant dystrophin or dystrophin-related protein under the control of regulatory elements directing its expression. Compositions containing such viral vectors formulated for delivery to human patients are also provided.)

1. A recombinant adeno-associated virus (rAAV) having an AAV capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a dystrophin superfamily mutein comprising a hybrid triple helical domain under the control of regulatory sequences which direct its expression

A first helix comprising the N-terminal part of helix A fused to the C-terminal part of helix A';

a second helix comprising the N-terminal part of helix B' fused to the terminal C-terminal part of helix B; and

a third helix comprising the N-terminal part of helix C fused to the C-terminal part of helix C';

wherein helices A, B and C are present in the first triple helix repeat that is not adjacent to the second triple helix repeat having helices A ', B ' and C ' in the native dystrophin superfamily protein.

2. The rAAV of claim 1, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin.

3. The rAAV according to claim 1 or 2, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 3 through helical repeat 21 of full-length dystrophin.

4. The rAAV according to claim 1 or 2, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 3 through helical repeat 23 of full-length dystrophin.

5. The rAAV of any one of claims 1 to 3, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 1.

6. The rAAV of any one of claims 1 to 3 and 5, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 2.

7. The rAAV of claim 1, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 2 through helical repeat 19 of full-length dystrophin.

8. The rAAV of claim 1 or 7, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 22.

9. The rAAV of claim 1, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin-related protein.

10. The rAAV of claim 1 or 9, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin-related protein and comprises a deletion in at least helical repeat 3 to helical repeat 19 of a full length dystrophin-related protein.

11. The rAAV of claim 10, wherein the triple mutein comprises SEQ ID NO: 3.

12. The rAAV according to any one of claims 1, 9, 10, or 11, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 4.

13. The rAAV according to any one of claims 1 or 10, wherein the triple mutein comprises SEQ ID NO: 20.

14. The rAAV of claim 13, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 19, or a variant of SEQ ID NO: 19 from about 95% to about 99% identical.

15. The rAAV of claim 1 or 9, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin-related protein and comprises a deletion in at least helical repeat 2 through helical repeat 17 of a full length dystrophin-related protein.

16. The rAAV of claim 15, wherein the triple mutein comprises SEQ ID NO: 21.

17. A rAAV having an AAV capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a triple splice mutein of the dystrophin superfamily under the control of regulatory sequences which direct its expression, wherein the triple-spliced mutein comprises one or more N-terminal helical repeats, a hybrid triple-helical repeat and one or more C-terminal helical repeats, wherein the total number of helical repeats including hybrid repeats in the triple-spliced mutant protein is selected from any integer from 1 to 1 less than the number of helical repeats of the full-length dystrophin superfamily protein, and wherein the hybrid triple helical repeat is formed from two helical repeats spliced on a plane bisecting the helical repeat perpendicular to its long axis as depicted in figure 2F.

18. The rAAV of claim 17, wherein one or more N-terminal helical repeats of the mutein is directly adjacent to helical repeat 1 in two repeats forming a hybrid triple helical repeat when in a full-length dystrophin superfamily protein; and wherein one or more C-terminal helical repeats of the mutein are directly adjacent to helical repeat 2 in two repeats forming a hybrid triple helical repeat when in the full length dystrophin superfamily protein.

19. The rAAV according to claim 17 or 18, wherein the full length dystrophin superfamily protein is a dystrophin protein.

20. The rAAV according to any one of claims 17 to 19, wherein one or more N-terminal helical repeats of the mutein comprises helical repeat 1 in full-length dystrophin, and/or wherein one or more C-terminal helical repeats comprises helical repeat 23 and helical repeat 24 in full-length dystrophin.

21. The rAAV according to any one of claims 17 to 20, wherein the N-terminal helical repeat consists of helical repeat 1 in full-length dystrophin, wherein the C-terminal helical repeat consists of helical repeat 23 and helical repeat 24 in full-length dystrophin, wherein helical repeat 1 in the two repeats forming the hybrid triple helical repeat is helical repeat 2 in full-length dystrophin, and wherein helical repeat 2 in the two repeats forming the hybrid triple helical repeat is helical repeat 22 in full-length dystrophin.

22. The rAAV according to any one of claims 17 to 21, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 1.

23. The rAAV of claim 22, wherein the sequence encoding the triple-spliced mutein comprises SEQ ID NO: 2.

24. The rAAV according to claim 17 or 18, wherein the full length dystrophin superfamily protein is a dystrophin related protein.

25. The rAAV of any one of claims 17, 18, and 24, wherein one or more N-terminal helical repeats of the mutein comprise helical repeat 1 in a full-length dystrophin-related protein, and/or wherein the C-terminal helical repeats of the mutein comprise helical repeats 21 and 22 in a full-length dystrophin-related protein.

26. The rAAV of any one of claims 17, 18, 24, and 25, wherein the N-terminal helical repeat of the mutein consists of helical repeat 1 in a full-length dystrophin-related protein, wherein the C-terminal helical repeat of the mutein consists of helical repeat 21 and helical repeat 22 in a full-length dystrophin-related protein, wherein helical repeat 1 in the two repeats forming the hybrid triple helical repeat is helical repeat 2 in a full-length dystrophin-related protein, and wherein helical repeat 2 in the two repeats forming the hybrid triple helical repeat is helical repeat 20 in a full-length dystrophin-related protein.

27. The rAAV according to any one of claims 17, 18, and 24-27, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 3.

28. The rAAV of claim 27, wherein the sequence encoding the triple-spliced mutein comprises SEQ ID NO: 4.

29. The rAAV according to any one of claims 17, 18, and 24-26, wherein the triple mutein comprises the amino acid sequence of SEQ ID NO: 20.

30. The rAAV of claim 29, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 19, or a variant of SEQ ID NO: 19 from about 95% to about 99% identical.

31. A rAAV having an AAV capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a dystrophin superfamily triple-spliced mutein under the control of regulatory sequences that direct its expression, wherein the triple-spliced mutein comprises a hybrid triple-helical repeat and a C-terminal helical repeat, wherein the total number of helical repeats including the hybrid repeat in the triple-spliced mutein is 5, and wherein the hybrid triple-helical repeat is formed from two helical repeats spliced on a plane that bisects the helical repeats perpendicular to their long axis as depicted in figure 2F.

32. The rAAV of claim 31, wherein the C-terminal helical repeat of the mutein consists of helical repeats 21, 22, 23, and 24 in full-length dystrophin, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 1 in full-length dystrophin, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 20 in full-length dystrophin.

33. The rAAV of claim 32, wherein the triple-spliced mutein comprises SEQ ID NO: 22.

34. The rAAV of any one of claims 31, wherein the C-terminal helical repeat of the mutein consists of helical repeats 19, 20, 21, and 22 in a full-length dystrophin-related protein, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 1 in the full-length dystrophin-related protein, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 18 in the full-length dystrophin-related protein.

35. The rAAV of claim 34, wherein the triple-spliced mutein comprises SEQ ID NO: 21.

36. The rAAV according to any one of claims 1 to 35, wherein the regulatory sequence comprises a promoter, wherein the promoter is a constitutive promoter, or wherein the promoter is a muscle-specific promoter.

37. The rAAV according to claim 36, wherein the muscle-specific promoter is a Muscle Creatine Kinase (MCK) promoter or SPc5-12 promoter.

38. The rAAV according to any one of claims 1 to 37, wherein the regulatory sequences comprise a chimeric CS-CRM4/SPc5-12 promoter.

39. The rAAV according to any one of claims 1 to 38, wherein the regulatory sequence comprises an enhancer.

40. The rAAV of claim 39, wherein the enhancer is a double or triple tandem MCK enhancer.

41. The rAAV of any one of claims 1 to 40, wherein the recombinant adeno-associated virus is selected from AAV1, AAV5, AAV6, AAV8, AAV8 triplets, AAV9, Anc80, Anc81, and Anc 82.

42. A recombinant dystrophin superfamily triple-spliced mutant protein comprising a hybrid triple-helical domain comprising

A first helix comprising the N-terminal part of helix A fused to the C-terminal part of helix A';

a second helix comprising the N-terminal part of helix B' fused to the terminal C-terminal part of helix B; and

a third helix comprising the N-terminal part of helix C fused to the C-terminal part of helix C';

wherein helices A, B and C are present in the first triple helix repeat that is not adjacent to the second triple helix repeat having helices A ', B ' and C ' in the native dystrophin superfamily protein.

43. The recombinant protein according to claim 42, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin.

44. The recombinant protein according to claim 42 or 43, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 3 through helical repeat 21 of full-length dystrophin.

45. The recombinant protein according to claim 42 or 43, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 3 through helical repeat 23 of full-length dystrophin.

46. The recombinant protein according to any one of claims 42-44, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 1.

47. The recombinant protein according to any one of claims 42-44 and 46, wherein a nucleic acid sequence encoding said triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 2.

48. The recombinant protein according to claim 42, wherein the triple-spliced mutein comprises a deletion in at least helical repeat 2 through helical repeat 19 of full-length dystrophin.

49. The recombinant protein according to claim 42 or 48, wherein said triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 22.

50. The recombinant protein according to claim 42, wherein said dystrophin superfamily mutein is a triple splice mutant dystrophin related protein.

51. The recombinant protein according to claim 42 or 50, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin related protein and comprises a deletion in at least helical repeat 3 to helical repeat 19 of a full length dystrophin related protein.

52. The recombinant protein according to claim 51, wherein the triple mutein comprises the amino acid sequence of SEQ ID NO: 3.

53. The recombinant protein according to any one of claims 42 and 50-52, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 4.

54. The recombinant protein according to any one of claims 42 or 51, wherein the triple mutein comprises the amino acid sequence of SEQ ID NO: 20.

55. The recombinant protein according to claim 54, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 19, or a variant of SEQ ID NO: 19 from about 95% to about 99% identical.

56. The recombinant protein according to claim 42 or 50, wherein the dystrophin superfamily mutein is a triple splice mutant dystrophin related protein and comprises a deletion in at least helical repeat 2 to helical repeat 17 of a full length dystrophin related protein.

57. The recombinant protein according to any one of claims 42, 50, or 56, wherein the triple mutein comprises the amino acid sequence of SEQ ID NO: 21.

58. A recombinant dystrophin superfamily triple-spliced mutein comprising one or more N-terminal helical repeats, a heterozygous triple-helical repeat and one or more C-terminal helical repeats, wherein the total number of one or more helical repeats, including the heterozygous repeats, in the triple-spliced mutein is selected from any integer from 1 to 1 less than the number of helical repeats of the full-length dystrophin superfamily protein, and wherein the heterozygous triple-helical repeat is formed from two helical repeats spliced on a plane bisecting the helical repeats perpendicular to their long axis as depicted in figure 2F.

59. The recombinant protein according to claim 58, wherein one or more N-terminal helical repeats of the mutein are directly adjacent to helix repeat 1 in two repeats forming a hybrid triple helix repeat when in a full length dystrophin superfamily protein; and wherein one or more C-terminal helical repeats of the mutein are directly adjacent to helical repeat 2 in two repeats forming a hybrid triple helical repeat when in the full length dystrophin superfamily protein.

60. The recombinant protein according to claim 58 or 59, wherein the full length dystrophin superfamily protein is a dystrophin protein.

61. The recombinant protein according to any one of claims 58-60, wherein one or more N-terminal helical repeats of the mutein comprise helical repeat 1 in full-length dystrophin, and/or wherein one or more C-terminal helical repeats comprise helical repeat 23 and helical repeat 24 in full-length dystrophin.

62. The recombinant protein according to any one of claims 58-61, wherein the N-terminal helical repeat consists of helical repeat 1 in full-length dystrophin, wherein the C-terminal helical repeat consists of helical repeat 23 and helical repeat 24 in full-length dystrophin, wherein helical repeat 1 in the two repeats forming the hybrid three-stranded helical repeat is helical repeat 2 in full-length dystrophin, and wherein helical repeat 2 in the two repeats forming the hybrid three-stranded helical repeat is helical repeat 22 in full-length dystrophin.

63. The recombinant protein according to any one of claims 58-62, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 1.

64. The recombinant protein according to claim 63, wherein the sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 2.

65. The recombinant protein according to claim 58 or 59, wherein the full length dystrophin superfamily protein is a dystrophin related protein.

66. The recombinant protein according to any one of claims 58, 59, and 65, wherein one or more N-terminal helical repeats of the mutein comprise helical repeat 1 in full-length dystrophin-related protein, and/or wherein the C-terminal helical repeat of the mutein comprises helical repeats 21 and 22 in full-length dystrophin-related protein.

67. The recombinant protein according to any one of claims 58, 59, 65, and 66, wherein the N-terminal helical repeat of the mutein consists of helical repeat 1 in full-length dystrophin-related protein, wherein the C-terminal helical repeat of the mutein consists of helical repeat 21 and helical repeat 22 in full-length dystrophin-related protein, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 2 in full-length dystrophin-related protein, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 20 in full-length dystrophin-related protein.

68. The recombinant protein according to any one of claims 58, 59, and 65-67, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 3.

69. The recombinant protein according to claim 68, wherein the sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 4.

70. The recombinant protein according to any one of claims 58, 59, and 65-67, wherein the triple mutein comprises the amino acid sequence of SEQ ID NO: 20.

71. The recombinant protein according to claim 70, wherein the nucleic acid sequence encoding the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 19, or a variant of SEQ ID NO: 19 from about 95% to about 99% identical.

72. A recombinant dystrophin superfamily triple-spliced mutein comprising a heterozygous triple-helical repeat sequence and a C-terminal helical repeat sequence, wherein the total number of helical repeats, including the heterozygous repeat sequence, in the triple-spliced mutein is 5, and wherein the heterozygous triple-helical repeat sequence is formed by two helical repeat sequences spliced on a plane bisecting the helical repeat sequence perpendicular to its long axis as depicted in figure 2F.

73. The recombinant protein according to claim 72, wherein the C-terminal helical repeat of the mutein consists of helical repeats 21, 22, 23 and 24 in full-length dystrophin, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 1 in full-length dystrophin, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 20 in full-length dystrophin.

74. The rAAV of claim 73, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 22.

75. The rAAV of any one of claims 72, wherein the C-terminal helical repeat of the mutein consists of helical repeats 19, 20, 21, and 22 in a full-length dystrophin-related protein, wherein helix repeat 1 of the two repeats forming the hybrid triple helix repeat is helix repeat 1 in the full-length dystrophin-related protein, and wherein helix repeat 2 of the two repeats forming the hybrid triple helix repeat is helix repeat 18 in the full-length dystrophin-related protein.

76. The rAAV of claim 75, wherein the triple-spliced mutein comprises the amino acid sequence of SEQ ID NO: 21.

77. A novel recombinant mutant dystrophin protein comprising SEQ ID NO: 1. 13, 14, 15, 16, 17, 18 or 22.

78. A novel recombinant mutant dystrophin-related protein comprising SEQ ID NO: 3. 20 or 21.

79. A recombinant nucleic acid sequence encoding a mutant dystrophin superfamily protein of any one of claims 42 to 78.

80. A plasmid comprising a nucleic acid sequence according to claim 79, under the control of regulatory sequences which direct its expression.

81. A pharmaceutical composition comprising the rAAV of any one of claims 1-41 in a formulation buffer.

82. A method of treating a subject diagnosed with Duchenne muscular dystrophy comprising administering the composition of claim 81.

83. The rAAV of any one of claims 1-41, for use in a method of treating MDS in a subject in need thereof.

84. Use of the rAAV according to any one of claims 1-41 in the manufacture of a medicament for treating duchenne muscular dystrophy.

Background

Duchenne Muscular Dystrophy (DMD) is a severely disabling systemic childhood-onset disease that progresses from early motor weakness to end-stage respiratory and cardiomyopathy characterized by very expensive caregiver and technical dependencies (Landfeldt, E. et al, The burden of Dual pulmonary dynamics: an international, cross-sectional study. Neurology, 2014.83 (6): pages 529-36; and Schreiber-Katz, O. et al, Comparative sample of bone analysis and assessment of health care of Dunne and Beer pulmonary muscular strategies in Germany study. Orny Dis, 2014.9: page 210). DMD is one of the most common single-gene lethal diseases in humans, with a historical global incidence of about 1:4000 male births. The molecular basis is the absence of the 427 kd isoform of the cytoskeletal protein dystrophin (Dp 427), which in most cases is caused by a multiple exon frameshift deletion of the DMD gene. The minor disease Becker Muscular Dystrophy (BMD) is allelic and most cases are caused by internal deletions or duplications of the dystrophin gene that alter the length of the rod domain of the encoded protein.

Dystrophin is the first protein discovered by "positional cloning" and this discovery provides initial proof of concept (proof of concept) on using the position of the human genetic map of the disease as the main basis for elucidating its molecular basis. Many proteins found by this method are very low in abundance in affected cells, thus complicating the determination of the physiological function of the protein. Dystrophin was discovered in 1987, and 30 years in the field faced a major gap in understanding the precise function of proteins in muscle cells. However, indirect evidence has been provided for the role of dystrophin in protecting muscle cell membranes from the forces generated during muscle contraction. The mechanical load characteristics of dystrophin are still poorly characterized.

In the United states, the incidence of DMD has been somewhat reduced by carrier testing and pre-natal consultation (Pegoraro, E. et al, SPP1 genetics is a family of disease safety in Duchenne molecular dynamics, Neurology, 2011.76 (3): pages 219-26). Current therapies that combine glucocorticosteroids, ACE inhibitors and mechanical ventilatory support may temporarily slow The rate of progression, but The final clinical course is irresistible (McDonald, C.M. et al, The cooperative organic neurological research group negative-a longitudinal induction in The medicine of clinical experience: Design of protocol and The method used. Muscan Nerve, 2013).

In the age of gene therapy, various AAV vectors have emerged as the least toxic and most widely spread platform for systemic gene delivery. Those gene therapies hold great promise for systemic biodistribution, but limitations include (a) the cloning capacity is limited to one-third of that required for full-length Dp427, (b) unresolved problems with immunogenicity and toxicity of vectors at doses potentially required for long-lasting therapy, and (c) the particular cost of routine manufacture on the scale required for human therapy. AAV vectors are structurally related to wild type members of the parvovirinae subfamily, encapsidating a single stranded DNA genome of about 5 kilobases. The mRNA for full-length dystrophin is 14 kilobases with an open reading frame of about 12 kb.

Most of the mutations that cause DMD are in>2.5 megabases of sporadic multiple exons, frameshift deletions in X-linked genes (Kunkel, L.M. et al, Analysis of reactions in DNA from tissues with Becker and Duchenne molecular dynamics. Nature, 1986.322 (6074): pages 73-7; Monaco, A.P. et al, Isolation of coding cDNAs for relations of The Duchenne molecular dynamics gene. Nature, 1986.323 (6089): pages 646-50; and Koenig, M.et al, The molecular basis for Dunnen veruss molecular dynamics. St reproduction of selectivity with type of deletion, Am J Hum Genet, 1989.45 (4): page 498-506). Recombinant dystrophins have the ability to induce an immune response in a host to heterologous proteins without central (thymic) tolerance to the defective full-length protein (Mendell, J.R. et al, Dystrophin immunity in Duchenne's molecular dynamics. N Engl J. Med., 2010.363 (15): p. 1429-37). The novel vectors and methods of vascular delivery have achieved promising regional and systemic Gene transfer in preclinical studies, suggesting rational approaches for Gene therapy of DMD (Greelish, J.P. et al, Stable restriction of the research complex with high efficiency and a recombinant adno-associated viral vector Nat. Med., 1999.5 (4): pages 439-43; Su, L.T. et al, Uniform scale-induced Gene transfer to transformed therapeutic Gene expression, 2005.112 (12): pages 1780-8; Gao, G. J.H. Van. and J.Willeberson, Willeber. J.S. Pat. No. Wilford. J.S. J.P. Wilford therapeutic Gene, AAV 3. V.285: thermal Gene therapy, AAV 3. see FIGS. 285-285, 2010.21 (4): p, 371-80). However, these advances have also emphasized the major vector discovery challenges and patient safety concerns in this area (Mendell, J.R. et al, Dystrophin immunity in Duchenne's molecular dynamics. N Engl J Med, 2010.363 (15): pages 1429-37; Mendell, J.R. et al, Myoblast transfer in the treatment of Duchenne's molecular dynamics. N Engl J Med, 1995.333 (13): pages 832-8; Moule, V.et al, Myoblast transfer: is thermal light at the end of the tunnel actaMyol, 2005.24 (2) pages 128-33; and Wang, Z. et al, Immunity to amplified virus-mediated Gene transfer in a random-branched cane model of Duchenne molecular dynamics, Hum Gene Ther, 2007.18 (1): pages 18-26). As another example of an AAV vector for the treatment of DMD, U.S. Pat. Nos. 7,771,993 provides a "micro-dystrophin-associated protein" (also recorded as "m-dystrophin-associated protein)", "mu-dystrophin-associated protein" or "mu-U") having a functional portion of the "actinin-binding domain" of about 270 amino acids relative to the human dystrophin-associated protein located within the N-terminal dystrophin-associated protein region, functional portions of at least proline rich hinge regions 1 and 4 (H1) and (H4) and a portion of the C-terminal dystrophin-associated protein. Micro-dystrophin related proteins contain internal deletions of the central rod repeat domain and truncations in the downstream C-terminal region.

There remains a need for the treatment of duchenne muscular dystrophy and related diseases.

Disclosure of Invention

The present invention provides compositions and methods useful for treating Muscular Dystrophy (MD), including Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), and other diseases. Provided herein are recombinant adeno-associated virus (rAAV) vectors having an AAV capsid and a vector genome. The vector genome comprises a nucleic acid sequence encoding a triple splice (triple splice) mutein of the dystrophin superfamily under the control of regulatory sequences directing its expression.

In certain embodiments, the dystrophin superfamily triple splice mutant protein comprises a hybrid helix domain comprising a first helix comprising the N-terminal portion of helix a fused to the C-terminal portion of helix a ', a second helix comprising the N-terminal portion of helix B' fused to the terminal C-terminal portion of helix B, and a third helix comprising the N-terminal portion of helix C fused to the C-terminal portion of helix C, wherein helices A, B and C are present in a first triple helix repeat that is not adjacent to a second triple helix repeat having helices a ', B', and C in a native dystrophin superfamily protein. In certain embodiments, the dystrophin superfamily mutein is a triple splice mutant dystrophin or a triple splice mutant dystrophin related protein.

In yet a further embodiment, a dystrophin superfamily triple-spliced mutein comprises one or more N-terminal helical repeats, a hybrid triple-helical repeat and one or more C-terminal helical repeats, wherein the total number of helical repeats including hybrid repeats in the triple-spliced mutein is selected from any integer from 1 to 1 less than the number of helical repeats of the full-length dystrophin superfamily protein, and wherein the hybrid triple-helical repeat is formed from two helical repeats spliced on a plane bisecting a helical repeat perpendicular to its long axis as depicted in fig. 2F. In certain embodiments, the dystrophin superfamily mutein is a triple splice mutant dystrophin or a triple splice mutant dystrophin related protein.

Provided are polypeptides having SEQ ID NO: 1 or 22, and further provides recombinant mutant dystrophins having an amino acid sequence selected from SEQ ID NOs: 3. 7 and 20. In certain embodiments, the triple-spliced mutein is a dystrophin-related protein and is encoded by a sequence comprising SEQ ID NO: 19 or a sequence that is about 95% to about 99% identical thereto.

In a further embodiment, the triple-spliced mutein comprises a hybrid triple-helical repeat sequence and a C-terminal helical repeat sequence, wherein the total number of helical repeat sequences comprising the hybrid repeat sequence in the triple-spliced mutein is 5, and wherein the hybrid triple-helical repeat sequence is formed by two helical repeat sequences spliced on a plane bisecting the helical repeat sequence perpendicular to its long axis as depicted in fig. 2F. In certain embodiments, the C-terminal helical repeat of the mutein consists of helical repeats 21, 22, 23 and 24 in full-length dystrophin, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 1 in full-length dystrophin, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 20 in full-length dystrophin. In yet another embodiment, the C-terminal helical repeat of the mutein consists of helical repeats 19, 20, 21 and 22 in the full-length dystrophin-related protein, wherein helical repeat 1 of the two repeats forming the hybrid triple helical repeat is helical repeat 1 in the full-length dystrophin-related protein, and wherein helical repeat 2 of the two repeats forming the hybrid triple helical repeat is helical repeat 18 in the full-length dystrophin-related protein.

In yet a further embodiment, there is provided a polypeptide comprising SEQ ID NO: 1. 13, 14, 15, 16, 17, 18 or 22. In certain embodiments, nucleic acids encoding mutant dystrophin superfamily proteins are provided. In yet a further embodiment, a plasmid comprising a nucleic acid encoding a mutant dystrophin superfamily protein is provided.

In certain embodiments, pharmaceutical compositions comprising a rAAV comprising a vector genome comprising a nucleic acid sequence encoding a triple-spliced mutant dystrophin superfamily protein are provided.

In yet a further embodiment, a method of treating a subject diagnosed with duchenne muscular dystrophy is provided comprising administering a pharmaceutical composition comprising a rAAV comprising a vector genome having a nucleic acid sequence encoding a triple-splice mutant dystrophin superfamily protein.

Other aspects and advantages of the invention will become apparent from the following detailed description of the invention.

Brief Description of Drawings

Fig. 1A to 1D provide illustrations of the formation of hybrid triple helical domains. Figure 1A shows components of exemplary dystrophic superfamily proteins. From the N-terminus of the protein (denoted NH) ₂-) to the C-terminal end (denoted-COOH), the composition is 1) two homeodomain of calmodulin (CH 1 and CH 2), 2) several triple helical domains (noted TH plus a number), 3) modular multidomain globular region (WW-EF-ZZ) and 4) the last C-terminal region. In the illustration, there are 24 triple helical domains. Gray arrows indicate splicing used in the previous formation of short dystrophin superfamily proteinsA point site (such as that described in U.S. patent No. 7,771,993, which is incorporated herein by reference in its entirety), while a black arrow indicates a splice point site for forming a hybrid triple helical domain. Figure 1B provides a closer look at triple helix domains (THn in white arrows and THn' in shaded arrows) spliced to form a hybrid triple helix as shown in figure 1C. Each white or shaded arrow in FIGS. 1B through 1D represents a helix in a domain (helices A, B and C for THn; helices A ', B ' and C ' for THn '; and helices A-A ', B ' -B and C-C ' in a hybrid triple-helical domain). Black arrow lines indicate splice sites. Figure 1D provides a linear display of the hybrid triple helical domain. The white parts of the bars/arrows indicate that they originally came from THn, while the shaded parts indicate that they originally came from THn'. As shown in fig. 1D, the first helix of the hybrid triple-helical domain comprises helix a at the N-terminus and helix a' at the C-terminus; the second helix of the hybrid triple helical domain comprises helix B' at the N-terminus and helix B at the C-terminus; and the third helix of the hybrid triple helical domain comprises helix C at the N-terminus and helix C' at the C-terminus.

Figures 2A to 2F illustrate the dystrophin-related protein modifications provided herein. The lighter colored residues depict tryptophan, which is the most conserved residue in Hidden Markov Models (HMMs) of the triple helical repeat domain shared by alpha-actinin, beta-and alpha-spectrin, dystrophin-related proteins, and spectoplakins. Sequence divergence at all other positions with extensive evolutionary coupling (evolution consensus) confirmed that these regions of each repeat sequence could not be interchanged between one repeat sequence and another without destabilizing the triple helix. Single splicing may not solve this problem, but triple splicing by leaving coupled amino acids to the left and right of the plane of the tryptophan residue through the plane of the tryptophan residue. Fig. 2A illustrates a portion of the triple helical repeat domain of alpha-actinin (TH 1). FIG. 2B illustrates the TH2 portion of alpha-actinin. FIG. 2C illustrates the TH3 portion of alpha-actinin. FIG. 2D illustrates the TH4 portion of alpha-actinin. FIG. 2E illustrates the beta-and alpha-spectrin tet sites. Figure 2F illustrates triple splicing through the plane of tryptophan residues by leaving coupled amino acids to the left and right of the plane of tryptophan residues (tryptophan is shown twice to aid orientation).

FIG. 3 shows a portion of the HMM identification (logo) of the spectrin family (PF 00435). More details can be found in pfam. xfam. org/family/PF00435# tabview = tab 4. The HMM identification provides a quick overview of HMM properties in graphical form. The relative conservation of each of the two anchor tryptophans, as shown by the HMM labeling of the spectrin-like triple helical repeat at position # 16. One skilled in the art can ascertain how to interpret the logo, as described, for example, in Schuster-B baby B et al, HMM logs for visualization of protein families, BMC biologics, 2004, 1/21/2004, 5: 7.

Figure 4 provides an overview of the formation of nano-dystrophin (nano-dystrophin) and a comparison with the formation of micro-dystrophin (micro-dystrophin) as described herein.

Figure 5 provides a grid showing possible splice points in helix B of dystrophin. A more detailed description of the figures can be found in the first paragraph of the detailed description of the invention, i. dystrophin, dystrophin-related proteins, etc., splice junctions in the a. helix. The variants described correspond to SEQ ID NOs: 13-18, the amino acid sequence of a nano-dystrophin protein.

Fig. 6A-6C show that the length of the dystrophin bar is established before the sarcomere appears. Figure 6A shows aligned human dystrophin gene, mRNA and protein domain structures to show intron position and phasing (phasing). 22 of the 24 spectrin repeats of human dystrophin contain a phase 0 intron at HMM position 46 (the point with the black outline). The spinor (Cnidarian) dystrophins share the rod domain phase 0 intron (for clarity, only the HMM position 46 phase 0 intron is depicted in the spinor mRNA). FIG. 6B provides a structure in the rod domain with triple helix Human β -ghost, dystrophin and MACF1 depicted with the consensus sequences of domain HMM aligned vertically. Superimposed are the intron positions and phases relative to the corresponding coding sequences, clearly showing the structural similarity of dystrophin and MACF1 genes. For clarity, only the intron shared with the orthologous gene from the farthest related species is shown, while the spectrin gene shows the remnant intron (arrow) of the distant partial gene repeat from the 13 repeats (spectrin-large reef sponge (ghost-large reef sponge) ((iii)A. queenslandica) (ii) a Dystrophin-asteroid sea anemone (A)N. vectensis) (ii) a MACF 1-Silk Panuliasis (T. adhaerens)). Figure 6C provides germline genomics distributions of members of the alpha-accessory actin superfamily of proteins in selected eukaryotic lineages. The number of rod domain spectrin repeats is indicated in parentheses. Members of the dystroplakin superfamily carry the phase 0 intron at position 46 of the HMM, while alpha-actinin and spectrin family proteins lack conservation of the phase 0 intron at that position. HMM 46 intron-driven expansion of the ortholog of ancestral MACF1 was observed between fungi and Pantoea (Placozoa). The ancestral MACF1 ortholog underwent partial gene duplication which contributed an N-terminal actin-binding domain and a full-length rod domain to the ancestral WW-EF-ZZ dystrophin ortholog.

Figures 7A to 7F demonstrate extensive transduction restoring dystrophin binding protein complexes, preventing myofibrosis, normalizing serum CK levels and improving muscle function in AAV9- μ dystrophin-associated protein treated mdx mice. As shown in fig. 7A, immunostaining of representative limb muscles for an epitope shared by native and recombinant dystrophin-related proteins (Utro _ N), an epitope unique to native dystrophin-related proteins (Utro _ C), gamma-sarcoleminophane, embryonic myosin heavy chain (eMHC), and laminin; and the proportional scale is 25 mu m. FIG. 7B provides H & E of representative limb muscles showing inhibition of muscle necrosis and mononuclear cell infiltration; the proportional scale is 100 mu m. FIG. 7C provides a Western blot analysis to detect expression of recombinant dystrophin-related protein (AAV 9- μ Utro) and γ -sarcoleminopolyn (γ -sarc). Fig. 7D provides the percentage of central nucleated muscle fibers (CNFs), a statistical measure, as defined in the method (color = different animals, shape = different muscles, same color/shape = technical repetition). Fig. 9E provides results in treated mice (n = 5), untreated mice (n = 12) (× p) <0.0001) and n.s. from measurement of serum CK levels in wild type (n = 7). Fig. 7F provides mice treated (n = 8), untreatedmdxQuantification of vertical activity one hour after grip strength (vertical activity) in mice (n = 11) and wild type mice (n = 6). Error bars indicate SD,. about.p<0.001; N.S for not significant; statistical significance was assessed by the Kruskal-Wallis test with multiple sets of comparisons.

Fig. 8A to 8G show systemic delivery of AAV9- μ dystrophin-related proteins in 7 week old GRMD dogs prevents muscle necrosis and results in a rapid decrease in serum CK levels. Fig. 8A provides an experimental schedule. Figure 8B provides representative H & E of the vastus lateralis and temporalis muscles showing abundant muscle necrosis fibers and mononuclear cellular infiltration in untreated muscles, while treated muscles resemble WT. Figure 8C provides alizarin red S staining showing calcified fibers indicating muscle degeneration (left panel, red) with corresponding quantification (figure 8E). Figure 10D provides immunofluorescence staining with F1.652 showing clusters of eMHC-positive fibers (right panel, red) with corresponding quantification (figure 8F). Figure 8G provides serum Creatine Kinase (CK) levels at various time points before/after systemic AAV9- μ dystrophin-related protein infusion. The proportional scale is 100 mu m.

Fig. 9A-9D show that extensive expression of μ dystrophin-related proteins rescues dystrophin binding protein complex proteins in treated GRMD dogs after systemic delivery at 7 weeks of age. Figure 9A and figure 9B provide immunofluorescent staining of representative limb muscles. Figure 9A shows native and recombinant dystrophin-related protein (Utro _ N), native dystrophin-related protein (Utro _ C), laminin. Figure 9B shows β -dystrophin glycans (green), β -sarcolemmal proteoglycans (green), and γ -sarcolemmal proteoglycans (red). The proportional scale is 100 mu m. Fig. 9C shows a western blot analysis showing the extensive bio-distribution of μ -dystrophin-related protein (— 135 kD) in striated muscle at necropsy. Fig. 9D shows western blot analysis showing expression of β -dystrophin glycans in muscle biopsies of the Vastus Lateralis (VL) and the sartorius Capitis (CS). Treated (H)/treated (B) represents tissues from Hann and beette of treated dogs.

Figures 10A to 10D show that localized expression of μ dystrophin but not μ dystrophin-related proteins elicits detectable peripheral and localized immune responses in the dystrophin deprivation-null dog model. Fig. 10A provides an experimental schedule. Dystrophin deficient-null dogs (Grinch and Ned) each received an equal dose (1 x 10) ¹²vg/kg) AAV9- μ dystrophin (right) and AAV9- μ dystrophin-related proteins (left) to their tibialis anterior compartment. As shown in figure 10B, PBMCs were collected before injection, 2, 4, 6, 8 weeks after injection and cultured with synthetic peptides spanning the entire μ dystrophin (Pool) a-D) and μ dystrophin-related protein (Pool E-J) peptide sequences, while vaccine peptides and PMA/Ion served as positive controls. Positive results were interpreted as ≧ 5 Spot Formation Units (SFU)/1E 5 PBMCs (dashed line). Figure 10C shows immunofluorescence (green) staining for utron N (upper row) and dystrophin (lower row) of muscle biopsies collected 4 weeks post-injection. Inset with red box-for reference, appearance of normal muscle stained green for dystrophin. FIG. 10D shows representative H & E of muscle biopsies collected 4 weeks after injection.

Figure 11 provides an overview of micro-dystrophin related proteins. The sites of deletion junctions (splice junctions) are shown. Hinges 1, 2 and 4 are labeled H1, H2 and H4. SR1, SR2, SR3 and SR22 correspond to TH1, TH3, TH3 and TH22 of full-length dystrophin-related proteins.

Fig. 12A to 12D provide the design of the hybridization assay: vertical activity monitoring and whole limb force testing. Fig. 12A provides a schematic diagram showing the experimental schedule. 12B shows c57 (n = 11) and before full limb strength testing mdxQuantification of vertical activity in open field cages between mice (n = 12) at c57 andmdxno significant differences were shown between mice (P)>0.05 Mann-Whitney test). Fig. 12C shows for C57 (n = 11) andmdxboth mice (n = 17) were tested for full limb strength in a series of seven stretches. C57 mice are represented by squares, andmdxmice are indicated by circles. The distribution of full limb force for each series of stretches is shown. Describing the equation (P)<0.0001, two-way ANOVA test). FIG. 12D provides an analysis of post-cumulative vertical activity (post-vertical activity) for 1 hour after the force test, shown at c57 andmdxthere was a significant difference between mice (P)<0.0001, two-way ANOVA).

FIG. 13 provides c57 and c5 minutes for the first 5 minutes (Pre) before full limb force testing, and the first 5 minutes and the second 5 minutes after force testingmdxVisual representation of the mouse's movements. Lines represent horizontal activity and dots represent vertical activity.

Fig. 14A to 14G show acute myofascial disruption in fasciculated muscle cells of BIO 14.6 hamster skeletal muscle after vigorous contraction. FIG. 14A provides intravenous (i.v.) Simultaneous observation of Evans blue dye and dystrophin counterstaining in muscle fibers 72 hours after injection of Evans blue (Evans blue) dye. In fig. 14B, there was clearly no dystrophin staining at all in evans blue positive fibers in the section from fig. 14A as observed by the FITC filter. Figure 14C shows that within 8 hours of a single spontaneous run, myocyte injury results in loss of dystrophin in most evans blue positive fibers. Figure 14D shows the tibialis anterior muscle 3 hours after tonic contracture with a lengthening rate of 0.75 muscle length per second. Acute injury was shown by the uptake of prasuan (procion) orange dye; dystrophin counterstaining indicates the absence of complete membrane breakdown and non-specific proteolysis in these fibers. Figure 14E shows dystrophin counterstaining of muscle cryosections after 1 hour of wheel (running wheel) exercise. Figure 14F shows evans blue dye fluorescence in the same section as in figure 14E. Figure 14G provides alizarin red S staining of the same area of serial cryosections. Original magnification: FIGS. 14A, 14B and 14E to 14G, 100X; fig. 14C and 14D, 200X.

Figure 15 provides an analysis of the dystrophin binding protein complex glycosylase, ligand, actinin superfamily, dynamin and titin-masking protein (obsurin) superfamily. More discussion about the figure can be found in example 3.

Figures 16A and 16B provide models of adjacent triple helical repeats of human dystrophin-related protein using templates derived from human β 2-spectrin (3 EDV, figure 16A) and human reticulin (5J 1G). More details can be found in example 3.

Figure 17 provides an overview of micro-dystrophin related proteins. The micro-dystrophin related protein has an unstructured, proline rich, inter-helix "hinge-2" domain (H2) juxtaposed to the last triple helix repeat (22 nd) of the full length dystrophin related protein. R1, R2, R3 and R22 correspond to TH1, TH3, TH3 and TH22 of full-length dystrophin-related proteins. More details can be found in section F of example 3.

Figure 18 provides the expression levels of micro-dystrophin related proteins optimised as described in example 3.

Figure 19 shows the stabilization of robust dose-dependent μ dystrophin-related protein expression and wild-type sarcolemcan expression levels in sarcolemmas six weeks after injection in GRMD dogs as described in example 3.

Figure 20 shows μ dystrophin-related protein specific T cell responses in injected GRMD dogs as discussed in examples 2 and 3. Peripheral Blood Mononuclear Cells (PBMCs) collected at 5 and 8 weeks post-injection were cultured with three synthetic peptide pools corresponding to AAV9 capsid (A, B and C) and five synthetic peptide pools spanning the entire μ dystrophin-related protein peptide sequence (A-B, C-D, E-F, G-H, I-J). Production of interferon-gamma was assessed by counting spot-forming units per million PBMCs, whereas none of the injected dogs responded above background to either AAV9 capsid or a dystrophin-related protein-derived peptide library. On the right, control assay after adenovirus-CMV-lacZ injection, based on the most conservative interpretation of positive results (dashed line), shows positive responses (asterisks) to both Had5 (1-4) and lacZ (5-8) peptide pools.

FIG. 21 shows AAV- μ -U injection as discussed in section G of example 3mdx79 kd strip in the weight bearing muscle of mice. Lanes 1, 2 and 4: non-weight bearing (e.g., flexor) muscles; 3. 5 and 7: weight bearing (e.g., extensor) muscles; 6, liver disease; 8 PBS-injectedmdxA muscle; and 9, molecular weight standard reference substance.

FIG. 22 shows a combination of Western blot, immunoaffinity purification and LC/MS-MS to identify a 79 kd fragment as the N-terminal portion of a micro-dystrophin-related protein. The box contains the sequence PPPPP, a portion of "hinge 2" immediately upstream of the missing connection, as shown.

Figure 23 shows that SH3 domain forms multiple high affinity contacts with compatible amino acid side chains from adjacent triplexes on both sides, a configuration with the potential to transmit longitudinal forces and resist stretching.

Figures 24A to 24C show that, in this sense, Anc80 achieves a global biodistribution of micro-dystrophin-related proteins similar to AAV9 in the case of strong transduction of cardiac and skeletal muscle (see micrographs in figures 24A and 24B, and western blot in figure 24C), as discussed in example 3.

FIG. 25 shows the data for the data at 2.5X 10¹²Qualitative comparison of AAV9 and Anc80 of the biodistribution of μ dystrophin-related proteins in the mdx mouse cohort (cohort) following systemic administration of equal doses of vg/mice with these vectors. Representative western blots of various muscles from two mice per vector are shown, demonstrating extensive and efficient transduction of striated muscle with both vectors. The uppermost band is a μ dystrophin-associated protein, as labeled with a polyclonal antibody that specifically recognizes an epitope corresponding to the N-terminus of the protein. The sample loading control is the lowest band labeled with antibody to the protein vinculin. Representative striated muscle: diaphragm, triceps, quadriceps, gastrocnemius, abdominal wall, pectoral muscle, tibialis anterior muscle, heart.

FIGS. 26A and 26B show AAV 9-mu dystrophin-related proteinsElimination of MuRF-1 (+), TUNEL (+) and Central nucleated muscle fibers andmdxin the muscle of the mouse. In FIG. 26A, the images as labeled represent 8-week old injected with AAV9 muU or PBS as neonatesmdxA mouse. Histological staining with MuRF-1 and TUNEL were used as biomarkers for active proteolysis and apoptosis, respectively. FIG. 26B provides a summary ofmdxPBS-treated,mdxTable of mean and standard deviation of central nucleated muscle fibers, MuRF-1, TUNEL and embryo positive muscle fibers in AAV9- μ dystrophin-related protein treated and c57 wild type PBS-treated groups (n = 3).mdxCentral nucleation of muscle fibers indicates at least one prior necrotic episode followed by a concomitant necrosismdxMice reached 8 weeks of age and were regenerated.

Figure 27 provides normal growth of GRMD dogs randomized to AAV9- μ dystrophin-related protein as evidence against immune-mediated myositis. Randomization to the highest dose (1 × 10) without immunosuppression^13.5vg/kg) of AAV9-cU (canine μ -dystrophin-associated protein) and related controls (including litter carrier females randomized to PBS and other litter carriers littermates GRMD males and females not littermates). For comparison, the relative weights of previously reported GRMD females receiving AAV9-hD (human μ -dystrophin) that showed rapid weight loss immediately prior to euthanasia and necropsy revealed signs of systemic myositis (Kornegay, J.N. et al, Wireless muscle expression of an AAV9 human mini-stressor vector after intragenic injection in neurological therapy-specific dogs. Mol Ther, 2010.18 (8): p.1501-8.) were also included.

Figure 28 shows a western blot from a study comparing the stability of micro-and nano-dystrophin-related proteins in vivo. Mdx mice were injected with AAV vectors encoding micro-or nano-dystrophin-related proteins, followed by detection of the protein in muscle tissue (including 79 kd N-terminal subfragments).

FIGS. 29A and 29B show alternative viewpoints (viropoints) for 3-D reproduction of mutant hybrid helical repeats formed by splicing TH 1 and TH 20 of full-length dystrophin. Splicing in the B antiparallel helix is localized in the plane with the W residue (yellow) on the parallel A and C helices.

FIGS. 30A and 30B show alternative viewpoints for 3-D reproduction of mutant hybrid helical repeats formed by splicing TH 1 and TH 18 of full-length dystrophin-related proteins. Splicing in the B antiparallel helix is localized in the plane with the W residue (yellow) on the parallel A and C helices.

Detailed Description

Compositions and methods are provided for treating Muscular Dystrophy (MD), including Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) and other related diseases, by dystrophin superfamily triple splice muteins, nucleic acid sequences encoding the same, or vectors comprising such nucleic acid sequences.

An unusual molecular evolution analysis of dystrophin and its supergene family was performed, resulting in paradigm shift. The inventors' analysis indicates that the primary effect of dystrophin is that of a strong, tractional, inextensible "strut" -like rod, rather than a shock absorber (shock absorber) as previously theorized; its length is the last time of importance to its strength; its strength depends on the interaction of amino acids at the boundaries between adjacent "triple helix", "spectrin-like" repeats, over the entire rod domain, which together account for 80% of the primary structure of the protein; dystrophin is only as strong as its weakest link; evolution has selected against internal deletions of attenuated proteins. Thus, to shorten without weakening the protein, the coding sequence for the polypeptide is cleaved through the central triple helical domain with the greatest sequence conservation at multiple sites that can be aligned in the folded protein (e.g., through the 2-dimensional plane bisecting the interacting tryptophan residues in the hydrophobic core in dystrophin). This is the strongest element in the hidden Markov model for all spectrin-like triple helix repeats and applies to most repeats of dystrophin, dystrophin-related proteins and spectoplakins (MACF, dystonin, etc.).

I. Dystrophin, dystrophin-related proteins, and the like.

As used herein, a dystrophin superfamily protein refers to a protein comprising "spectrin-like" and "rod-like" domains that consist of three alpha-helices and occur in the protein as a single copy or in a tandem arrangement of multiple repeats, such as dystrophin, a dystrophin-related protein, alpha-actinin, alpha-spectrin, beta-spectrin, or other members of the spectrin family, plaques, spectraplatin (i.e., spectroplasins). The triple-alpha-helical domain comprises two similarly (helices a and C) and one oppositely (helix B) oriented alpha-helices connected by a non-helical linker. Many aromatic residues in the hydrophobic core of a domain are generally conserved. See, e.g., Parry DA et al, Analysis of the same-alpha-helix motif in the protein superfamily of proteins, biophysis J.1992, month 4, 61(4): 858-67; and Djinovic-Carugo K et al, The spectrin repeat: a structural platform for cellular protein architectures, FEBS Lett.2002, 20/2/513 (1): 119-23. Examples of such repetitive sequences can be found in fig. 2A to 2E. In certain embodiments, a full-length dystrophin superfamily protein refers to a dystrophin superfamily protein or isoform thereof, which may be present in a healthy control. In certain embodiments, a full-length dystrophin superfamily protein refers to a dystrophin superfamily protein or isoform thereof that is considered by those of skill in the art to be a canonical sequence. Such canonical sequences are available, for example, at www.uniprot.org.

Spectrin is a cytoskeletal protein that intercalates into the intracellular side of the plasma membrane of eukaryotic cells. Spectrin forms into a pentagonal or hexagonal arrangement, thereby forming a scaffold and playing an important role in maintaining plasma membrane integrity and cytoskeletal structure. See, for example, Huh GY et al, Calpain proteins of alpha II-spectrin in the normal adult human brain, Neurosci Lett. 2001, 12/4, 316(1): 41-4. The proteins in the superfamily can be defined by two features: (1) an N-terminal actin-binding domain; and (2) alpha-helical spectrin weightA complex sequence portion. See, e.g., R baby K et al, The 'spectra' of cytological giants with characteristics of both spectra, and plasma family, J Cell Sci, 2002, 11/15, 115(Pt 22): 4215-25. Members of the spectrin superfamily include, but are not limited to, alpha-actinin (e.g., alpha-actin-1, see, e.g., UniProtKB-P12814 and www.genecards.org/cgi-bin/carddis. plgene = ACTN 1; α -actin-2, see, e.g., UniProtKB-P35609 and www.genecards.org/cgi-bin/carddisgene = ACTN 2; α -actin-3, see, e.g., UniProtKB-Q08043 and www.genecards.org/cgi-bin/carddis. pl gene = ACTN 3; and alpha-actin-4, see, e.g., UniProtKB-O43707 and www.genecards.org/cgi-bin/carddisgene = ACTN4, each of which is incorporated in its entirety by reference), alpha-spectrin (e.g., spectrin alpha chain, erythrocyte 1, see, e.g., UniProtKB-P02549 and www.genecards.org/cgi-bin/carddisgene = SPTA 1; and spectrin alpha chain, non-erythrocyte 1, see, e.g., UniProtKB-Q13813 and www.genecards.org/cgi-bin/carddisgene = SPTAN1, each of which is incorporated in its entirety by reference), β -spectrin (e.g., spectrin β chain, red blood cells, see, e.g., UniProtKB-P11277 and www.genecards.org/cgi-bin/carddisgene = SPTB; and spectrin beta chain, non-erythrocyte 1, see, e.g., UniProtKB-Q01082 and www.genecards.org/cgi-bin/carddis. plgene = SPTBN1, each incorporated in its entirety by reference), dystrophin and dystrophin-related proteins.

Plaque proteins are cell-associated (cytoskeletoner) proteins that bind to cytoskeletal elements and connective complexes. See, for example, Leung Cl et al, plankins: a family of versatile cytolinker proteins. Trends Cell biol. 2002, 1 month, 12(1): 37-45. Seven plaque family members have been identified: desmoplakin (UniProtKB-P15924 and www.genecards.org/cgi-bin/carddis. pl gene = DSP, which includes the sequences listed therein, herein attached in their entirety), reticulin (UniProtKB-P15924 and www.genecards.org/cgi-bin/carddisgene = PLEC, which includes the sequences listed therein, herein attached in their entirety), bullous pemphigus antigen 1 (BPAG 1, Dystonin) (UniProtKB-Q03001 and www.genecards.org/cgi-bin/carddis. plgene = DST, which includes the sequences listed therein, herein attached in their entirety), microtubule-actin cross-linking factor (MACF) (UniProtKB-Q9 UPN3 and www.genecards.org/cgi-bin/carddisgene = MACF1, which includes the sequences listed therein, herein attached in their entirety), coat plaque proteins (UniProtKB-Q92817 and www.genecards.org/cgi-bin/carddis. plgene = EVPL, which includes the sequences listed therein, herein attached in their entirety), paucin (UniProtKB-O60437 and www.genecards.org/cgi-bin/carddisgene = PPL, which includes the sequences listed therein, herein attached in their entirety) and epiplakin (UniProtKB-P58107 and www.genecards.org/cgi-bin/carddisgene = EPPK1, which includes the sequences listed therein, herein attached in its entirety). The protein family is defined by the presence of a plausin domain and/or a Plausin Repeat Domain (PRD). In addition to these two domains, the plaques carry other domains that are common in some but not all members: actin Binding Domain (ABD), coiled coil rods, rods containing spectrin repeats, and microtubule binding domain.

spectraplatin belongs to both the spectrin and plaque superfamily and is an abnormally long intracellular protein with a rare ability to bind all three cytoskeletal elements: actin, microtubules and intermediate filaments. spectraplasins are critical to tissue integrity and function, acting with and coordinating individual cytoskeletal elements. See, for example, R baby K et al and Huelsmann S et al, Spectraplakins, Curr biol 2014 4-month and 14-day, 24(8) R307-8. doi: 10.1016/j. cub 2014.02.003, as referenced above. Members of spectraplasins include, but are not limited to, BPAG1 and MACF as described above. Dystrophin anchors the extracellular matrix to the cytoskeleton via F-actin and is a ligand for dystrophin glycans. Components of the dystrophin-binding glycoprotein complex accumulate in the neuromuscular junction (NMJ) and various synapses of the peripheral and central nervous systems and have structural functions in stabilizing the sarcolemma. It is also implicated in signaling events and synaptic transmission.See, e.g., www.uniprot.org/uniprot/P11532 and www.genecards.org/cgi-bin/carddisgene=DMD&keywords = prostrophin. There are 10 isoforms of dystrophin. Isoform 4 is considered a canonical sequence and contains the amino acid sequence with the UniProtKB identifier P11532-1, which is incorporated herein. Other isoforms, including those with UniProtKB identifiers: isoform 1 of P11532-2, having the UniProtKB identifier: isoform 2 of P11532-3, having the UniProtKB identifier: isoform 3 of P11532-4, having the UniProtKB identifier: isoform 5 of P11532-5, having the UniProtKB identifier: isoform 6 of P11532-6, having the UniProtKB identifier: isoform 7 of P11532-7, having the UniProtKB identifier: isoform 8 of P11532-8, having the UniProtKB identifier: isoform 9 of P11532-9 and a peptide having the UniProtKB identifier: isoform 10 of P11532-10, the sequence of each isoform being incorporated herein. The term "full-length dystrophin" as used herein may refer to any dystrophin isoform. In certain embodiments, the term "full-length dystrophin" refers to isoform 4 (Dp 427). Homologues of dystrophin (homologs) have been identified in a variety of organisms, including mice (UniProt P11531), rats (UniProt P11530) and dogs (UniProt O97592). Possible nucleic acid sequences encoding Dp427 or any other isoform or homologue of dystrophin are publicly available, see, e.g., NCBI reference sequence: NM _000109.3, NM _004006.2, NM _004009.3, NM _004010.3, NM _004011.3, NM _004012.3, NM _004013.2, NM _004014.2, NM _004015.2, NM _004016, 2 NM _004017.2, NM _004018.2, NM _004019.2, NM _004020.3, NM _004021.2, NM _004022.2, NM _004023.2, NM _004007.2, XM _006724468.2, XM _006724469.3, XM _006724470.3, XM _006724473.2, XM _006724474.3, XM _006724475.2, XM _011545467.1, XM _011545468.2, XM _011545469.1, XM _017029328.1, XM _017029329.1, XM _017029330.1, and XM _017029331.1, each of which is incorporated herein. Can be translated by Tools for reverse translation, e.g., www.ebi.ac.uk/Tools/st/, www.ebi.ac.uk/Tools/st/embos _ trans/, www.ebi.ac.uk/Tools/st/embos _ sixpack/, www.ebi.ac.uk/Tools/st/embos _ backstranseq/, and www.ebi.ac.uk/Tools/st/embos _ backstranmbig/, additional sequences encoding Dp427 or any other isoform or homologue of dystrophin were generated. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

Dp427 is 3685 amino acids in length. Also, see, e.g., US7892824B2 and GenBank: AAA 53189.1. The N-terminal 240 amino acids of Dp427 are folded into two homeodomain Calpain proteins (CH 1 & 2) which have the ability to bind with high affinity to the cytoskeletal actin filaments. The central region from about amino acids 340 to 3040 consists of 24 serially linked domains identifiable as triple helices by hidden markov modeling (TH 1-24) with measurable structural homology to the crystalline repeats of the rod-like protein spectrin and alpha-actinin. The region from 3057 to 3352 comprises a modular multidomain globular region (WW-EF-ZZ) which has a high affinity for the transmembrane complex of proteins centred on β -dystrophinan. The last C-terminal region from 3353 to 3685 has a seemingly depleting high affinity binding domain for the proteins small dystrophin and dystrophin binding protein. Dp427 has been modeled as a rod-like protein in the striated muscle cell cortex, binding to the outermost edge of the cytoskeletal F-actin via the N-terminal calmodulin homeodomain, and to the transmembrane member of DGC via the C-terminal WW-EF-ZZ domain. The central rod domain consists of 24 domains with low levels of homology to the triple helical repeat hinges 1-4. In the allelic disease becker md (bmd), internal deletions of spectrin-like repeats are often associated with a slower rate of disease progression. The main challenge for gene therapy for DMD is to safely, effectively and durably replace Dp427 in most skeletal and cardiac myocytes. Ideally, the substitution would be compatible with the functionality of Dp 427; there is a concern that proteins significantly less than 427 kd may only convert DMD to a severe BMD phenotype. An illustration of Dp427 can be found in fig. 1A. Hidden markov modeling can be performed by conventional methods and its parameters can be adjusted by those skilled in the art. See, e.g., en. wikipedia. org/wiki/Hidden _ Markov _ model. Deletion of the 19-20 contiguous TH domain and the region of 3353-3685 results in an "AAV-sized" miniaturized dystrophin in the sense that the synthetic coding sequences for these recombinant proteins are within the cloning capabilities of AAV vectors. All such recombinant proteins share the rod-like domain of rod length 1/5 to 1/6 in Dp427, thereby raising the concern that such shortening gradually impairs the ability of the recombinant protein to "absorb" as much "shock" as the full-length protein of the 24-repeat sequence.

Dystrophin-related proteins are substantially homologous to dystrophin, with significant divergence existing in the rod domain where dystrophin-related proteins lack repeats 15 and 19 and two hinge regions (see, e.g., Love et al, Nature 339:55 [1989 ]](ii) a Winder et al, FEBS Lett., 369:27 [1995 ]](ii) a www.uniprot.org/uniprot/P46939; pl. www.genecards.org/cgi-bin/carddisgene=UTRN&keywords = Utrophin). Four isoforms of dystrophin-related proteins were found. Isoform 1 is considered the canonical sequence and contains the amino acid sequence with UniProt identifier P46939-1, which is incorporated herein. Other isoforms include isoform 2 with UniProt identifier P46939-2; isoform UP71 with UniProt identifier P46939-3; and isoform Up140 with UniProt identifier P46939-4. The full-length dystrophin-related protein may refer to any dystrophin-related protein isoform. In certain embodiments, the full-length dystrophin-related protein is dystrophin-related protein isoform 1, which contains 22 spectrin-like repeats (SR 1-SR22, or TH1-TH 22) and two hinge regions. Homologs of dystrophin-related proteins have been identified in a variety of organisms, including mice (Genbank accession numbers Y12229 and UniProt E9Q6R 7), rats (Genbank accession numbers AJ002967 and UniProt G3V7L 1), and dogs (Genbank accession number NW-139836). Nucleic acid sequences of these or additional homologues may be related to human dystrophin related proteins using any suitable method The nucleic acid sequences of (a) are compared. Nucleic acid sequences encoding isoform 1 or any other isoform or any homologue of the dystrophin related protein are available. See, for example, NCBI reference sequence: NM _007124.2, XM _005267127.4, XM _005267130.2, XM _005267133.2, XM _006715560.3, XM _011536101.2, XM _011536102.2, XM _011536106.2, XM _011536107.2, XM _011536109.2, XM _017011243.1, XM _017011244.1, XM _ 017011245.1; genbank accession No. X69086 and Genbank accession No. AL357149, each of which is incorporated herein. Additional sequences encoding a dystrophin-related protein isoform 1 or any other isoform or any homolog may be generated by means for reverse translation, e.g., www.ebi.ac.uk/Tools/st/, www.ebi.ac.uk/Tools/st/embos _ trans/, www.ebi.ac.uk/Tools/st/embos _ sixpack/, www.ebi.ac.uk/Tools/st/embos _ backstrans/, and www.ebi.ac.uk/Tools/st/embos _ backstranmbig/. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

In one aspect, provided herein are triple splicing muteins of the dystrophin superfamily. In one embodiment, the triple-spliced mutant protein comprises internal deletions of multiple helical repeats, and a hybrid helical domain formed by joining portions of helical repeats of a full-length dystrophin superfamily protein. In certain embodiments, the dystrophin superfamily triple splice muteins contain a first helix having an N-terminal portion of helix a fused to a C-terminal portion of helix a ', a second helix comprising an N-terminal portion of helix B ' fused to a terminal C-terminal portion of helix B, and a third helix comprising an N-terminal portion of helix C fused to a C-terminal portion of helix C, wherein helices A, B and C are present in the first triple helix repeat and helices a ', B ' and C ' are present in the second triple helix domain in a native dystrophin superfamily protein. In certain embodiments, the first and second triple helical domains are non-adjacent, and thus a mutant dystrophin superfamily protein is provided that has a hybrid triple helical domain and a deletion of one or more triple helical domains present in the native dystrophin superfamily protein. Thus, the total number of helical repeats in the triple-spliced mutant protein is selected from any integer from 3 to 1 less than the number of helical repeats of the full-length dystrophin superfamily protein, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23. In certain embodiments, provided herein are mutant dystrophin proteins having deletions in at least the helical repeat 3 through the helical repeat 21 of a full length dystrophin protein. In yet another embodiment, the mutein has a deletion in at least the helical repeats 3 to 23 of the full-length dystrophin. In a still further embodiment, the mutein has a deletion in at least helical repeat 2 through helical repeat 19 in the full length dystrophin. In certain embodiments, mutant dystrophin-related proteins are provided having deletions in at least the helical repeat 3 through helical repeat 10 of the full length dystrophin protein. In a further embodiment, the mutant dystrophin-related protein has a deletion in at least helical repeat 2 through helical repeat 17 of the full length dystrophin-related protein.

As used herein, the terms "triple helical domain", "triple helical repeat" and "TH" are interchangeable and refer to the rod-like and spectrin-like repeats of a dystrophin superfamily protein, which consist of three alpha helices, i.e. two similarly (helices a and C) and one oppositely (helix B) oriented alpha helix, connected by a non-helical linker. Those repeated sequences can be identified by hidden markov modeling. Examples of such repeating sequences can be found in fig. 2A to 2E. The hybrid triple helical repeat is formed from two helical repeats spliced on a plane bisecting the helical repeat perpendicular to its long axis. Such planes are discussed further below and are illustrated in the examples and in fig. 2F. An illustration of the formation of such hybrid triple helical repeats is provided in fig. 1, while an illustration of hybrid triple helical domains (also recorded herein as hybrid triple helical repeats) is shown in fig. 1C and 1D. As used herein, "splice point" refers to a location in a nucleic acid sequence, amino acid sequence, or protein having a secondary, tertiary, or quaternary structure at which an internal deletion begins or ends in either a full-length dystrophin superfamily protein or in the two triple helical repeats forming a hybrid triple helical domain; or sequences at said positions in the full-length protein whose corresponding sequences are not directly adjacent to each other but are joined in a hybrid triple-helical domain or in a dystrophin superfamily triple-spliced mutant protein. The term "directly adjacent" means that two sequences, domains or repeats are not separated by any other sequence, domain or repeat, respectively. The terms "join," "re-join," or any grammatical variations thereof, mean that two sequences, domains, or repeated sequences become immediately adjacent. The term "form" or any grammatical variation thereof refers to a spliced or joined sequence. The correspondence of sequences or positions in a sequence can be determined by sequence alignment or Hidden Markov Models (HMMs).

As used herein, "N-terminal portion" refers to the amino acid sequence flanking the amino terminus of the splice junction for the selected helix. In certain embodiments, the "N-terminal portion" of the selected helix refers to the full-length amino acid sequence from the initial Met through to the last amino acid sequence preceding the splice junction (on the N-terminal side). In certain embodiments, there may be amino acid substitutions, deletions, truncations, and/or insertions in the N-terminal portion. In certain embodiments, such substitutions are conservative amino acid changes. In certain embodiments, the deletion, truncation, or insertion is 1-5 amino acids in length, which does not affect the folding of the helix.

The term "C-terminal portion" refers to the amino acid sequence at the carboxy-terminal side of the splice junction for the selected helix. In certain embodiments, the "C-terminal portion" of the selected helix refers to the full-length amino acid sequence from the first amino acid sequence after (on the C-terminal side of) the splice junction. In certain embodiments, there may be amino acid substitutions, deletions, truncations, and/or insertions in the N-terminal portion. In certain embodiments, such substitutions are conservative amino acid changes. In certain embodiments, the deletion, truncation, or insertion is 1-5 amino acids in length, which does not affect the folding of the helix.

For example, in certain embodiments, a triple mutant hybrid helical domain has three spliced helices formed by joining segments of non-adjacent helical domains, wherein each helix comprises the N-terminal portion and the C-terminal portion of the helix in the helical repeat of a native dystrophin superfamily protein. Because the triple-helical domains that are joined to form the mutant junction (mutant junction) each have parallel a and C helices and antiparallel B helices, the N-terminal portions of the a and C helices in the triple-helical mutant are from the same triple-helical repeat sequence in the native dystrophin superfamily protein, while the C-terminal portions of these helices are from another triple-helical repeat sequence in the native dystrophin superfamily protein. As a result of the positioning of the linkage to form the triple-helical mutant domain, the N-terminal portion and the C-terminal portion may have different lengths, but together form the helix of the mutant triple-helical domain.

Throughout this specification, ordinal numbers such as "first," "second," "third," "fourth," or the term "additional" are used as reference terms to distinguish various forms and components of compositions and methods. Unless specified, if ordinal numbers are used to indicate TH, amino acid sequence or nucleic acid sequence, the numbers are counted from N-terminus to C-terminus in the amino acid sequence or protein or from 5 'to 3' in the nucleic acid sequence.

As used herein, unless otherwise specified, a protein repeat plus a number refers to the number of repeats in all repeats in a reference protein or reference amino acid sequence counted from the N-terminus. For example, triple helical domain 2 refers to TH2 illustrated in fig. 1A. When the repeat sequence and the reference sequence are polynucleotides, the ordinal number of the repeat sequence is counted from the 5 'end to the 3' end unless otherwise specified.

In certain embodiments, the dystrophin superfamily triple splice muteins comprise one or more N-terminal helical repeats, a hybrid triple helical repeat, and one or more C-terminal helical repeats. In certain embodiments, the dystrophin superfamily triple splice mutant proteins comprise a hybrid triple helix repeat and a C-terminal helix repeat. The total number of one or more helical repeats in the triple-spliced mutant protein is selected from any integer from 3 to 1 less than the number of helical repeats of the full-length dystrophin superfamily protein, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23. In certain embodiments, one or more sequences in the full length dystrophin superfamily protein corresponding to one or more N-terminal helical repeats in the mutein are directly adjacent to the sequence corresponding to the first of the two helical repeats forming the hybrid triple helical repeat. In certain embodiments, one or more sequences in the full length dystrophin superfamily protein corresponding to one or more C-terminal helical repeats in the mutein are directly adjacent to a sequence corresponding to the second of two helical repeats forming the hybrid triple helical repeat. In further embodiments, the one or more N-terminal helical repeats may comprise TH1, TH2, TH3, TH4, TH5, TH6, TH7, TH8, TH9, TH10, TH11, TH12, TH13, TH14, TH15, TH16, TH17, TH18, and TH 19. In still further embodiments, the one or more C-terminal helical repeats may comprise TH1, TH2, TH3, TH4, TH5, TH6, TH7, TH8, TH9, TH10, TH11, TH12, TH13, TH14, TH15, TH16, TH17, TH18, and TH19, each TH ordinal number being counted from the C-terminus. In addition, the mutant protein may have only one helical repeat, wherein the helical repeat is a hybrid triple helical domain formed by helical domain 1 and the last helical domain of the full-length dystrophin superfamily protein. In addition, there may be two helical repeats in the mutein. Such a 2-TH mutein may comprise a helical repeat 1 of the full-length protein, and a hybrid triple helical domain formed by helical repeat 2 and the last helical repeat of the full-length protein. In another embodiment, the 2-TH mutein may comprise the last helical repeat in the full-length protein, as well as a hybrid triple-helical domain formed by helical repeat 1 and the penultimate helical repeat of the full-length protein. As used herein, the terms "dystrophin superfamily triple spliced muteins", "triple spliced muteins" and "muteins" are used interchangeably. Likewise, the repeats and sequences in the mutant proteins are directly adjacent to identical repeats and sequences, respectively, as they are in the full-length dystrophin superfamily protein, except for one or more splice junctions in the hybrid triple helical repeat.

In certain embodiments, the dystrophin superfamily triple-splicing muteins may be truncated at the C-terminus, e.g., any integer up to 1 to 500 amino acids. This truncation does not comprise any triple helix repeat sequence. In certain embodiments, such truncation may occur at a position corresponding to the beginning or end of an exon of the full-length dystrophin superfamily protein.

Also provided herein are nucleic acid sequences encoding triple splice muteins of the dystrophin superfamily. Such a coding sequence can be generated by means for reverse translation. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

In one embodiment, the dystrophin superfamily mutein is a triple splice mutant dystrophin. In a further embodiment, the triple-spliced mutant dystrophin comprises a deletion in at least helical repeat 3 through helical repeat 21 of a full-length dystrophin. In another embodiment, the triple-spliced mutant dystrophin comprises a deletion in at least helical repeat 3 through helical repeat 23 of a full-length dystrophin. In yet another embodiment, the one or more N-terminal helical repeats of the mutant dystrophin protein comprise helical repeat 1 in full length dystrophin protein. The one or more C-terminal helical repeats of the mutein comprise helical repeat 23 and helical repeat 24 in full-length dystrophin. In a further embodiment, the N-terminal helical repeat of the mutein consists of helical repeat 1 in full-length dystrophin. The C-terminal helical repeat of the mutein consists of helical repeat 23 and helical repeat 24 in full-length dystrophin. The first of the two helical repeats that form the hybrid triple helical repeat is helical repeat 2 in full-length dystrophin. The second of the two helical repeats that form the hybrid triple helical repeat is the helical repeat 22 in the full length dystrophin. In yet another embodiment, the triple splice mutant dystrophin protein comprises a deletion in at least helical repeat 2 through helical repeat 19 of the full length dystrophin protein. The C-terminal helical repeats comprise helical repeats 21, 22, 23 and 24 in full-length dystrophin. The first of the two helical repeats forming the hybrid triple helical repeat is helical repeat 1 in full-length dystrophin and the second of the two helical repeats forming the hybrid triple helical repeat is helical repeat 19 in full-length dystrophin. In certain embodiments, the triple-spliced mutant dystrophin protein may further comprise amino acids (aa) from about 1 to about aa 338 of dystrophin isoform 4 at the N-terminus. In certain embodiments, the triple-splice mutant dystrophin may further comprise at the C-terminus about aa 3041 to about aa 3352, about aa 3041 to about aa 3054, about aa 3041 to about aa 3056, about aa 3041 to about aa 3057, about aa 3041 to about aa 3088, about aa 3041 to about aa 3408, or about aa 3041 to about aa 3685 of dystrophin isoform 4. In certain embodiments, further truncations of these C-terminal non-TH sequences of triple-spliced mutant dystrophins may be present. As used herein, truncation refers to the deletion of consecutive amino acids from the C-terminus. In certain embodiments, such truncation may occur at a position corresponding to the beginning or end of an exon of the full-length dystrophin. In certain embodiments, the truncated length can be 1, 2, 3, 4, 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 100, about 150, about 200, about 250, about 300, about 400, about 500, or about 600 aa. Also provided herein are nucleic acid sequences encoding triple-spliced mutant dystrophins. Such a coding sequence can be generated by means for reverse translation. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

In one embodiment, the triple mutant dystrophin protein is a variant having the amino acid sequence of SEQ ID NO: 1 (also recorded as n-dystrophin). In yet a further embodiment, there is provided a polypeptide having the sequence of SEQ ID NO: 2 encoding a triple-spliced mutant dystrophin protein. In one embodiment, the nucleic acid encoding SEQ ID NO: 1 is codon optimized for expression in a subject. In a further embodiment, the polynucleotide encoding SEQ ID NO: 1 is codon optimized for expression in humans. Conventional tools for codon optimization are either public or commercially available to those skilled in the art. See, for example, Fuglting A (Codon optimizer: a free tool for coding optimization. Protein Expr purify. 10. 2003; 31(2):247-9,) www.genscript.com/code-opt. html, www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/gene-synthesis/gene optimization. html, and www.idtdna.com/Codon Opt.

In yet another embodiment, the triple mutant dystrophin protein is a variant having the amino acid sequence of SEQ ID NO: 13. 14, 15, 16, 17 or 18. In yet a further embodiment, provided herein is a nucleic acid encoding a polypeptide having the sequence of SEQ ID NO: 13. 14, 15, 16, 17 or 18, or a nucleic acid sequence of a triple-splicing mutant dystrophin protein. In certain embodiments, the nucleic acid sequence encoding SEQ ID NO: SEQ ID NO: 13. 14, 15, 16, 17 or 18 is codon optimized for expression in a subject. In a further embodiment, the polynucleotide encoding SEQ ID NO: SEQ ID NO: 13. 14, 15, 16, 17 or 18 is codon optimized for expression in humans.

In yet another embodiment, the triple mutant dystrophin protein is a variant having the amino acid sequence of SEQ ID NO: 22.

The term "subject" as used herein means a male or female mammal, including a human, veterinary or farm animal, livestock or pet, and animals commonly used in clinical studies. In one embodiment, the subject of these methods and compositions is a human. In one embodiment, the subject of these methods and compositions is prenatal, neonatal, infant, toddler, preschool child, elementary school child, adolescent, young adult, or adult. Neonatal humans are humans 0 to 12 months of age; toddlers have an age of 1 to 3 years; preschool children have an age of 3 to 5 years; primary human schoolchildren have an age of 5 to 12 years; human adolescents have an age of 12 to 18 years; young adults have an age of 18 to 21 years; whereas human adults have an age over 18 years. "healthy subject" refers to a subject without a disease. As used herein, the term "disease" may refer to DMD and/or BMD. In certain embodiments, the term "disease" may refer to another disease caused by an abnormal dystrophin superfamily protein. In certain embodiments, "disease" refers to von willebrand disease.

In one embodiment, the dystrophin superfamily mutein is a triple splice mutant dystrophin related protein and comprises a deletion in at least helical repeat 3 and helical repeat 19 of the full length dystrophin related protein. In one embodiment, the one or more N-terminal helical repeats of the mutein comprise helical repeat 1 in the full-length dystrophin-related protein. The one or more C-terminal helical repeats of the mutein comprise helical repeat 21 and helical repeat 22 in the full-length dystrophin-related protein. In a further embodiment, the N-terminal helical repeat of the mutein consists of helical repeat 1 in the full-length dystrophin-related protein. The C-terminal helical repeat of the mutein consists of helical repeat 21 and helical repeat 22 in the full-length dystrophin-related protein. The first of the two helical repeats that form the hybrid triple helical repeat is helical repeat 2 in the full length dystrophin-related protein. The second of the two helical repeats that form the hybrid triple helical repeat is the helical repeat 20 in the full-length dystrophin-related protein. In yet another embodiment, the dystrophin superfamily mutein is a triple splice mutant dystrophin related protein and comprises a deletion in at least helical repeat 2 to helical repeat 17 of the full length dystrophin related protein. The C-terminal helical repeat of the mutein comprises helical repeats 19, 20, 21 and 22 in the full-length dystrophin-related protein. The first of the two helical repeats forming the hybrid triple helical repeat is helical repeat 1 in the full length dystrophin-related protein and the second of the two helical repeats forming the hybrid triple helical repeat is helical repeat 18 in the full length dystrophin-related protein. In certain embodiments, the triple-spliced mutant dystrophin-related protein may further comprise from about aa 1 to about aa 311 of isoform 1 of the dystrophin-related protein at the N-terminus. In certain embodiments, the triple-spliced mutant dystrophin-related protein may further comprise at the C-terminus about aa 2797 to about 2811, about aa 2797 to about 2845, about aa 2797 to about 3124, about aa 2797 to about 3134, about aa 2797 to about 3165, about aa 2797 to about 3168, or about 27aa 97 to about 3433 of dystrophin-related protein isoform 1. In certain embodiments, further truncations of these C-terminal non-TH sequences of triple-spliced mutant dystrophin-related proteins may be present. In certain embodiments, such truncation may occur at a position corresponding to the beginning or end of an exon of the full-length dystrophin-related protein. In certain embodiments, the truncated length can be 1, 2, 3, 4, 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 100, about 150, about 200, about 250, about 300, about 400, about 500, or about 600 aa. Also provided herein are nucleic acid sequences encoding triple-spliced mutant dystrophin-related proteins. Such a coding sequence can be generated by means for reverse translation. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

In one embodiment, the triple mutant dystrophin-related protein is a nano-dystrophin-related protein (also recorded as n-dystrophin-related protein or n-U with or without dashes) comprising the amino acid sequence: SEQ ID NO: 3. in further embodiments, provided herein are compositions encoding triple-spliced mutant dystrophin-related proteins and comprising SEQ ID NO: 4. In certain embodiments, the triple mutant dystrophin-related protein is a protein having the amino acid sequence of SEQ ID NO: 5 (also recorded as n-dystrophin-related protein or n-U with or without a dash). In further embodiments, provided herein are nucleic acids encoding SEQ ID NOs: 5 and having the amino acid sequence of SEQ ID NO: 6 or a nucleotide sequence identical to SEQ ID NO: 6 about 95% to about 99% identical to the nucleic acid sequence. In certain embodiments, the triple mutant dystrophin-related protein is a protein having the amino acid sequence of SEQ ID NO: 7 in a pharmaceutically acceptable carrier or vehicle. In further embodiments, provided herein are nucleic acids encoding SEQ ID NOs: 7 and having the amino acid sequence of SEQ ID NO: 8 or a nucleotide sequence identical to SEQ ID NO: 8 from about 95% to about 99% identical to the sequence of the nucleic acid sequence. In one embodiment, the nucleic acid sequence encoding SEQ ID NOs: 3. 5 or 7 is codon optimized for expression in a subject. In certain embodiments, the triple mutant dystrophin-related protein is a protein having the amino acid sequence of SEQ ID NO: 20, or a derivative thereof. In further embodiments, provided herein are nucleic acids encoding SEQ ID NOs: 20 and has the amino acid sequence of SEQ ID NO: 19 or a nucleotide sequence identical to SEQ ID NO: 19 from about 95% to about 99% identical to the sequence of the nucleic acid sequence. In one embodiment, the nucleic acid sequence encoding SEQ ID NOs: 3. 5, 7 or 20 is codon optimized for expression in a subject. In a further embodiment, the nucleic acid sequence encoding SEQ ID NOs: 3. 5, 7 or 20 is codon optimized for expression in humans. In yet another embodiment, the triple mutant dystrophin-related protein is a protein comprising SEQ ID NO: 21, or a derivative thereof.

In certain embodiments, the disclosure includes amino acids and all encoding synthetic nucleic acid sequences for both human nano-dystrophin-related proteins and human nano-dystrophin characterized as "triple spliced". In certain embodiments, a hybrid triple helix may link the middle of triple helix repeat 2 to the middle of the third last triple helix repeat domain (# 20 in 22 of dystrophin-related proteins, # 22 in 24 of dystrophin), giving a total of four repeat domains in both recombinant proteins, as described herein and as shown in SEQ ID NOs: 1 and 3 are illustrated. These amino acid sequences define the maximum strength of the recombinant protein that can be encoded within the coding capacity of a single AAV vector genome. See figure 4 and examples 2 and 3.

The mechanics biology (mechanobiology) of dystrophins is well known, but indirect studies of the physiological role of proteins suggest that the rod domain of proteins may be longitudinally loaded during muscle contraction. The examples provided herein provide the earliest direct evidence for this. In childhood and skeletal maturation of micro-dystrophin-related proteins mdxIn a comparison between (dystrophin null) mice, western blot analysis using antibodies directed against the N-terminus of the recombinant protein revealed evidence of rod destruction at the location of the single splice junction, as the process of muscle maturation and myosin isoform switching increases the mechanical load across the muscle membrane. This strongly supports the following assumptions: the strength of the rod domain is compromised at the precise location of the individual shear junctions. The design principles underlying the development of nano-dystrophin-related proteins and nano-dystrophin proteins compensate for said previous disadvantages by eliminating the juxtaposition of incompatible subdomains of the rods.

A. Shear contact in a spiral

The hybrid triple helical repeat consists of two helical repeats spliced on a plane bisecting the helical repeat perpendicular to its long axis as shown in fig. 1A-1D and 2F. The choice of splice points in the antiparallel "B" helix is derived in an indirect manner. Only one X-ray crystal structure has been determined for a single repeat sequence of the dystrophin triple helix TH1 (i.e. so far there is no structure for TH 2-24). The adjacent triple helices of dystrophin overlap more than those of spectrin and alpha-actinin, stabilizing the helix during longitudinal weight bearing, possibly accounting in part for the lack of structural information, as there may be difficulties in subregions of the crystalline rod. Nevertheless, the conservation of tryptophan residues at the center of the hydrophobic core provides "anchor points" for two of the three splices ("those in the" a "and" C "helices). Note the prominence of W at position 16 in the HMM identification shown in fig. 3, for example (Wheeler et al, BMC Bioinformatics 2014). All crystal structures were analyzed for triple helix repeats containing two interacting tryptophan residues and HMMscan analysis on the HMMer portal (web portal) was used to define the probability that each position within the "B" helix would correspond to a cross-sectional plane bisecting tryptophan (i.e., a plane bisecting the helix repeat perpendicular to its long axis).

LQGEI E AHTDVY (N-terminal to C-terminal, amino acid sequence in full-length dystrophin TH 22) Q EQV (N-to C-terminal, amino acid sequence in full-length dystrophin TH 2) is a sequence around the splice junction of two TH helices B (helix 2, the second of the three in TH) of the full-length dystrophin forming hybrid TH in nano-dystrophin. Underlined letters E and Q represent splice junctions in helix B, while both E and Q remain as set forth in SEQ ID NO: 1, as exemplified by the resultant hybrid TH. However, one skilled in the art will appreciate that triple-spliced mutant dystrophins for use herein may have a sequence other than as set forth as SEQ ID NO: 1, the splice point in helix B outside the EQ interval. Such a shear connection may be located, for example, at LQGEI E AHTDVY…QEDLE Q EQV and fig. 5 and SEQ ID NOs: 13-18, at the N-terminus or C-terminus of any amino acid in helix B shown in the corresponding amino acid sequence of TH, or at another corresponding position in helix B in another TH. Correspondence of positions in a sequence can be determined by amino acid sequence alignment or Hidden Markov Models (HMMs) between any two or more dystrophin TH.

Similarly, triple-spliced mutant dystrophin-related proteins for use herein may have a splice junction in helix B that is found in the amino acid sequence as set forth as SEQ ID NO: 3, or may be located between HQ as shown by the characteristics of AEIDA HNDIFKS (N-terminal to C-terminal, amino acid sequence in full-Length dystrophin-related protein TH 20) … DL EAEQVKV（N-terminal to C-terminal, amino acid sequence in full-length dystrophin-related protein TH 2) or the N-terminal or C-terminal end of any amino acid in helix B, or another corresponding position of helix B in another dystrophin-related protein TH. Correspondence of positions in a sequence can be determined by amino acid sequence alignment or Hidden Markov Models (HMMs) between any two or more dystrophin-related proteins TH.

In certain embodiments, the splice junctions in helix a or C, which is helix 1, i.e., the first helix, and helix 3, i.e., the third helix, of the three helices in the three-helix consensus sequence, can be located at tryptophan (W) at the core of the Hidden Markov Model (HMM) and all crystal structures of the proteins in the superfamily. In further embodiments, the splice junction in helix a or C may be located at a position from w(s) to the C-terminal side or to the N-terminal side of the protein of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids or 5 amino acids.

B. Human nano-dystrophin related protein sequences

The larger capital letters in the following sequences indicate the same portions as the N-terminal region of the full-length human dystrophin-related protein. The smaller capital letters indicate the same region as the C-terminal region of the full-length human dystrophin-related protein. W in italics corresponds to tryptophan residues in the "a" and "C" helices located at the core of the Hidden Markov Model (HMM) and all crystal structures of the proteins in the superfamily, and HQ in italics corresponds to a position within the superfamily HMM of the "B" helix flanked by hypothetical cross-sectional planes as depicted in figure 2F. The expected secondary and tertiary structures of the folded protein correspond to the hybrid TH depicted in figure 4.

In certain embodiments, the nano-dystrophin-related proteins provided herein comprise an amino acid sequence comprising a triple splice mutation in the full length dystrophin-related protein joining repeats 2 and 20:

detailed design of nano-dystrophin-related proteins to address structural constraints is described herein. Phylogenetic analysis suggests that the ancestral triple helix repeat sequence is present in proteins that are orthologous to alpha-actinin, as this is the only protein in the majority of the single cell eukaryotic proteome that matches the spectrin consensus sequence. The training set (training sets) comprising alpha-actinin, alpha-and beta-spectrin and dystroplakins produced HMMs whose identity showed an abnormal conservation of tryptophan residues. Among the available high resolution crystal structures, the position of the side chains and the aromatic interactions are highly conserved, as is the structure of the third "B" alpha helix. In the above sequence, please note the underlined positions of the W and B helix amino acids H and Q. Rearrangement or "splicing" of the polypeptide sequences corresponding to these subdomains is depicted in different font sizes, while the three locally discontinuous (focal discontinuity) sites correspond to the cross-sectional plane illustrated in fig. 2F, thereby creating a 3-D hybrid (hybrid) between dystrophin-related protein repeats 2 and 20.

Also provided herein are nano-dystrophin-related proteins having five spectrin-like triple-helical repeats, comprising a hybrid triple-helical domain formed by splicing TH 1 and 18 in a full-length human dystrophin-related protein. In certain embodiments, the nano-dystrophin-related protein has the following sequence:

C. human nano-dystrophin sequence:

the following larger capital letters indicate the same portion as the N-terminal region of full-length human dystrophin. The smaller capital letters indicate the same region as the C-terminal region of full-length human dystrophin. Underlined W corresponds to tryptophan residues in the "a" and "C" helices located at the core of the Hidden Markov Model (HMM) and all crystal structures of the proteins in the superfamily. The underlined EQs correspond to positions within the superfamily HMM of "B" helices flanked by hypothetical cross-sectional planes as depicted in FIG. 2F. The expected secondary and tertiary structures of the folded protein correspond to the hybrid TH depicted in figure 4.

Also provided herein are nano-dystrophins having five spectrin-like triple helical repeats, including hybrid triple helical domains formed by splicing TH 1 and 20 in full-length human dystrophin. In certain embodiments, the nano-dystrophin-related protein has the following sequence:

D. Others

Many high molecular weight proteins have repeating internal domains. Dystrophin is an example of a large class of proteins for which limited three-dimensional structural information is available for repeated internal domains. The same approach in concept may be applicable to other genetic diseases. For example, common inherited coagulation disorders von willebrand disease are caused by mutations in the 8 kilobase coding sequence of proteins with multiple repetitive sequences ("vWF"). Recently published studies have revealed that transgenic expression of recombinant proteins in the liver is sufficient to treat the disease, but the coding sequences are too large for a single AAV genome, and trans-splicing between two AAV genomes is too inefficient to achieve therapeutic levels of recombinant protein expression. There is no crystal structure of the entire protein, but analysis by the methods outlined herein immediately suggests an opportunity to generate miniaturized nano-vWF proteins that can replace full-length vWF.

Von Willebrand disease is generally caused byVWFGenetic diseases caused by variations (mutations) in genes.VWFThe genes provide instructions for the preparation of coagulation proteins called von Willebrand factor (von Willebrand factor), which are important for the formation of blood clots and to prevent further blood loss following injury. If von willebrand factor does not function properly or too little protein is available, blood clots will not form properly. Reducing the amount of von Willebrand factor or causing the protein to function abnormally (or not at all) VWFGenetic mutations are responsible for the diseases and symptoms associated with the condition. These variations may be inherited in an autosomal dominant or autosomal recessive manner, or may first occur in diseased humans without any other cases in the family (referred to as de novo mutations). See, e.g., ghr.nlm.nih.gov/condition/von-willebrand-disease. Those of skill in the art will understand that any of the compositions, approaches (regions), aspects, embodiments, and methods described herein throughout the specification are intended to apply to, or comprise, von willebrand disease, mutant von willebrand factor, a nucleic acid sequence encoding mutant von willebrand factorA vector for the seed coding sequence.

Von willebrand factor (vWF) is important in maintaining hemostasis. It promotes the adhesion of platelets to the vascular injury site by forming a molecular bridge between the endothelial collagen matrix and the platelet surface receptor complex GPIb-IX-V. It may also act as a chaperone for coagulation factor VIII, delivering it to the site of injury, stabilizing its heterodimeric structure and protecting it from premature clearance from plasma. There are 2 isoforms of von willebrand factor. Isoform 1 is considered a canonical sequence and contains the sequence with the UniProtKB identifier: the amino acid sequence of P04275-1, which is incorporated herein. Another isoform is the one with the UniProtKB identifier: isoform 2 of P04275-2, the sequence of which is incorporated herein. "full-length" vWF can refer to isoform 1. In certain embodiments, "full-length" vWF can refer to isoform 2 of vWF or other homologues of vWF. Homologs of von willebrand factor have been identified in a variety of organisms, including mice (UniProt Q8CIZ 8), rats (UniProt Q62935), pigs (UniProt Q28833), and dogs (UniProt Q28295). Possible nucleic acid sequences encoding von willebrand factor or any other isoform or homolog thereof are publicly available, see, e.g., NCBI reference sequence: NM _000552.4, X04385.1, M10321.1, X04146.1, AK128487.1, AK297600.1, AK292122.1, BC069030.1, BC022258.1, U81237.1, K03028.1, M17588.1, AF086470.1, and X02672.1, each of which is incorporated herein.

As used herein, "mutant von willebrand factor" or "mutant vWF" refers to von willebrand factor having an internal deletion of one or more repeat sequences and a splice junction connecting the two repeat sequences, wherein the splice junction site is within the repeat sequences but not between the repeat sequences. In certain embodiments, the splice point is determined by a method similar to the splice point in the a. helix, i. dystrophin, dystrophin-related protein, or the like, or any of the examples. The identification or characterization of one or more repeated sequences in vWF may be determined by Hidden Markov Models (HMMs) or any other conventional method. See, e.g., Zhou YF et al, Sequence and structure relationships with in von Willebrand factor, blood. 2012, 7/12, 120(2) 449-58, doi 10.1182/blood-2012-01-405134, Epub 2012, 4/6; sadler JE. Biochemistry and genetics of von Willebrand factor, Annu Rev Biochemistry, 1998, 67: 395-424; and Perkins SJ et al, The second structure of The von Willebrand factor type A domain in factor B of human composition by fashion transform in specific specificity, Its cure in collagen types VI, VII, XII and XIV, The integrins and other proteins by extracted structure predictions, J Mol biol.1994, 4, 22, 238(1) 104-19. The nucleic acid sequence encoding the mutant vWF can be generated by means for reverse translation. In addition, the coding sequence can be codon optimized for expression in a subject, e.g., human, mouse, rat, or dog.

It should be understood that any compositions described herein are intended to apply to other compositions, aspects, embodiments, and methods described throughout the specification.

II. expression cassette

Provided herein are expression cassettes comprising a nucleic acid sequence encoding a dystrophin superfamily triple splice mutant protein under the control of regulatory sequences that direct its expression.

As used herein, the term "expression" or "gene expression" refers to the process of using information from a gene for the synthesis of a functional gene product. The gene product may be a protein, peptide or nucleic acid polymer (e.g., RNA, DNA or PNA). In certain embodiments, the functional gene product is a dystrophin superfamily triple splice mutant protein. In certain embodiments, the terms "gene," "minigene," and "transgene" refer to a sequence encoding a dystrophin superfamily triple splice mutant protein, such as a nano-dystrophin or a nano-dystrophin related protein.

As used herein, "expression cassette" refers to a nucleic acid polymer comprising a coding sequence, a promoter, and may include other regulatory sequences thereto, which cassette may be packaged into a vector.

As used herein, the term "regulatory sequence" or "expression control sequence" refers to a nucleic acid sequence, such as an initiator sequence, enhancer sequence, and promoter sequence, that induces, blocks, or otherwise controls the transcription of a protein-encoding nucleic acid sequence to which it is operably linked.

As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with the coding sequence and expression control sequences that act in trans or remotely to control the coding sequence. In certain embodiments, the coding sequence encodes a dystrophin superfamily triple splice mutein.

The term "heterologous" when used in reference to a protein or nucleic acid means that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, having two or more sequences from unrelated genes arranged to produce new functional nucleic acids. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct expression of coding sequences from a different gene. Thus, the promoter is heterologous with respect to the coding sequence.

Identity or similarity with respect to sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues from the same group based on common side chain properties, see below) to the peptide and polypeptide regions provided herein after aligning the sequences and introducing gaps, if necessary, to achieve a maximum percent sequence identity. Percent (%) identity is a measure of the relationship between two polynucleotides or two polypeptides, as determined by comparing their nucleotide or amino acid sequences, respectively. Typically, two sequences to be compared are aligned to give the maximum correlation between the sequences. The alignment of the two sequences is examined and the number of positions giving the exact amino acid or nucleotide correspondence between the two sequences is determined, divided by the total length of the alignment and multiplied by 100 to give a% identity value. The% identity values can be determined over the full length of the sequences to be compared, which is particularly suitable for sequences of the same or very similar length and which are highly homologous, or over a shorter determined length, which is more suitable for sequences of unequal length or which have a lower level of homology. There are many algorithms and computer programs based thereon that are available to be used in the literature and/or publicly or commercially available for performing alignments and percent identities. The choice of algorithm or program is not a limitation of the present invention.

Examples of suitable alignment programs include, for example, the software CLUSTALW under Unix and then imported into the Bioedit program (Hall, T.A. 1999, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. acids. Symp. Ser. No. 41: 95-98); clustal Omega available from EMBL-EBI (Sievers, Fabian et al, "Fast, scalable generation of high-quality protein multiple sequence alignment Using Clustal Omega." Molecular systems biology 7.1 (2011): 539 and Goujon, Mickael et al, "A new Bioinformation analysis tools frame at EMBL-EBI." Nucleic acids research 38. sup.2 (2010): W695-W699); the Wisconsin Sequence Analysis Package (Wisconsin Sequence Analysis Package), version 9.1 (Devereux J et al, Nucleic Acids Res., 12:387-395, 1984, available from Genetics Computer Group, Madison, Wis., USA). The programs BESTFIT and GAP can be used to determine the% identity between two polynucleotides and the% identity between two polypeptide sequences.

Other programs for determining identity and/or similarity between sequences include, for example, the BLAST program series available from the National Center for Biotechnology Information (NCB) of Bethesda, Md., usa and accessible through the NCBI homepage located at www.ncbi.nlm.nih.gov, the ALIGN program (version 2.0) as part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weight residue table (weight residual table), a gap length penalty of 12, and a gap penalty of 4 can be used; and FASTA (Pearson W. R. and Lipman D. J., Proc. Natl. Acad. Sci. USA, 85: 2444-. SeqWeb software (GCG Wisconsin Package: web-based interface to Gap program).

In one embodiment, the expression cassette is designed for expression and secretion in a subject such as a human, rat, mouse, or dog. In one embodiment, the expression cassette is designed for expression in muscle, including cardiac muscle, skeletal muscle, and smooth muscle.

In certain embodiments, the regulatory control elements include a promoter sequence that is part of the expression control sequence, e.g., located between the selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [ see, e.g., WO 2011/126808 and WO 2013/04943], tissue-specific promoters (see, e.g., www.invivogen.com/tissue-specific-promoter), or promoters responsive to a physiological cord may be utilized in the vectors described herein. In certain embodiments, a muscle-specific promoter, e.g., a Muscle Creatine Kinase (MCK) promoter, a desmin promoter, an Mb promoter, or a promoter for myosin-heavy polypeptide 2, myosin, troponin T type 3, troponin C type 2, myosin binding protein C, fast skeletal myosin (fast skeletal myosin) light chain 2, actin α 2, synaptic vesicle associated membrane protein 5, thyroid hormone receptor interacting protein (interctor) 10, tropomyosin 3, tropomyosin γ, myogenic differentiation 1, myogenic factor 6 (force protein) or calcium channel, voltage-dependent γ 1 may be used. Another useful promoter is the synthetic SPc5-12 promoter, which allows robust expression in skeletal and cardiac muscle (see, e.g., Rasowo et al, European Scientific Journal, 6.month edition 2014, vol. 10, vol. 18, and U.S. patent application publication Nos. 20040192593 and 2017/0275649, which are incorporated herein by reference in their entirety).

In certain embodiments, the regulatory control element comprises a cardiac-specific cis regulatory module (CS-CRM), which comprises any one of CS-CRM elements 1-8. In certain embodiments, the regulatory sequence comprises a CS-CRM4 element or a CS-CRM4 element in combination with an SPc5-12 promoter, such as the chimeric synthetic CS-CRM4/SPc5-12 promoter previously described (Rincon et al, Genome-wide computer analysis systems catalysts-specific transcriptional transport Cis-regulatory movable bed heat gene expression. Mol Ther. 2015, 1/23 (43-52), which is incorporated herein by reference).

Examples of constitutive promoters suitable for controlling expression of therapeutic products include, but are not limited to, the chicken β -actin (CB) promoter, the human Cytomegalovirus (CMV) promoter, the ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV 40), the U6 promoter, the metallothionein promoter, the EFl α promoter, the ubiquitin promoter, the Hypoxanthine Phosphoribosyltransferase (HPRT) promoter, the dihydrofolate reductase (DHFR) promoter (Scharfmann et al, Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), the adenosine deaminase promoter, the phosphoglycerate kinase (PGK) promoter, the pyruvate kinase promoter phosphoglycerate mutase promoter, the β -actin promoter (Lai et al, Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989), the long terminal repeat sequences (LTR) of Moloney leukemia virus and other retroviruses, Thymidine kinase promoter of herpes simplex virus and other constitutive promoters known to those skilled in the art. Examples of tissue or cell specific promoters suitable for use in the present invention include, but are not limited to, endothelin-I (ET-I) and Flt-I, which are specific for endothelial cells, FoxJ1 (which targets ciliated cells).

Inducible promoters suitable for controlling expression of the therapeutic product include promoters responsive to exogenous agents (e.g., pharmacological agents) or to a physiological cord. These response elements include, but are not limited to, binding to HIF-I α andβhypoxia Responsive Element (HRE), metal ion responsive element, such as those provided by Mayo et al (1982, Cell 29: 99-108); as described by Brinster et al (1982, Nature 296: 39-42) and Searle et al (1985, mol. cell. biol. 5: 1480-1489); or Heat Shock responsive elements as described by Nouer et al (in Heat Shock Response, eds Nouer, L., CRC, Boca Raton, Fla., pp.167-220, 1991).

In one embodiment, expression of a coding sequence is controlled by a regulatable promoter that provides tight control over transcription of the coding sequence, e.g., a pharmacological agent, or a transcription factor activated by a pharmacological agent, or in alternative embodiments, a physiological cue. Promoter systems that are not leaky and can be tightly controlled are preferred. Examples of regulatable promoters that are ligand-dependent transcription factor complexes useful in the present invention include, but are not limited to, nuclear receptor superfamily members activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysones, and analogs and mimetics thereof (mimetics)) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch (gene switch) is an EcR-based gene switch. Examples of such systems include, but are not limited to, the systems described in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. published patent application Nos. 2006/0014711, 2007/0161086, and International published application No. WO 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. published patent application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457 and 2006/0100416 and International published application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075 and WO 2005/108617, each of which is incorporated herein by reference in its entirety. An example of a non-steroidal ecdysone agonist regulated System is the Rheoswitch Mammalian Inducible Expression System (Rheoswitch Mammalian Expression System) (New England Biolabs, Ipswich, Mass.).

Still other promoter systems may include responsive elements including, but not limited to, tetracycline (Tet) responsive elements (such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89: 5547-); or hormone responsive elements such as described by Lee et al (1981, Nature 294: 228-) -232); Hynes et al (1981, Proc. Natl. Acad. Sci. USA 78: 2038-) -2042); Klock et al (1987, Nature 329: 734-) -736); and Israel & Kaufman (1989, Nucl. Acids Res. 17: 2589-) -2604) and other inducible promoters known in the art using such promoters may be used, for example, by the Tet-off system (Gossen et al, 1995, Sci: 268: 6-9; Gossen et al, Acad. 17617647. Nature 51. Nature R. USA 51.: 51. USA 51.; 51. Uutr 51. multidot. KR., 2003, Genome biol., 4(10): 231; deuschle U et al, 1995, Mol Cell biol. (4): 1907-14); mifepristone (RU 486) regulated systems (Generswitch; Wang Y et al, 1994, Proc. Natl. Acad. Sci. USA, 91(17): 8180-4; Schillinger et al, 2005, Proc. Natl. Acad. Sci. U S A.102(39): 13789-94); a humanized tamoxifen-dep regulatable system (Roscilli et al, 2002, mol. Ther. 6(5): 653-63) controls the expression of the transgene. The gene switch may be based on heterodimerization of FK506 binding protein (FKBP) with FKBP Rapamycin Associated Protein (FRAP) and is regulated by rapamycin or a non-immunosuppressive analog thereof. Examples of such systems include, but are not limited to, ARGENT-transciptional Technology (ARGENT-transgenic technologies) (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757 and 6,649,595, U.S. publication No. 2002/0173474, U.S. publication No. 200910100535, U.S. Pat. No. 5,834,266, U.S. Pat. No. 7,109,317, U.S. Pat. No. 7,485,441, U.S. Pat. 7,485,441, U.S. Pat. No. 7,485,441, the systems described in U.S. Pat. No. 6,509,152, U.S. Pat. No. 6,015,709, U.S. Pat. No. 6,117,680, U.S. Pat. No. 6,479,653, U.S. Pat. No. 6,187,757, U.S. Pat. No. 6,649,595, U.S. Pat. No. 6,984,635, U.S. Pat. No. 7,067,526, U.S. Pat. No. 7,196,192, U.S. Pat. No. 6,476,200, U.S. Pat. No. 6,492,106, WO 94/18347, WO 96/20951, WO 96/06097, WO 97/31898, WO 96/41865, WO 98/02441, WO 95/33052, WO 99110508, WO 99110510, WO 99/36553, WO 99/41258, WO 01114387, ARGENT ™ Regulated Transcription retroviral Kit (ARGENT. Regulated Transcription retroviruses Kit), version 2.0 (9109102) and ARGENT ™ Regulated Transcription Plasmid Kit (ARGENT. Regulated Transcription Plasmid Kit), version 2.0 (9109/02), each of which is incorporated herein by reference in its entirety. The Ariad system was designed to be induced by rapamycin and its analogs known as "rapalogs". Examples of suitable rapamycins are provided in the documents listed above in the description of the ARGENT system. In one embodiment, the molecule is rapamycin [ e.g., commercially available as rapalmone (Rapamune) by Pfizer ]. In another embodiment, rapalog, designated AP21967 [ ARIAD ], is used. Examples of such dimerizer molecules useful in the present invention include, but are not limited to, rapamycin, FK506, FK1012 (homodimers of FK 506), rapamycin analogues ("rapalogs") which can be readily prepared by chemical modification of natural products to add "bumps" that reduce or eliminate affinity for endogenous FKBP and/or FRAP. Examples of rapalogs include, but are not limited to, for example, AP26113 (Ariad), AP1510 (Amara, J.F. et al, 1997, Proc Natl Acad Sci USA, 94(20): 10618-23) AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692 and AP1889, with "bumps" designed to minimize interaction with endogenous FKBP 1881881881881889. Still other rapalogs may also be selected, for example, AP23573 [ Merck ].

Other suitable enhancers include those suitable for the desired target tissue indication. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may be different from each other. For example, the enhancer may include the CMV immediate early enhancer. The enhancer may be present in two copies located adjacent to each other. Alternatively, double copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further comprises an intron, for example, the chicken β -actin intron. Other suitable introns include those known in the art, for example, as described in WO 2011/126808. Examples of suitable polyA sequences include, for example, rabbit binding globulin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone and synthetic polyAs. Optionally, one or more sequences may be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence that can be engineered upstream of polyA and downstream of the coding sequence [ see, e.g., MA Zanta-Boussif et al, Gene Therapy (2009) 16: 605-619. In one embodiment, the enhancer is a double or triple tandem MCK enhancer.

In one embodiment, the regulatory sequence further comprises a polyadenylation signal (polyA). In a further embodiment, the polyA is rabbit globin polyA. See, for example, WO 2014/151341. Alternatively, another polyA, such as a human growth hormone (hGH) polyadenylation sequence, SV40 polyA or a synthetic polyA may be included in the expression cassette.

It is to be understood that the compositions in the expression cassettes described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described throughout the specification.

III. vector

In certain embodiments, the nucleic acid sequence encoding the dystrophin superfamily triple splice muteins is engineered in a vector, including viral vectors and non-viral vectors.

As used herein, a "vector" is a biological or chemical moiety that comprises a nucleic acid sequence, which can be introduced into an appropriate target cell for replication or expression of the nucleic acid sequence. Examples of carriers include, but are not limited to, recombinant viruses, plasmids, Lipoplexes, polymersomes (polymersomes), Polyplexes, dendrimers, cell-penetrating peptide (CPP) conjugates, magnetic particles, or nanoparticles. Such vectors preferably have one or more origins of replication, and one or more sites into which a coding sequence or expression cassette can be inserted. Vectors often have means by which cells with the vector can be selected from those cells that do not have the vector, e.g., they encode a drug resistance gene. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods for the production, characterization or quantification of vectors are available to those skilled in the art.

As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., a recombinant AAV) is produced. The host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect or yeast) containing exogenous or heterologous DNA that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Examples of host cells can include, but are not limited to, isolated cells, cell cultures, Escherichia coli (e.g., Escherichia coli) cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, liver cells, kidney cells, muscle cells, smooth muscle cells, cardiac muscle cells, or skeletal muscle cells.

The term "exogenous" as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the location where it is found in the chromosome or host/target cell. Exogenous nucleic acid sequences also refer to sequences that are derived from and inserted into the same host cell or host but which are present in a non-native state (e.g., different copy numbers) or under the control of different regulatory elements.

As used herein, the term "target cell" refers to any target cell in which expression of a dystrophin superfamily triple splice mutant protein is desired. In certain embodiments, the term "target cell" is intended to refer to a cell of a subject being treated for MD (including DMD and BMD). Examples of target cells may include, but are not limited to, liver cells, kidney cells, muscle cells, smooth muscle cells, cardiac muscle cells, or skeletal muscle cells. In certain embodiments, the vector is delivered ex vivo to the target cell. In certain embodiments, the vector is delivered to the target cell in vivo.

The non-viral vector may be a plasmid carrying an expression cassette comprising at least a nucleic acid sequence encoding a dystrophin superfamily triple splice mutant protein and optionally a promoter or other regulatory elements, which is delivered to the heart. Non-viral delivery of nucleic acid molecules to smooth muscle and the cardiac muscle system may include chemical or physical methods. Chemical methods include the use of cationic liposomes ("lipoplex"), polymers ("polyplex"), combinations of both ("lipoplex"), calcium phosphate and DEAE dextran. Additionally or optionally, such nucleic acid molecules may be used in compositions further comprising one or more agents including, for example, liposomal agents, such as, for example, DOTAP/DOPE, Lipofectin, Lipofectamine, and the like, and cationic polymers, such as PEI, effect, and dendrimers. This reagent is effective for transfecting smooth muscle cells. In addition to chemical methods, there are many physical methods that facilitate the direct entry of uncomplexed DNA into cells. These methods may include microinjection, hydration, electroporation, ultrasound, and biolistic delivery of individual cells (i.e., gene gun).

In certain embodiments, the expression cassette comprising a nucleic acid sequence encoding a dystrophin superfamily triple splice mutein is carried by a viral vector, e.g., a recombinant adenovirus, lentivirus, bocavirus (bocavirus), hybrid AAV/bocavirus (see, e.g., Yan Z et al, A novel chimeric adeno associated virus 2/human bocavirus 1 partial virus vector effective transfer human air epithelial. Mol The 12.2013; 21(12):2181-94. doi: 10.1038/mt.2013.92. Epub 2013.7.30), herpes simplex virus or adeno associated virus. In such embodiments, the viral vector may be a replication-defective virus.

"replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which a vector genome comprising an expression cassette is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, they are unable to produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome may be artificially engineered to be "content-free" -containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes may be provided during the production process. Thus, progeny virions are considered safe for use in gene therapy because they cannot replicate and infect unless in the presence of the viral enzymes required for replication.

The vector may be any vector known in the art or disclosed above, including naked DNA, plasmids, phages, transposons, cosmids, episomes, viruses, and the like. Introduction of the vector into the host cell may be accomplished by any means known in the art or as disclosed above, including transfection and infection. One or more adenoviral genes can be stably integrated into the genome of the host cell, stably expressed as episomes, or transiently expressed. The gene products may be expressed all transiently, episomally or stably integrated, or some gene products may be stably expressed while others are transiently expressed. In addition, the promoter of each adenoviral gene can be independently selected from a constitutive promoter, an inducible promoter, or a native adenoviral promoter. Promoters may be regulated by a particular physiological state of the organism or cell (i.e., by differentiation state or in replicating or dormant cells) or by factors added exogenously, for example.

Introduction of the molecule (as a plasmid or virus) into the host cell can also be accomplished using techniques known to the skilled artisan and as discussed throughout the specification. In a preferred embodiment, standard transfection techniques are used, e.g., CaPO ₄Transfection or electroporation. The assembly of the selected DNA sequences of the adenovirus (as well as the transgene and other vector elements into various intermediate plasmids, and the use of plasmids and vectors to produce recombinant viral particles is accomplished using conventional techniques, including conventional cDNA Cloning techniques, such as, for example, textbooks [ Sambrook et al, Molecular Cloning: A Laboratory Manual]Those described in (1), use of overlapping oligonucleotide sequences of the adenovirus genome, and polymerizationThe synthase chain reaction and any suitable method of providing the desired nucleotide sequence. Using standard transfection and co-transfection techniques, e.g. CaPO₄Precipitation techniques. Other conventional methods used include homologous recombination of viral genomes, plaque of the virus in agar overlays, methods of measuring signal production, and the like.

The dosage of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and thus may vary from patient to patient. For example, a therapeutically effective adult or veterinary dose of viral vectors is typically in the range of about 100 μ L to about 100 mL of vector containing about 1X 10⁶To about 1X 10¹⁵Particles, about 1X 10¹¹To 1X 10 ¹³Per particle or about 1X 10⁹To 1X 10¹²Concentration of individual particle virus. The dosage will vary depending on the size of the animal and the route of administration. For example, for a single site, a suitable human or veterinary dosage for intramuscular injection (for an animal of about 80 kg) is about 1X 10 per mL⁹To about 5X 10¹²Within the range of individual particles. Optionally, multiple sites of administration may be delivered. In another example, for a formulation, a suitable human or veterinary dosage may be at about 1 × 10¹¹To about 1X 10¹⁵Within the range of individual particles. Those skilled in the art can adjust these dosages depending on the route of administration and the therapeutic or vaccination application in which the recombinant vector is employed. The expression level of the transgene can be monitored to determine the frequency of dose administration. Still other methods of determining the timing of the frequency of administration will be apparent to those skilled in the art.

As used herein, "vector genome" refers to a nucleic acid sequence packaged within a vector.

A. Replication-defective adenovirus vectors

In one embodiment, a replication-defective adenovirus vector is used. Any of a number of suitable adenoviruses may be used as a source of adenoviral capsid sequences and/or for production. See, for example, U.S. patent nos. 9,617,561; 9,592,284, respectively; 9,133,483, respectively; 8,846,031, respectively; 8,603,459, respectively; 8,394,386, respectively; 8,105,574, respectively; 7,838,277, respectively; 7,344,872, respectively; 8,387,368, respectively; 6,365,394, respectively; 6,287,571, respectively; 6,281,010, respectively; 6,270,996, respectively; 6,261,551, respectively; 6,251,677, respectively; 6,203,975, respectively; 6,083,716; 6,019,978, respectively; 6,001,557, respectively; 5,872,154, respectively; 5,871,982, respectively; 5,856,152, respectively; 5,698,202. Still other adenoviruses are available from the American Type Culture Collection. In one embodiment, the adenovirus particle is made replication-deficient by a deletion in the E1a and/or E1b genes. On the other hand, the adenovirus is made replication-defective by another means, optionally while retaining the E1a and/or E1b genes. The adenoviral vector may also contain other mutations to the adenoviral genome, for example, temperature sensitive mutations or deletions in other genes. In other embodiments, it is desirable to retain the entire E1a and/or E1b region in the adenoviral vector. Such an intact E1 region may be located in its natural location in the adenovirus genome or placed at a deletion site in the natural adenovirus genome (e.g., in the E3 region).

In constructing useful adenoviral vectors for delivering genes to human (or other mammalian) cells, a range of adenoviral nucleic acid sequences can be used in the vectors. For example, all or part of the adenovirus delayed early gene E3 may be eliminated from the adenoviral sequences forming part of the recombinant virus. It is believed that the function of E3 is irrelevant to the function and production of recombinant viral particles. Adenoviral vectors having deletions of at least the ORF6 region of the E4 gene can also be constructed and are more desirable due to the functional redundancy of the entire E4 region of the region. Yet another adenovirus vector contains a deletion in the delayed early gene E2 a. Deletions may also be made in any of the late genes L1 to L5 of the adenovirus genome. Similarly, metaphase genes IX and IVa₂May be useful for some purposes. Other deletions may be made in other structural or non-structural adenovirus genes. The deletions discussed above may be used individually, i.e., the adenoviral sequences for use as described herein may contain deletions in only a single region. On the other hand, deletions of the entire gene or portions thereof effective to destroy its biological activity may be used in any combination. For example, in one exemplary vector, adenopathy The toxin sequence may have deletions of the E1 gene and the E4 gene, or the E1, E2a and E3 genes, or the E1 and E3 genes, or the E1, E2a and E4 genes, with or without deletion of E3, and the like. As discussed above, such deletions may be used in combination with other mutations (e.g., temperature sensitive mutations) to achieve the desired results.

Adenoviral vectors lacking any essential adenoviral sequences (e.g., E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4, and L5) can be cultured in the presence of the missing adenoviral gene product required for viral infectivity and propagation of the adenoviral particle. These helper functions may be provided by culturing the adenoviral vector in the presence of one or more helper constructs (e.g., plasmids or viruses) or a packaging host cell. See, for example, the techniques described in International patent application WO96/13597, published 5/9 1996 and incorporated herein by reference, for the preparation of "minimal" human Ad vectors.

a. Helper virus

Thus, depending on the adenoviral gene content of the viral vector used to carry the expression cassette, a helper adenovirus or non-replicating viral fragment may be necessary to provide sufficient adenoviral gene sequences necessary for the production of infectious recombinant viral particles containing the expression cassette. Useful helper viruses contain selected adenoviral gene sequences that are not present in the adenoviral vector construct and/or are not expressed in the packaging cell line into which the vector is transfected. In one embodiment, the helper virus is replication-defective and contains multiple adenoviral genes in addition to the sequences described above. It is desirable to use such helper viruses in combination with a cell line expressing E1.

Helper viruses can also form polycationic conjugates, such as Wu et al, J. biol. chem., 264:16985-16987 (1989); fisher and J.M. Wilson, biochem J., 299:49 (1/4 of 1994). The helper virus may optionally contain a second reporter minigene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus that is different from the transgene on the adenoviral vector allows the Ad vector and helper virus to be monitored independently. The second reporter gene is used to enable separation of the resulting recombinant virus and helper virus upon purification.

b. Complementary cell lines

In order to produce a recombinant adenovirus (Ad) deleted in any of the above genes, the function of the deleted gene region (if necessary for viral replication and infectivity) must be provided to the recombinant virus by a helper virus or cell line, i.e., a complementing or packaging cell line. In many cases, cell lines expressing human E1 can be used for trans-complementary (trans-complementary) Ad vectors. However, in some cases it will be desirable to utilize a cell line expressing the E1 gene product, which can be used to generate an E1-deleted adenovirus. Such cell lines have been described. See, for example, U.S. Pat. No. 6,083,716.

If desired, one can use the sequences provided herein to generate a packaging cell or cell line that expresses at least the adenovirus E1 gene under the transcriptional control of a promoter for expression in the parental cell line of choice. Inducible or constitutive promoters can be used for this purpose. Examples of such promoters are described in detail elsewhere in this specification. The parental cells are selected for the generation of new cell lines expressing any desired adenoviral genes. Such parental cell lines may be, without limitation, HeLa [ ATCC accession number CCL 2], A549 [ ATCC accession number CCL 185], HEK 293, KB [ CCL 17], Detroit [ e.g., Detroit 510, CCL 72] and WI-38 [ CCL 75] cells, among others. These cell lines are all available from the American type culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-. Other suitable parental cell lines may be obtained from other sources.

This cell line expressing E1 can be used to generate recombinant adenoviral E1 deletion vectors. In addition or alternatively, cell lines expressing one or more adenoviral gene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructed using essentially the same procedures as used to produce recombinant viral vectors. Such cell lines can be used to transcomplement adenoviral vectors deleted in essential genes encoding those products, or to provide helper functions necessary for packaging helper-dependent viruses (e.g., adeno-associated viruses). Preparation of the host cell includes techniques such as assembly of the selected DNA sequence. The assembly may be accomplished using conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and described in Sambrook et al cited above, the use of overlapping oligonucleotide sequences of the adenoviral genome, the incorporation of polymerase chain reaction, synthetic methods and any other suitable method that provides the desired nucleotide sequence.

In yet another alternative, the essential adenoviral gene products are provided in trans by an adenoviral vector and/or a helper virus. In this case, suitable host cells may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, as well as eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Particularly desirable host cells are selected from any mammalian species, including, but not limited to, cells such as A549, WEHI, 3T3, 10T1/2, HEK 293 cells or PERC6 (both expressing functional adenovirus E1) [ Fallaux, FJ et al, (1998), Hum Gene Ther, 9: 1909-. The choice of mammalian species from which the cells are provided is not a limitation of the present invention; the types of mammalian cells, i.e., fibroblasts, hepatocytes, tumor cells, etc., are also not.

c. Assembly of viral particles and transfection of cell lines

Typically, when the vector comprising the minigene is delivered by transfection, the vector is delivered to about 1X 10 in an amount of about 5 to about 100 μ g DNA, and preferably about 10 to about 50 μ g DNA ⁴Cell to about 1X 10¹³A cell, and preferably about 10⁵And (4) cells. However, factors such as the vector of choice, the method of delivery, and the host cell of choice may be considered to regulate the relative amount of vector DNA to host cell.

B. Lentiviral system

A variety of different lentiviral systems are known in the art. See, for example, WO2001089580 a1 for a method for obtaining stable cardiovascular transduction using a lentiviral system. See, for example, U.S. patent 6,521,457. See also The discussion of NB Wasala et al, The evolution of heart gene delivery vectors, J Gen Med., 2011.10,; 13(10): 557-.

C. Recombinant AAV

In certain embodiments, a vector genome refers to a nucleic acid sequence packaged within a vector, such as a rAAV. For rAAV, such nucleic acid sequences may contain AAV Inverted Terminal Repeats (ITRs) and an expression cassette. In one example, the vector genome comprises, from 5 'to 3', at least AAV 5 'ITRs, nucleic acid sequences encoding triple splice muteins of the dystrophin superfamily, and AAV 3' ITRs. In one example, the vector genome comprises, from 5 'to 3', at least an AAV 5 'ITR, an expression cassette and an AAV 3' ITR. The ITRs may be from AAV2 or from a different source AAV than AAV 2. In other embodiments, the vector genome may contain Terminal Repeats (TRs) required for a self-complementary AAV vector.

In one embodiment, provided herein is a recombinant adeno-associated virus (rAAV) having an AAV capsid and a vector genome, wherein the vector genome comprises an expression cassette as described herein or a nucleic acid sequence encoding a dystrophin superfamily triple splice mutein (i.e., cDNA as used herein) under the control of regulatory sequences that direct its expression.

In some embodiments, the dystrophin superfamily triple splice muteins are designed for expression from recombinant adeno-associated viruses, and the vector genome further contains AAV Inverted Terminal Repeats (ITRs). In one embodiment, the rAAV is pseudotyped, i.e., the AAV capsid is from a different source AAV than the AAV providing the ITRs. In one embodiment, ITRs of AAV serotype 2 are used. However, ITRs from other suitable sources may be selected. Optionally, the AAV may be a self-complementary AAV.

The abbreviation "sc" means self-complementary. "self-complementary AAV" refers to a construct in which the coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. Upon infection, the two complementary halves of the scAAV will combine to form one double stranded dna (dsdna) unit that can replicate and transcribe immediately at any time, rather than waiting for cell-mediated second strand synthesis. See, for example, D M McCarty et al, Self-complementary vector addition-assisted virus (scAAV) vectors promoter transformation index of DNA synthesis, Gene Therapy (8.2001), Vol.8, No. 16, p.1248-1254. Self-complementary AAVs are described, for example, in U.S. patent nos. 6,596,535; 7,125,717, respectively; and 7,456,683, each of which is incorporated by reference herein in its entirety.

Where the gene is to be expressed from an AAV, the expression cassettes described herein include an AAV 5 'Inverted Terminal Repeat (ITR) and an AAV 3' ITR. However, other configurations of these elements may be suitable. Shortened forms of 5' ITR, called Δ ITR, have been described in which the D-sequence and terminal resolution site (trs) are deleted. In other embodiments, full length AAV 5 'and/or 3' ITRs are used. Where pseudotyped AAV is to be produced, the ITRs in expression are selected from a source different from the AAV source of the capsid. For example, AAV2 ITRs may be selected for use with AAV capsids having a particular efficiency for targeting muscle. In one embodiment, the ITR sequence from AAV2 or its deleted form (Δ ITR) is used for convenience and to speed up regulatory approval. However, ITRs from other AAV sources may be selected. Where the source of ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. However, other sources of AAV ITRs may be utilized.

As used herein, "recombinant AAV viral particle" or "AAV viral particle" refers to a nuclease-resistant particle (NRP) having a capsid and packaged therein a heterologous nucleic acid molecule (vector genome) comprising an expression cassette for a dystrophin superfamily triple splice mutant protein. Such expression cassettes typically contain AAV 5 'and/or 3' inverted terminal repeats flanking a gene sequence operably linked to an expression control sequence. Such capsids in which the vector genome is packaged may also be referred to as "solid" AAV capsids. Such rAAV viral particles are referred to as "pharmacologically active" when they deliver a transgene to a host cell capable of expressing the desired gene product carried by the expression cassette.

In many cases, rAAV particles are referred to as "dnase-resistant". However, in addition to the endonucleases (dnases), other endonucleases and exonucleases can be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded DNA and/or double-stranded DNA as well as RNA. Such a step may contain a single nuclease, or a mixture of nucleases directed against different targets, and may be an endonuclease or an exonuclease.

The term "nuclease-resistant" means that the AAV capsid has been completely assembled around an expression cassette designed to deliver a transgene to a host cell, and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present from the production process.

As used herein, an "AAV 9 capsid" is an automatically assembled AAV capsid composed of multiple AAV9 vp proteins. AAV9 vp protein is typically expressed as a protein from SEQ ID NO: 9 (GenBank accession: AAS 99264) of the amino acid sequence vp1 (SEQ ID NO: 10 or an alternative splice variant of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of the sequence therewith. These splice variants result in SEQ ID NO: 9, or a variant length protein. In certain embodiments, the "AAV 9 capsid" comprises a capsid having a nucleotide sequence 99% identical to SEQ ID NO: 9 (i.e., less than about 1% variation from a reference sequence). See also US7906111 and WO 2005/033321. Such AAVs may include, for example, natural isolates (e.g., hu31, whose vp1 is encoded by SEQ ID NO: 11; or hu32, whose vp1 is encoded by SEQ ID NO: 12), or variants of AAV9 having amino acid substitutions, deletions, or additions, e.g., including, but not limited to, amino acid substitutions selected from the group of replacement residues "recruited" from the corresponding position in any other AAV capsid aligned with AAV9 capsid; for example as described in US 9,102,949, US 8,927,514, US 8,734,809 and WO 2016/049230a 1. However, in other embodiments, other variants of the AAV9 or AAV9 capsid having at least about 95% identity to the above sequences may be selected. See, for example, U.S. published patent application No. 2015/0079038. Methods of producing capsids, coding sequences therefor, and methods of producing rAAV viral vectors have been described. See, for example, Gao et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A 1.

In addition to AAV9, other AAV vectors may be used, such as AAV1, AAV5, AAV6, AAV8, AAV8 triplets, AAV9, Anc80, Anc81, and Anc 82. See, e.g., Santiago-Ortiz et al, Gene ther, 22(12) 934-46 (2015); US20170051257a 1; and Zinn et al, Cell Rep., 12(6): 1056-.

The sequences of any AAV capsid can be readily produced synthetically or using a variety of molecular biological and genetic engineering techniques. Suitable production techniques are well known to those skilled in the art. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively, oligonucleotides encoding peptides (e.g., CDRs) or the peptides themselves can be synthetically produced, e.g., by well-known solid phase peptide synthesis methods (Merrifield, (1962) J.Am. Chem. Soc.2149, 85: 85; stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pages 27-62). These and other suitable production methods are within the knowledge of one skilled in the art and are not limiting of the present invention.

Methods for preparing AAV-based vectors are known. See, for example, U.S. published patent application No. 2007/0036760 (2/15/2007), which is incorporated herein by reference. The use of AAV capsids having tropism for muscle cells and/or heart cells is particularly well suited for the compositions and methods described herein. However, other targets may be selected. The sequences of AAV9 and methods for producing AAV9 capsid-based vectors are described in US 7,906,111; US 2015/0315612; WO 2012/112832; and WO2017160360A3, which is incorporated herein by reference. In certain embodiments, the sequences of AAV1, AAV5, AAV6, AAV9, AAV8 triplets, Anc80, Anc81, and Anc82 are known and can be used to generate AAV vectors. See, for example, US 7186552, WO 2017/180854, US 7,282,199B 2, US 7,790,449 and US 8,318,480, which are incorporated herein by reference. Many such AAV sequences are provided in the above-referenced U.S. Pat. Nos. 7,282,199B 2, US 7,790,449, US 8,318,480, US 7,906,111, WO/2003/042397, WO/2005/033321, WO/2006/110689, US 8,927,514, US 8,734,809; WO2015054653A3, WO-2016065001-A1, WO-2016172008-A1, WO-2015164786-A1, US-2010186103-A1, WO-2010138263-A2 and WO 2016/049230A1, and/or are available from GenBank. Corresponding methods for AAV1, AAV8, and AAVrh 10-like vectors have been described. See WO2017100676 a 1; WO2017100674a 1; and WO2017100704a 1.

The recombinant adeno-associated viruses (AAV) described herein can be produced using known techniques. See, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772B 2. Such methods comprise culturing a vector comprising a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette consisting of at least AAV Inverted Terminal Repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the expression cassette into the AAV capsid protein. The host cell may be a 293 cell or a suspension 293 cell. See, e.g., Zinn, E et al, as cited herein; joshua C Grieger et al, Production of Recombinant Adeno-associated viral Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vectors From the Culture Media for GMP FIX and FLT1 Clinical Vectors, Mol Ther.2016.2.24 (287) 297. online 2015 11/3. online 2015 6/2015 6. doi 10.1038/mt 2015.187; laura Adamson-Small et al, Sodium Chloride enzymes Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing plant Using the Herpes Simplex Virus System, Hum Gene tools method, 2017, 2.1.28 (1): 1-14, Online publication 2.1.2017, doi: 10.1089/hgtb.2016.151; US20160222356a 1; and Chahal PS et al, Production of adono-associated viruses (AAV) by transformed transformation of HEK293 cell subsets for gene delivery. J Virol methods, 2 months 2014, 196:163-73. doi: 10.1016/j.jviromet.2013.10.038. 11 months and 13 days in Epub 2013.

Other methods of producing rAAV available to those skilled in the art may be utilized. Suitable methods may include, but are not limited to, baculovirus expression systems (e.g., baculovirus infected insect cell systems) or by yeast production. See, for example, WO2005072364a 2; WO2007084773a 2; WO2007148971a 8; WO2017184879a 1; WO2014125101a 1; US6723551B 2; bryant, L.M. et al, Lessons left from the Clinical Development and Market Authorization of Glybera. Hum Gene Ther Clin Dev, 2013; robert M, Kotin, Large-scale recombinant adono-associated virus production, Hum Mol Gene, 2011, 4/15 days, 20(R1), R2-R6, on-line publication, 2011, 4/29 days, doi, 10.1093/hmg/ddr 141; aucoin MG et al, Production of involved viral vectors in involved cells using triple introduction, optimization of bacterial concentration ratios, Biotechnol Bioeng, 2006, 12, 20 days, 95(6), 1081-92; SAMI S. THAKUR, a paper of reduction Adeno-associated viral vectors in year, supplied to the Graduate School of the University of Florida, 2012; kondratov O et al, Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human subjects institute Cells, Mol Ther.2017, 8.10.p.8.pii.S 1525-0016(17)30362-3. doi. 10.1016/j.ymhe.2017.08.003. [ Epub precedes printing ]; mietzsch M et al, OneBac 2.0 Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Forein DNA. Hum Gene tools methods, 2.2017, 28(1) 15-22. doi 10.1089/hgtb.2016.164; li L et al, Production and characterization of novel recombinant adenosine-associated viral regenerative-form genes, a an eukaryotic source of DNA for gene transfer, PLoS one, 2013, 8(8), e69879, doi: 10.1371/joural. point.0069879.2013; galibert L et al, last definitions in the large-scale production of adono-assisted viruses in isolated cells heated the treatment of neurological diseases J Invertebr Pathol, 7 months 2011, 107 Suppl, S80-93, doi, 10.1016/j.j.ip.2011.05.008; and Kotin RM, Large-scale recombinant adeno-associated virus production, Hum Mol Gene 2011, 15/4/15/20 (R1), R2-6, doi, 10.1093/hmg/ddr 141/Epub 2011, 4/29/4.

To calculate the content of empty and full particles, VP3 band volumes (band volumes) of selected samples (e.g., in the examples herein, iodixanol gradient purified formulations, where GC # -particles #) were plotted against the loaded GC particles. The resulting linear equation (y = mx + c) was used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μ L of load was then multiplied by 50 to give particles (pt)/mL. Pt/mL divided by GC/mL gives the ratio of particles to genomic copies (Pt/GC). Pt/mL-GC/mL gives empty Pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.

Generally, methods for assaying empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al,Gene Therapy(1999) 6: 1322-1330; the method of Sommer et al,Molec. Ther.(2003) 7:122-128. To test for denatured capsids, the method involves subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins (e.g., a gradient gel containing 3-8% Tris-acetate in buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. The anti-AAV capsid antibody is then used as the first antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al, J. Virol.(2000) 74:9281-9293). Then using a second antibody which binds to the first antibody and contains a second antibody for detectionAn antibody binding means, more preferably an anti-IgG antibody comprising a detection molecule covalently bound thereto, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. The binding between the first and second antibodies is determined semi-quantitatively using a method of detecting binding, preferably a detection method capable of detecting radioisotope emissions, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, a sample from the column fraction can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid proteins separated on a pre-graded polyacrylamide gel (e.g., Novex). Silver staining can be performed using a silver xpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO ruby or coomassie stain. In one embodiment, the concentration of AAV vector genomes (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or other suitable nuclease) to remove the foreign DNA. After nuclease inactivation, the samples were further diluted and amplified using primers and TaqMan-fluorescent probes specific for the DNA sequence between the primers. The number of cycles (threshold cycles, Ct) required to reach a defined fluorescence level was measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing sequences identical to those contained in the AAV vector was used to generate a standard curve in the Q-PCR reaction. Cycle threshold (Ct) values obtained from the samples were used to determine vector genome titers by normalizing them to the Ct values of the plasmid standard curve. Digital PCR-based endpoint assays can also be used.

In one aspect, an optimized q-PCR method is used that utilizes a broad-spectrum serine protease, e.g., proteinase K (as commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay except that after dnase I digestion, the samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably, the sample is diluted with an amount of proteinase K buffer equal to the size of the sample. Proteinase K buffer can be concentrated to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but can vary from 0.1 mg/mL to about 1 mg/mL. The treatment step is typically carried out at about 55 ℃ for about 15 minutes, but may be carried out at a lower temperature (e.g., about 37 ℃ to about 50 ℃) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60 ℃) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, heat inactivation is typically at about 95 ℃ for about 15 minutes, but the temperature may be reduced (e.g., about 70 to about 90 ℃) and the time extended (e.g., about 20 minutes to about 30 minutes). The samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in standard assays.

In addition or alternatively, droplet digital pcr (ddpcr) may be used. For example, methods have been described for determining the genomic titer of single stranded and self-complementary AAV vectors by ddPCR. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Therapy Methods, 4 months 2014, (25) (115-25. doi: 10.1089/hgtb.2013.131. Epub.2014, 14 months 2.

Briefly, a method for isolating rAAV particles with packaged genomic sequences from genomically defective AAV intermediates comprises subjecting a suspension comprising recombinant AAV viral particles and AAV capsid intermediates to fast liquid chromatography (fast performance liquid chromatography), wherein the AAV viral particles and AAV intermediates are bound to a strong ion exchange resin at high (e.g., pH 10.2 for AAV 9) equilibrium, and subjected to a salt gradient while monitoring the eluate for uv absorption at about 260 and about 280. Although less optimal for rAAV9, the pH may be in the range of about 10.0 to 10.4. In the method, AAV solid capsids are collected from fractions that elute when the a260/a280 ratio reaches the inflection point. In one example, for an affinity chromatography step, the diafiltered product can be applied to a Capture Select port-AAV 2/9 affinity resin (Life Technologies) that effectively captures AAV2/9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and protein flows through the column, while AAV particles are effectively captured.

As used herein, the term "treatment" or "treating" refers to one or more compositions and/or one or more methods for the purpose of improving one or more symptoms of MD, including DMD and BMD, restoring desired full-length dystrophin function, or improving biomarkers of disease. In some embodiments, the terms "treatment" or "treating" are defined to include administering one or more of the compositions described herein to a subject for the purposes shown herein. Thus, "treating" may include one or more of preventing a disease, reducing the severity of a disease symptom, arresting the progression thereof, eliminating a disease symptom, delaying the progression of a disease, or increasing the efficacy of a treatment in a given subject. As used herein, the term disease refers to MD, including DMD and BMD, or any other dystrophin-related disease.

It is to be understood that the compositions in the carriers described herein are intended to apply to other compositions, aspects, embodiments, and methods described throughout the specification.

Methods and kits

In other embodiments, methods for targeting muscle, including skeletal muscle, cardiac muscle, and/or smooth muscle, are desired. This may include intravenous or intramuscular injection. However, other routes of delivery may be selected.

In certain embodiments, the compositions of the invention are specifically targeted (e.g., by direct injection) to the heart. In certain embodiments, the composition is specifically expressed in the heart (e.g., cardiomyocytes). Methods for preferentially targeting cardiac cells and/or minimizing off-target non-cardiac gene transfer have been described. See, for example, Matkar PN et al, Cardiac gene therapy: are thermal layerGene ther, 2016, 8 months, 23(8-9):635-48. doi: 10.1038/gt.2016.43. Epub 2016, 4 months, 29 days; patent publications US20030148968a1, US20070054871a1, WO2000038518a1, US7078387B1, US6162796A and WO1994011506a 1.

In certain embodiments, a method such as that of U.S. patent 7,399,750 is used to increase the residence time of a vector carrying a gene of interest in the heart by inducing hypothermia, cardiac isolation from circulation, and near or complete cardiac arrest. Permeabilizing agents are an essential component of the method and are used to increase viral uptake by cardiac cells during viral administration. The methods are particularly well suited for viral vectors where gene expression may be highly specific to myocardium and, particularly in the case of rAAV vectors, expression can be maintained for long periods without signs of myocardial inflammation. Yet another system and technique may be used, including, but not limited to, for example, a "bio-pacemaker" such as described in U.S. patent 8,642,747, US-2011-0112510.

In one embodiment, delivery is accomplished by the general myocardial perfusion method described in international publication No. WO2005027995a 2. In another embodiment, delivery is accomplished by the gene transfer method described in International patent application No. PCT/US2004/031322 filed 24.9.2004. Briefly, the method comprises transferring a micro-dystrophin-related protein of the invention to muscle cells by exsanguinating a microvascular region of a subject and delivering the complex to the region under high hydrostatic pressure using a configuration such as a perfusion cannula and balloon required to protect the heart and lungs from protein organs during perfusion. A balloon catheter for systemic delivery of a vector is provided having a balloon extending substantially the entire length of the aorta or blood vessel inserted into the body. In yet another embodiment, the invention provides for delivery via a perfusion circuit and provides a surgical method for delivering a substance to the heart of a subject in situ during cardiopulmonary bypass surgery. The perfusion circuit defines a pathway for recirculating the solution containing the macromolecular complex through the heart of the subject through the coronary circulation circuit during the surgical procedure, wherein the substance is prevented from being delivered to other organs of the subject.

In one aspect, provided herein is a pharmaceutical composition comprising a dystrophin superfamily triple-spliced muteins, a nucleic acid sequence encoding a dystrophin superfamily triple-spliced mutein, an expression cassette, or a vector comprising such a nucleic acid sequence in a formulation buffer (i.e., vehicle). In one embodiment, the formulation further comprises a surfactant, preservative, excipient and/or buffer dissolved in the aqueous suspending liquid. In one embodiment, the buffer is PBS. Various suitable solutions are known, including those comprising one or more of the following: buffered saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7 or pH 7.2 to 7.8.

Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, difunctional block copolymer surfactants terminated in primary hydroxyl groups are selected, for example, as Pluronic F68 [ BASF ], also known as Poloxamer (Poloxamer) 188, having a neutral pH, having an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e. nonionic triblock copolymers consisting of a central polyoxypropylene (polypropylene oxide) hydrophobic chain flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (glyceryl polyhydroxyoctanoate), polyoxyethylene 10 oleyl ether (polyoxyethylene 10 oleyl ether), tween (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually designated by the letter "P" (for poloxamers) plus three digits: the first two numbers x 100 give the approximate molecular weight of the polyoxypropylene core and the last number x 10 gives the percent polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

In one example, the formulation may contain, for example, a buffered saline solution comprising one or more of sodium chloride, sodium bicarbonate, glucose, magnesium sulfate (e.g., magnesium sulfate 7H 2O), potassium chloride, calcium chloride (e.g., calcium chloride 2H 2O), disodium hydrogen phosphate, and mixtures thereof in water. Suitably, for intrathecal delivery, the osmolarity is in a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, for example, emidicine.mediscape.com/article/2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspending agent, or in combination with another suspending agent and other optional excipients. See, for example, Elliotts B solutions [ Lukare Medical ].

In other embodiments, the formulation may contain one or more penetration enhancers (permentation enhancers). Examples of suitable penetration enhancers may include, for example, mannitol, sodium glyceryl cholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the invention into a suitable host cell. In particular, rAAV vectors can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like. In one embodiment, a therapeutically effective amount of the carrier is included in the pharmaceutical composition. The choice of the carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject during a course of treatment, or the dose or amount delivered in a single unit (or multiple units or separate doses) administration.

Likewise, the carrier composition may be formulated to contain at about 1.0 x10 in dosage units⁹To about 1.0 x10¹⁸Amount of carrier within the range of individual particles (to treat one subject), including all whole or fractional amounts within said range, and preferably 1.0 x10 for human patients¹²Particle size to 1.0 x10¹⁴And (4) granules. In one embodiment, the composition is formulated to contain at least 1x10 per dose⁹、2x10⁹、3x10⁹、4x10⁹、5x10⁹、6x10⁹、7x10⁹、8x10⁹Or 9x10⁹Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹⁰、2x10¹⁰、3x10¹⁰、4x10¹⁰、5x10¹⁰、6x10¹⁰、7x10¹⁰、8x10¹⁰Or 9x10¹⁰Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹¹、2x10¹¹、3x10¹¹、4x10¹¹、5x10¹¹、6x10¹¹、7x10¹¹、8x10¹¹Or 9x10¹¹Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹²、2x10¹²、3x10¹²、4x10¹²、5x10¹²、6x10¹²、7x10¹²、8x10¹²Or 9x10¹²Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹³、2x10¹³、3x10¹³、4x10¹³、5x10¹³、6x10¹³、7x10¹³、8x10¹³Or 9x10¹³Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹⁴、2x10¹⁴、3x10¹⁴、4x10¹⁴、5x10¹⁴、6x10¹⁴、7x10¹⁴、8x10¹⁴Or 9x10¹⁴Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹⁵、2x10¹⁵、3x10¹⁵、4x10¹⁵、5x10¹⁵、6x10¹⁵、7x10¹⁵、8x10¹⁵Or 9x10¹⁵Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹⁶、2x10¹⁶、3x10¹⁶、4x10¹⁶、5x10¹⁶、6x10¹⁶、7x10¹⁶、8x10¹⁶Or 9x10¹⁶Particles, including all whole or fractional amounts within the stated ranges. In another embodiment, the composition is formulated to contain at least 1x10 per dose¹⁷、2x10¹⁷、3x10¹⁷、4x10¹⁷、5x10¹⁷、6x10¹⁷、7x10¹⁷、8x10¹⁷Or 9x10¹⁷Particles, including all whole or fractional amounts within the stated ranges. In one embodiment, for human use, the dosage may be from 1x10 per dose ¹⁰To about 1x10¹²The individual particles are not equal and include all whole or fractional amounts within the stated ranges. In one embodiment, a therapeutically effective amount of a vector described herein is delivered to a subject. As used herein, "therapeutically effective amount" refers to a group comprising nucleic acid sequences encoding triple splice muteins of the dystrophin superfamilyAn amount of the compound that delivers and expresses the enzyme in the target cell in an amount sufficient to achieve efficacy. Or "therapeutically effective amount" refers to the amount of a composition comprising a dystrophin superfamily triple splice mutant protein delivered to a subject. In one embodiment, the dose of the carrier is about 1x10 per kg body weight⁹From one particle (e.g., genomic copy, GC) to about 1x10 per kg body weight¹⁶And (b) particles, including all whole or fractional amounts and endpoints within the stated ranges. In another embodiment, the dose is 1x10 per kg body weight¹⁰To about 1x10 per kg body weight¹³And (4) granules.

The dosage of the carrier will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and thus may vary from patient to patient. For example, a therapeutically effective human dose of the carrier will generally be in the range of about 1 ml to about 100 ml of a solution containing about 1X10 ⁷To 1X 10¹⁶Concentration of individual genomic or particulate carriers. The dosage will be adjusted to balance the therapeutic benefit against any side effects, and such dosage may vary depending on the therapeutic application in which the recombinant vector is used. The expression level of the transgene can be monitored to determine the frequency of doses resulting in a vector, preferably an AAV vector containing a minigene. Optionally, dosage regimens similar to those described for therapeutic purposes can be utilized for immunization with the compositions of the invention.

Optionally, therapy with a dystrophin superfamily triple-spliced mutein (e.g., a nano-dystrophin-related protein or a nano-dystrophin) or a vector expressing a dystrophin superfamily triple-spliced mutein may be combined with other therapies.

Expression of dystrophin superfamily triple spliced muteins (e.g., nano-dystrophin related proteins or nano-dystrophin) can be detected by immunofluorescence staining and immunoblotting (western blotting). Dystrophin superfamily triple splice mutein (e.g., nano-dystrophin related protein or nano-dystrophin) therapy can be monitored by measuring the absence of DAP complexes (including the sarcolemetanan complex) on the myofibrillar membrane, which are generally not found in untreated dystrophic muscle due to primary dystrophin deficiency. On the other hand, dystrophin superfamily triple splice mutein (e.g., nano-dystrophin related protein or nano-dystrophin) therapy can be monitored by assessing muscles that are protected from pathological phenotypes.

In one aspect, the invention provides kits for use by a clinician or other person. Typically, such kits will include a mutein of the invention or a vector, and optionally instructions for its reconstitution and/or delivery. In another embodiment, the kit will include the mutein or carrier in a physiologically compatible salt solution, and optionally instructions for diluting and performing the methods described herein.

The kits of the invention may further comprise a balloon catheter to facilitate somatic gene transfer, as described (international patent application No. PCT/US2004/030463, or by the gene transfer method described in international patent application No. PCT/US2004/031322 filed 24.9.2004), an oxygen delivery agent and/or at least one disposable element of an extracorporeal circulation support and oxygenation system. For example, the at least one disposable element may be an oxygenator having a hollow body, a liquid inlet in fluid communication with the interior of the body, a liquid outlet in fluid communication with the interior of the body, a gas inlet for providing gas to the interior of the gas chamber, at least one gas permeable membrane separating the gas chamber from the interior of the body, and a gas outlet allowing gas to exit from the gas chamber, thereby enabling gas exchange between the fluid inside the body and the gas in the gas chamber. The oxygenator may be constructed as described in U.S. patent No. 6,177,403, wherein the gas permeable membrane comprises a PTFE tube stretched within at least a portion of the tube, and wherein the gas chamber comprises an interior of the PTFE tube.

It is to be understood that the compositions in the methods and kits described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described throughout the specification.

The terms "a" or "an" mean one or more. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The words "comprise", "comprises" and "comprising" are to be interpreted non-exclusively and not exclusively. The words "consisting of … … (continst)", "consisting of … … (continuations)" and variations thereof are to be construed exclusively, rather than exclusively. While various embodiments in the specification have been presented using the term "comprising," in other instances, it is also intended that the related embodiments be interpreted and described using the term "consisting of … …" or "consisting essentially of … ….

Unless otherwise specified, the term "about" includes within ± 10% and includes variations of ± 10%.

Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the disclosure that provides those skilled in the art with a general guidance to many terms used in this application.

Examples

The primary structure of dystrophin and the molecular basis of disease BMD, which is lighter than DMD, suggest a conceptually simple method of constructing smaller, partially functional proteins for therapy in DMD. Internal deletions of a single contiguous portion of the dystrophin long repeat domain are used to achieve BMD-like partial length dystrophin variants that map to the cellular address normally occupied by full length dystrophin and thereby partially replace one or more critical physiological functions of dystrophin. Based on the idea that dystrophin acts as a molecular "shock absorber", it is widely assumed that the full length of the protein will be required for normal function in the effect. The expected result is that, under appropriate testing, the entire class of recombinant proteins will confer a BMD-like phenotype on DMD patients. None of the partial-length recombinant dystrophins delivered by somatic cells completely normalized the most sensitive pathology assay in preclinical studies, suggesting that vectors prepared for clinical development by multiple teams would at best, but temporarily, "becker (Beckerize)" the rate of disease progression in DMD. Both shorter and longer than wild-type dystrophin may be associated with severe disease in Becker Muscular Dystrophy (BMD), suggesting that the mechanical action of dystrophin is not as simple as a length-dependent "shock absorber". This genotype/phenotype association in duchenne and becker muscular dystrophy serves as a starting point for the development of low molecular weight substitutions for Dp427, including derivatives of potentially non-immunogenic dystrophin paralogs (paralogs) dystrophin-related proteins. Based on a new understanding of the mechanics biology of dystrophin and dystrophin-related proteins, we have developed dystrophin-related proteins or dystrophin variants that can be delivered to patients to replace the efficacy and safety of gene therapy previously investigated in addressing the DMD-specific limitations noted above.

Example 1 evolution of titin instead of dystrophin is associated with the tunability of motor kinetics (scalability)

A. Results and discussion

In large animals, rapid movement is always powered by sarcomeric myosins, while The fastest moving unicellular eukaryotes and The earliest branching animal lineages use ciliated dynein as The primary source of motive power (Colin, S. P. et al, Stealth prediction and The prediction summary of The innovative liver Mnemiopsis leidentis. Proc Natl Acad Sci U A107, 17223-, l, et al, The chrysene genome and The evolution orientations of neural systems Nature 510, 109-. The selection pressure driving the evolutionary switch from dynein to myosin must reflect the geometric constraints imposed by the organelles in which these dynamin proteins achieve the maximum energy density (power density), while the sarcomere, but not the cilia, are suitable for three-dimensional regulation. The molecular basis of this critical switch is poorly understood. Here we show that the appearance of sarcomere is related to the appearance of germ-line genomics remodeling of a large number of poly-IgG-repeat containing proteins orthologous to chordin, while dystrophin and its associated membrane-bound glycoprotein complexes appear gradually before the earlier branching lineage divergences. We have identified invertebrate species that retain the deduced ancestral titin supergene structure, providing a unified view of gene rearrangement that previously made gene orthologies and the common origin of animal sarcomeres with radial and bilateral symmetry unintelligible. Surprisingly, the gene structure provides convincing evidence that the abnormal size of the rod domain of dystrophin reflects the historical heritage of a paralogue class of microtubule-binding proteins, with selection for progressively increasing lengths occurring before the sprouting of sarcomere. These findings are of crucial importance for the mechanical biology of dystrophin and the design of miniaturized proteins for therapeutic use in muscular dystrophy (examples 2 and 3). Our reconstruction suggests that geometric constraints on cell morphology and body developmental layout require strong and flexible connections between the cortical cytoskeleton and the extracellular matrix before myosin can be safely aligned into sarcomeres at the density required to impart rapid, scale-independent motion with the motive force.

Myoconjins are the largest proteins in the human proteome, acting as the primary scaffold for sarcomere formation in monomeric form (Zoghbi, M. E., Woodhead, J. L., Moss, R.L. & Craig, R.Three-dimensional structure of transformed cardiac Muscle proteins, Proc Natl Acad Sci U S A105, 2386. conq. 2390, doi:10.1073/pnas.0708912105 (2008); and Kontrogitanni-Konstanoploios, A.Ackekonn, M. A., Bowman, A. L., Yap, S.V. & Bloo. V., R.J. Muscal viruses: molecular viruses in virology. 89, Physics. 7, Redox. 2009/12117 (2009/12117); 2009). In vertebrates, adiponectin is composed primarily of immunoglobulin (IgG) and fibronectin type III (Fn 3) domains organized into "super-repeats" that form polarized filaments spanning the hemisarcomere, with unique N-and C-termini located within the Z-disc and M-line, respectively. However, the previously identified Titin-like proteins in invertebrate species widely diverged in number, primary structure, domain composition and length, complicating the description of functional direct homology (Tskhovrebova, L. & Trinick, J. tin: properties and family relationships. Nat Rev Mol Cell Biol 4, 679-. Our findings suggest that the "ancestral titin supergene" of the general structure has undergone extensive lineage-specific genomic rearrangements and modular repeat expansion.

In vertebrates, the viability of striated muscle fibers under workload depends on membrane protection conferred by a dystrophin-dependent mechanical linkage between the outermost sarcomere and the extracellular matrix (Hoffman, e.p., Brown, r.h., Jr).& Kunkel, L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919-928, doi:0092-8674(87)90579-4 [pii](1987)). Approximately 75% of the molecular weight of dystrophin is contributed by a large central "rod domain" consisting of 24 spectrin repeat domains, while the flanking domains establish adhesive contacts at both ends (FIG. 6A; and data not shown). Patients with Becker Muscular Dystrophy (BMD) may have truncated deletions or lengthened repeats limited to the exon encoding the rod domain, raising the question of whether the physiological function of dystrophin has been optimized in the 24 repeat sequence. We investigated whether the number of spectrin-like repeats in dystrophin orthologs increased under selection pressure throughout metazoan phylogeny, which may be related to the increasing energy output of selected taxa during the evolution of the rank predation food chain. As shown (figure 6C), the ancestral dystrophin that was predicted to exist before the spinosyn-bilatalan division had a rod domain of the same length as in humans, but we could not find evidence of a significantly shorter rod in the earlier ortholog. Interestingly, our phylogenetic analysis provides a powerful tool Evidence suggests that the transmembrane dystrophin binding protein complex occurs much earlier than the multicellular nature of metazoans, and that orthologs of nearly all disease-implicated components are present in a single-celled sister population of metazoans (data not shown). The earliest ancestral dystrophin orthologues lacked both the N-terminal actin-binding domain (ABD) and the entire rod domain, and consisted of only the putative dystrophin glycan-binding C-terminal "WW-EF-ZZ" domain (FIG. 6A, FIG. 6E; and data not shown). The earliest branched lineage with "modern" dystrophin orthologs (i.e., N-terminal ABD and extended rod domain) was the platyhelminth species Spilaria ((A))T. adherens) Wherein the rod domain is similar in size to human (data not shown). Thus, dystrophin is "full length" before IgG expansion and sarcomere appearance of titin; however, the evolutionary lineage of the rod domain has not been addressed to date, because significant sequence divergence between homologous proteins causes the "long branch attraction" artifact.

We addressed this problem by identifying ancestral feature states that evolve more slowly than individual amino acids or nucleotides in the sequence: position and phase of the intron of the hidden markov model relative to the encoded protein domain. We found that the spectrin repeats of dystrophin and MACF1 share a conserved phase 0 intron at HMM common position 46 in a visually distinct pattern, in sharp contrast to the randomly distributed introns of the spectrin gene (fig. 6A and 6B). An illustration of the evolutionary arrest of the relevant intron positions is highlighted by we that depict only those shared among orthologous genes of remotely related species (e.g., note the evidence of the ancestral partial gene repeats that extend the β -spectrin ORF by 13 repeats) (fig. 6B). The rod domain signature status analysis identified MACF orthologs, but not β -spectrin, as the closest donors for dystrophin CH and rod domain, suggesting the name "dysplakin" for the branched taxa (fig. 6C). The gene structure constitutes strong evidence that dystrophin appears evolutionarily when partial repeats of the gene encoding the N-terminal part of the ancestral MACF 1-like spectoplakin become linked in cis to the gene encoding the ancestral Dp 71-like (WW-EF-ZZ) dystrophin ortholog. In the giant MACF ortholog of the selected lineage, there is evidence of the most recent tandem repeat of the exons encoding the spectrin repeats. This supports the reconstitution in which the selection pressure for the stepwise lengthening of the rod domain occurs in the cellular context of the microtubule-actin cross-linking "struts" (and persists in some lineages) rather than in the context of the dystrophin protein itself.

Comparing the molecular evolution of the repeat domains in both myosin and dystrophin reveals important differences. The dot matrix revealed evidence of lineage specific "treadmill" (data not shown) of the regions of the adiponectin IgG and Fn3 repeats, presumably on the basis of interchangeable regional tandem multiplication, provided that the total protein was long enough to promote sarcogenesis (sarcogenesis). In the dystrophin orthologs, similar transitions of individual spectrin repeats appear to have been almost absent, suggesting a strong negative selection for it for at least 6 million years (data not shown). Based on these results, we presented a model for the non-interchangeability of spectrin repeats of dystrophins, reflecting the role of the proteins in longitudinal force transmission, whereby amino acid interactions between adjacent spectrin repeats must be maintained by evolutionary coupling (Hopf, T. A. et al, Mutation effects predicted from sequence co-variation. Nat Biotechnol 35, 128-materials 135, doi:10.1038/nbt.3769 (2017)) to maintain tensile strength (data not shown). In this model, purification selection has offset the new juxtaposition (by internal gene deletion or duplication) of divergent spectrin-like repeats that have previously undergone coupled amino acid evolution with adjacent partners of their ancestors (as can be observed in BMD pathogenesis). Reconstruction of the ancestral event was further supported by alternative homology models comparing adjacent triple helices of the dystrophin rod domain based on the divergent templates provided by spectrin and bannin (data not shown). In other words, molecular evolution of dystrophin is consistent with the proposal that the tensile strength of the rod domain is more important than its length, the latter being a byproduct of its historical heritage. Since the proteins are localized to the ultrathin edges of the cytoskeletal cortex, the metabolic cost of maintaining a structurally redundant ancestral solution to the transmembrane force transmission problem is inconsequentially small. This concept is of crucial importance for the design of transgenes for the treatment of diseases, as demonstrated in detail in examples 2 and 3.

B. Materials and methods:

RNA-Seq: starlike sea anemone (A)Nematostella vectensis) The reference transcript set of (a) was a set of primary clustered ESTs published by JGI genome Assembly (Putnam, N.H. et al, Sea anerone genome reales and genetic organization Science 317, 86-94 (2007)) and a transcript set produced by Finnerty Lab, derived from the cloning lineage of New Jersey, USA (line NJ 3) [ Lubinski et al, amendments]And (4) assembling. Redundant contigs were removed from the pooled assemblies using CD-HITs with a cut-off value of 100% sequence identity.

Basic Local Alignment Search Tool (BLAST) search: the genome is blastd using the BLASTp and/or tBLASTn algorithms with predetermined parameters (BLOSUM 62 matrix, expected E value threshold: 10, gap cost present: 11, gap cost extension: 1). Where full-length homologues are not identified, the highest scoring partial length hits are downloaded into the surrounding genomic region and are subjected to de novo gene modeling (see methods for gene modeling). The BLAST transcriptome was again set using the tBLASTn algorithm from the NCBI BLAST server against the Transcriptome Shotgun Assembly (TSA) database using the predetermined parameters BLAST transcriptome.

Gene modeling: protein-encoding gene models were obtained from the procedure of the FGENESH kit (www.softberry.com) using biospecific gene discovery parameters of the listed organisms most closely related to the species under study, without corresponding RNAseq data.

Protein domain analysis: protein domains were analyzed by running primary amino acid sequences against the Pfam, TIGFAM, CATH-Gene3D, Superfamily (Superfamily) and PIRSF protein family HMM databases using the HMMER software package's HMMscan function (www.ebi.ac.uk/Tools/HMMER/-search/hmmsscan) of the European Bioinformatics Institute (European Bioinformatics Institute) (www.ebi.ac.uk/Tools/hmmcer) protein family (Finn, R. D. et al, HMMER web server: 2015 update. Nucleic Acids Res 43, W30-38, doi:10.1093/nar/gkv397 (2015397)).

Spectrin repeat alignment for intron position/phase identification: all Pfam profile-HMM identifiable spectrin repeat domains were aligned to the Pfam spectrin repeat consensus sequence in HMMscan. These spectrin repeats are aligned sequentially into multiple sequence alignments based on their alignment relative to the consensus sequence.

Intron position/phase identification: ORF-annotated cDNA sequences were aligned to their encoding genomic scaffolds using the dot matrix function in MacVector (v 15.1) (macvector.com) with a 94% sequence identity cutoff. The position and phase of the intron are identified as the break in the alignment. Each intron position and phase was confirmed by the presence of a consensus splice site donor (-GT) and acceptor site (AG-) in the genomic DNA sequences immediately after and before each block aligned for 100% identity, respectively.

Inferred ancestral intron identification: the deduced ancestral introns are those shared between homo sapiens (h. sapiens) and orthologous proteins of the large barrier sponges or asteroid sea anemones.

Dot matrix: cDNA/DNA, protein/genomic DNA and protein/protein dot matrices were generated in MacVector (v 15.1) (MacVector.

Homology modeling of dystrophin spectrin repeats: phere 2 was used to model adjacent spectrin repeats from human dystrophin (www.sbg.bio.ic.ac.uk/Phyre 2/html/pageid=index）（Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10845, 858, doi:10.1038/nprot.2015.053 (2015)). The crystal Structure of β 2-spectrin (PDB-ID = 3 EDV) (Davis, L. et al, Localization and Structure of The andkyrin-binding site on beta2-spectrin. J Biol Chem 284, 6984-6987, doi:10.1074/jbc. M809245200 (2009)) or The network protein (PDBID = 5J 1G) (Ortega, E. et al, The Structure of The Plakin Domain of The Plectin-derived Extended Rod-like shape. J Biol Chem 291, 18643-18662, doi:10.1074/jbc. M116.739 (2016)) was used as a template for homology models to generate homology models with different properties.

Data availability: sequence data to support the findings in this article is provided in supplementary information. All other data is available from the corresponding author upon request.

Example 2-effective Gene therapy for muscular dystrophy Using AAV-mediated delivery of micro-dystrophin-related proteins

A. Results and discussion

The essential protein product of The Duchenne Muscular Dystrophy (DMD) gene is Dystrophin (Hoffman, E.P., Brown, R.H., Jr. & Kunkel, L.M. Dystrophin: The protein product of The Duchenne muscular dystrophy, Cell 51, 919-928, doi:0092-8674(87)90579-4 [ pii ] (1987)), rod-like 427 kd protein (Koenig, M., monoaco, A.P. & Kunkel, L.M. The complex sequence of muscular precursors a rod-shaped cellular protein, Cell 53, 219) which is protected from extracellular matrix by linking The cortical cytoskeleton to The extracellular matrix (Ibreagkishinv-branched, Pearls-branched-Cell 355, Pearls-0, Pearls-branched-Cell 35, Pethromycin of The protein strain 0, Pearlington-35, Pearls-branched Cell strain, 0, Pearls-branched Cell strain, 0, Pearls-branched protein, Pearls, No. 9, D-9-one, j., Shrager, J.B., Stedman, H.H., Kelly, A.M. & Sweeney, H.L. Dystrophin technologies the saturated from the structures depleted music contract music Proceedings of the National Academy of Sciences of the United States of America 90, 3710-in 3714 (1993)). Most DMD patients have multiple exon frameshift deletions, while many patients with The less allelic disease, Becker MD, have frame-preserving mutations that alter The length of The 150nm rod domain of dystrophin (monoaco, A. P., Bertelson, C. J., Liechti-Gallati, S., Moser, H. & Kunkel, L.M. extension for The phenolic genetic two genes bearing partial protocols of The DMD logic. Genomics 2, 90-95 (1988); and Koenig, M. et al, The molecular basis for Dunner channels molecular dynamics regulation: correlation of section. Our analysis of the history of in-depth evolution of dystrophins suggests that the rod domain is recruited from longer cytoskeletal proteins and appears before the appearance of strong striated muscle (example 1). Here we show codon optimised synthetic transgenes encoding non-immunogenic 25 nm substitutes for dystrophin rationally designed from the paralogous protein dystrophin-related protein (Tinsley, J. M. et al, Primary structure of dystrophin-related protein Nature 360, 591-593, doi:10.1038/360591a0 (1992)) to maintain the tensile strength of the minirod domain, preventing the most detrimental histological and physiological aspects of muscular dystrophy in animal models. All histological and biochemical markers of muscle necrosis and regeneration were completely inhibited throughout growth by adult weight after systemic administration of AAV vectors to neonatal dystrophin deficient mdx mice. In dystrophin deficient dogs similarly treated at up to 4 kg body weight, systemic distribution and expression of the transgene prevents muscle necrosis without cell mediated immune recognition of the protein product, suggesting protection by central immune tolerance to full length dystrophin-related proteins. These findings support a model where tensile strength is an essential feature of dystrophin and dystrophin-related protein rods, while their 150nm length is maintained in most lineages by purification selection for mutations that reduce length at the expense of strength.

Although somatic transfer of 12 kb cDNAs encoding full-length dystrophin proteins has been achieved using internal deletion vectors derived from human adenovirus, the approach has been abandoned due to the immunogenicity and limited biodistribution of the complex vector capsid (Clemens, P. R. et al, In vivo multiple Gene transfer of full-length regulatory with an adoptive vector that lack viral genes. Gene therapy 3, 965 972 (1996)). Various vectors derived from human Adeno-associated viruses (AAVs) have been shown to promote Systemic gene transfer (Wang, B. et al, Adeno-associated viruses vector cloning human minor genes expression in mdx mouse model, Proc Natl Acad Sci U A97, 13714. 13719. (2000); Harper, S. Q. et al, modulation of molecular dynamics: plasmids for gene therapy of Duchen molecular dynamics. Nat. 8, 253. 11. (2002); Gregodynamic, P. et al, systematic of molecular dynamics-encoding genes-expression 834; G. dynamic, P. et al, wild type genes clone 6, wild type rAAV 120. 78. and wild type rAAV 120. 10. 78.); wild type rAAV 120. Ser. 10, wild type rAAV 120. 834; wild type genes clone No. 10. Ser. 7, wild type rAAV 828, wild type rAAV 120. 10. 11.), about 5 kb. A second constraint of equal importance for gene therapy for DMD is the deletion property of protein deficiency in most patients, whereas recombinant Dystrophin has the potential to act as a "non-self" protein (Mendell, J. r. et al, dysphin immunity in Duchenne's molecular dynamics, N Engl J Med 363, 1429-. We hypothesized that a detailed analysis of the molecular evolution of dystrophins might report synthetic biology methods to both constraints by revealing previously unrecognized aspects of the historical heritage of the protein. Our remote history of dystrophin reconstitution suggests that at the beginning of the protein, its rod domain contains a repeat of 24 "spectrin-like" triple helical domains, complemented from the N-terminal part of another much larger strut-like cytoskeletal protein (example 1). The crystal structure of triple helix repeats from dystrophin, dystrophin-related proteins and closely related spectoplakin suggests that amino acid side chain interactions between adjacent repeats create an interlocking interface that is critical to the strength of the rod. The principle may explain the phenotype caused by in-frame deletions and duplications in BMD patients and the rarity of chordin paralogs such as Lamprey deletion, since most disruptions of the native sequence of the triple helical repeats have the potential to locally weaken the rod domain. To minimize the risk of creating the "weakest link", we focused on deletions flanked by unordered domains traditionally labeled "hinge 2" on one side, and also deleted the C-terminal sequence beyond about the end of the ZZ domain (Ishikawa-Sakurai, M., Yoshida, M., Imamura, M., Davies, K.E. & Ozawa, E. ZZ domain is addressed for the phylogenetic binding of dystrin and thiophanatin beta-dystrialac. Hum Mol Gene 13, 693-. To exploit the central immune tolerance acquired by early developmental expression in the thymus (Mesnard-Rouiller, L. et al, Thymic myoid cells express high levels of muscle genes, J Neurommunel 148, 97-105, 2003), we mapped these deletions in dystrophin to the paralogous dystrophin-related protein, which diverged from dystrophin early in vertebrate evolution. Based on these considerations, we synthesized a transgene based on the wild-type dystrophin-related protein mRNA sequence, and then improved expression using an artificially engineered version of the sequence. Here, we report on the results obtained in a blinded preclinical study using AAV 9-based vectors and a derived ancestral capsid "Anc 80" to deliver a 3.5 kb synthetic transgene (AAV 9- μ U, AAV9- μ dystrophin-related protein) systemically to all striated muscles.

We initially performed up to 2.5X 10 in neonatal mdx mice weighing about 5 gm¹²Intraperitoneal injection of dose of vg AAV- μ dystrophin-related proteins and study of the degree of muscle protection (myoprotection) throughout muscle development. In these randomized blind studies, we observed an equal overall biodistribution to muscle with both AAV9 and Anc80, and both without any signs of toxicityIn all cases well tolerated in mice (FIGS. 7A-7C). At 2.5X 10¹²vg/mouse dose, recombinant μ dystrophin-related proteins were expressed at levels sufficient to qualitatively completely inhibit all of the histological signs tested for muscular dystrophy, including central nucleation of muscle fibers (fig. 7A and 7D), embryonic myosin heavy chain expression (fig. 7A and 7B), native dystrophin-related protein upregulation (fig. 7A and 9A), MURF1 expression as a marker of protein degradation, myonuclear apoptosis (data not shown), ongoing muscle necrosis and mononuclear cellular infiltration (fig. 7B). In these conditions, central nucleation is the quantitatively most sensitive indicator of muscle protection in mdx mice, since it reflects previous regeneration cycles. We show for the first time the normalization (or prevention) of the central nucleation phenomenon to a level that is biologically indistinguishable from the wild-type (fig. 7D). The observed muscle protection was associated with a sustained normalization of the dystrophin-binding glycoprotein complex (DGC) in the sarcolemma of cardiac and skeletal muscles (fig. 7A). Western blot analysis further confirmed that expressed μ dystrophin-related protein was sufficient to stabilize DGC (fig. 7C). Sustained expression of μ dystrophin-related proteins in skeletal and cardiac muscle was observed during the 4 month period after the entire vector delivery (end of study), indicating a sustained level of muscle protection conferred by single dose treatment. Strikingly, creatine kinase, a biomarker reflecting the permeability of the sarcolemma, was not statistically distinguishable from wild-type mice (FIG. 7E), suggesting that early codon optimization in development and recombinant protein overexpression improved the response relative to administration of a selectable transgene by tail vein injection after onset of a myopathy (myelopathology) (Gregographic, P. et al, systematic delivery of genes to targeted multiple use adono-localized viral vectors Nat Med 10, 828- f Severely Affected D2/mdx Mice. Mol Ther Methods Clin Dev 11, 92-105, 2018）。

To test whether AAV 9-mu dystrophin-related proteins were conferredmdxImprovement of function in mice, we used an established hybrid assay that combines a grip force test (force grip testing) with mental components, where we measured the post-grip vertical activity of the animal without injury (Song, Y. et al, Suite of clinical relevant functional activities to address thermal efficacy and disease mechanism in the same dystric mdx mouse. J Appl Physiol 122, 593-. Our previous studies show that the mixing test provides a distinctionmdxAnd one of the most sensitive, clinically relevant parameters of wild-type mice, thereby capturing the behavior causally linked to the exaggerated fatigue response of dystrophin deficient muscles (Kobayashi, y. m. et al, Sarcolemma-localized nNOS is required to obtain activity after mill muscle tissue. Nature 456, 511-. The test demonstrated objective, dramatic and statistically significant differences between untreated and AAV9- μ dystrophin-related protein treated mdx mice, whereas the latter group was indistinguishable from wild type mice (fig. 7F). Also, treated versus untreated mice mdxNot only did mice show increased distance between the voluntary wheel (8 weeks) and the downhill treadmill (16 weeks), but their ex vivo isolated EDL muscles also showed increased resistance to eccentric contraction induced injury and increased in vivo muscle performance by grip strength test. These findings suggest that early overexpression of μ dystrophin-related proteins can completely improve phenotype in the absence of full-length dystrophin, despite the short rod-like linkage length of the reverse engineered protein to the actin cytoskeleton and the lack of the R16-17 nNOS-binding motif.

These results led to the hope of achieving complete, but not BMD-like partial reversal of DMD pathophysiology through systemic muscle transduction; however, it is not clear whether size-dependent differences between small and large malnourished animals will reveal limitations of the method. Histological and immunological consequences of the μ dystrophin-related protein gene transfer were further studied in a blinded study in which 5 dogs with age 4-7 days of Golden Retriever Muscular Dystrophy (GRMD) were randomized to 1 × 10 at injection without immunosuppression ¹³And 3.2X 10¹³AAV9- μ dystrophin-related proteins were administered intravenously at a dose of vg/kg. Six weeks after injection, we observed robust μ dystrophin-associated protein expression in the sarcolemma and stabilization of wild-type sarcolemcan proteoglycan expression levels (data not shown). Furthermore, in contrast to the previously reported weight loss associated with immune myositis following systemic administration of xenogenous human dystrophin in the same GRMD model, these treated dogs achieved a Kornegay, j. n. et al, a wide spread multiple expression of an AAV9 human mini-dystrophin vector after-treatment in a neogenetic dystrophin-targeting dogs, Mol Ther 18, 1501-1508, 2010), similar to a four-fold increase in weight of carrier females. The sustained μ dystrophin-associated protein expression was associated with a significantly reduced level of muscle necrosis, normalization of mononuclear infiltration of myofiber minimum Feret diameter (data not shown). At 5 and 8 weeks after vector administration, the canine interferon- γ ELISpot assay revealed no cell-mediated immunity against AAV capsid or μ dystrophin-related protein transgene products in our non-immunosuppressive treated GRMD dogs (data not shown). The main limitation of the proof-of-concept study was attributed to a 1000-fold difference between adult weights of 25g and 25kg mdx mice and GRMD dogs, respectively, limiting the dose of AAV9 we could achieve in this dog to 2.0 x 10 based on the expected adult weight ¹²vg/kg. At this dose, dogs will inevitably "grow faster" than "vehicle", as for a 5 gm newborn with 2.15X 10¹¹Same for vg treated mdx mice (data not shown). We therefore focused on relatively early histological analysis to detect recombinant μ dystrophin-related protein expression, myocyte protection, and immune responses to systemic vector administration.

Our neonatal method provides both irreversible muscle damage prior to onsetThe possibility of early prophylactic treatment, yet minimizing the risk of immune response to vector capsid antigens, since memory T cells recognizing wild-type AAV develop Nichols, t. et al, through continuous environmental exposure, Translational Data from AAV-Mediated Gene Therapy of Hemophilia B in dogs. Hum Gene Ther Clin Dev, 2014; calcedo, R. et al, Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol 18, 1586-. However, most DMD patients are generally diagnosed after two years of age, before which time massive muscle fibrosis, necrosis with mononuclear cell invasion and increased fiber size variability have occurred (Yiu, e.m). &Kornberg, A.J. Duchenne musculature. Journal of paediatrics and child health 51, 759-764, 2015). To explore the feasibility of our approach in young boys with DMD, prednisone was used at a daily anti-inflammatory dose of 1 mg/kg for a brief period of time, up to 1.25 x 10 upon injection¹⁴Dose of vg/kg two 7.5 week old young GRMD dogs (Hann and Beetle) were injected intravenously with AAV9- μ dystrophin-related proteins (Liu, J. M. et al, Effects of prednisone in cancer musculature. Muscan Nerve 30, 767. minus 773, 2004) (FIG. 8A). Immunostaining of muscle biopsies taken four weeks after injection showed uniform myomembrane expression of μ dystrophin-related proteins (fig. 9A and data not shown), inhibition of native dystrophin-related proteins (fig. 9A), and rescue of DGC (fig. 9B). This was further confirmed by western blotting (fig. 9D).

Histopathological characterization of limb muscles from treated GRMD dogs showed almost complete inhibition of ongoing muscle damage as evidenced by a high proportion of muscle necrotic fibers, excessive calcium accumulation (fig. 8C and 8E), fasciculated regenerating muscle fibers (fig. 8D and 8F), abundant inflammatory cell infiltration and fat infiltration (fig. 10B and data not shown) in untreated age-matched controls. Impressively, the AAV 9-mu dystrophin-related protein treated dogs also showed almost complete prevention of muscle degeneration and regeneration in the chewing muscles (FIG. 8B and data not shown) They are severely affected in untreated dogs because they express the uniquely strong MYH16 Myosin isoform (Stedman, H. et al, Myosin gene mutation with atomic changes in the human linkage. Nature 428, 415-418 (2004); Toniolo, L. et al, Mastication muscle unbound: first determination of connective parameters of muscle fibers from calcium salt of beta. Cell Physiol 154295, C1535-2 (2008)). Further western blot analysis at necropsy (3.5 months of age) showed persistent widespread expression of μ dystrophin-related proteins in skeletal and cardiac muscle (fig. 9C). Consistent with our previous GRMD neonatal dog study, the interferon- γ ELISpot assay did not reveal a signal above background for μ dystrophin-related proteins (data not shown). Furthermore, in contrast to previous studies in GRMD dogs and non-Human Primates, no signs of Severe acute Toxicity were seen (Kornegay, J. N. et al, Wireless spread muscle expression of an AAV9 Human mini-dystrin Vector inhibitor in a neuronal dynamic-specific records. Mol. The 18, 1501-1508 (2010); Hinder, C. et al, Sever sensitivity in non-Human Primates and Pigles Follow High-Dose Administration of an Adeno-Associated Virus Vector expression Human SMN. m. Gene, (2018); Homeeaux, J. et al, Hot therapy of P. J. et al, M. J. E.S. J. E. Importantly, an 80% decrease in serum CK levels was measured 1 week after infusion with AAV9- μ dystrophin-related protein in both dogs (fig. 8G), a finding consistent with the observed histological improvement. In order to achieve durable muscle protection throughout muscle growth from infancy to skeletal maturation, dystrophic dogs and boys with DMD may require systemic administration of AAV vectors at doses proportional to those required for mdx mouse pups to maintain robust, uniform expression in The most severely affected muscle diaphragm (Stedman, h. et al, The mdx mouse diaphragmm processes The degenerative changes of Duchenne molecular dynamics. Nature 352, 536-539, (1991)), e.g., 1 × 10 ¹⁵vg/kg neonatal body weight (data not shown).

For a rigorous preclinical assessment of vector and/or transgenic immunotoxicity (immunotoxicity), we utilized the unique German Short Haired Pointg (GSHPMD) Deletion null dog Model (Schatzberg, S. J. et al, Molecular analysis of a specific animal dysstrophin 'knockout' dog. neuron Disord 9, 289 295. (1999); VanBelzen, D. J. et al, Mechanism of Deletion removal al dynamics Exons in a cancer Model for DMD Implicated evaluation of X Chromosome emulsions. GSHPMD provides an excellent platform for central tolerance studies because alternative splicing in the GRMD model allows for detectable near-full-length dystrophin readthrough of potentially tolerant levels, as might be expected to facilitate therapeutic, Long-term systemic expression of the AAV-encoded canine micromorphoproteins previously shown (Schatzberg, S. J. et al, Alternative chromosomal gene transcripts in gold retriever molecular dynamics. Muscan Nerve 21, 991. 998. (1998); Yue, Y. et al, Safe and body tissue transfer in young adun double sieve molecular dynamics genes. Hum Mol Gene 24, 5880. 5890, (1612015); Le Guiner, C. et al, Long-m chromosomal gene engineering protein nutrient protein a model of Nature 05). Adult GSHPMD dogs (Ned and Grinch) each received an equal dose (2 × 10) via intramuscular injection into the contralateral tibial compartment ¹²vg/kg) AAV9- μ dystrophin and AAV9- μ dystrophin-related proteins (fig. 10A). Interferon- γ detection via ELISPOT revealed that as early as 2 weeks post injection, there was a strong systemic cell-mediated immune response against μ dystrophin, but not for μ dystrophin-related proteins despite expression from the constitutive CMV promoter, indicating the strength of central immune tolerance (fig. 10B). Immunostaining of muscle biopsies collected 4 weeks after injection revealed persistent expression of the dystrophin-associated protein, but only in rare small amounts (a)Fig. 10C). H & E shows severe inflammation and mononuclear cellular infiltration on the side of μ dystrophin injection compared to its actual absence on the side of μ dystrophin-associated protein injection (fig. 10D and data not shown). These findings indicate that the observed immune response is driven by μ dystrophin, not by the vector capsid, as equal doses of vector were injected into both limbs.

In summary, after using comparative germline genomics approaches to identify evolutionary constraints, we have reverse engineered a highly therapeutic 3.5 kb synthetic transgene for safe systemic delivery to muscle in murine and canine models of DMD. Our blinded studies revealed surprisingly complete muscle protection as long as the initial level of gene delivery was sufficient to accommodate subsequent muscle growth. Taken together, these findings may refocus the field to use functionally optimized, non-immunogenic dystrophin-related protein-based gene therapy approaches as a treatment for duchenne muscular dystrophy.

B. Materials and methods

a. Bioinformatics and phylogenetic analysis

Publicly available genomic DNA sequences of the species listed in the legends and script text were queried by various blast algorithms, particularly tBLASTn26,27, to identify coding sequences homologous to full-length human dystrophin and dystrophin-related proteins. For most species, supportive evidence from mRNA sequences is available to define intron/exon boundaries. In the absence of this evidence, FGENESH + (Softberry) was used with biospecific gene discovery parameters and hidden markov models plus similar protein-based gene predictions to identify putative coding sequences from assembled contigs. As an internal test of this approach, virtually all transcriptome-defined coding sequences were correctly identified by the FGENESH + program 28. We recognize that these mRNA and protein sequence files miss sporadic exons in indeterminate regions of publicly available genomic DNA. HMMER (HMMER. janelia. org/search/hmmscan) was used with a cut-off defined by the E-value to define protein coding domains that match the hidden Markov model of calponin homology, spectrin-like repeats, WW, EF hand and ZZ domains. All deduced peptide sequence files were aligned by ClustalW using default settings in MacVector version 13.5.1: a Gonnet Series Matrix (Gonnet Series Matrix) with parameters for a pairwise alignment open gap penalty of 10, an extension gap penalty of 0.1, and parameters for a multiple alignment open gap penalty of 10, an extension gap penalty of 0.2, and a delay divergence of 30%. The reconstruction occurs using a neighbor tree construction method with both full-length and truncated sequences to generate the system, and where gaps are scaled or ignored, the connections (ties) in the tree are resolved randomly and distances are poisson corrected to determine if the selection affects the topology of the tree. In all such cases, the topology of the tree is insensitive to the management of gaps when the "best tree" mode is selected. The alternative use of the introductions pattern with 10000 repetitions confirms all branching points in the distance phylogenetic graph. Protein and DNA matrix analysis was based on the pam250 scoring matrix. Abbreviations used: purple sea urchin, purple ball sea urchin (Strongylocentrotus purpuratus), s.pur; amphioxus, florida amphioxus (Branchiostoma floridae), b.flo; basking (elephat shark), chimonochys mackerel (Callorhinchus miliii), c.mil; chinese Alligator (Chinese aligner), Chinese Alligator (Alligator sinensis), A.sin; mouse, mouse (Mus musculus), m.mus; dog, domestic dog (cantis familiaris), c.fam; human, Homo sapiens (Homo sapiens), h.sap; carrelanaena rapana (Carolina anole), north american green exendin (Carolina carolinansis), a.car; common chimpanzees (common chimpanzee), chimpanzees (Pan troglodytes), p.tro; duckbills, duckbills (ornithochida anatinus), o.ana; japanese puffer fish (Japanese puffer fish), red-fin eastern puffer fish (Takifugu rubripes), t.rub; xenopus laevis, Xenopus tropicalis (Xenopus tropicalis), x.tro; d-dystrophin; u-dystrophin related proteins.

b. Transgenic design and vector production of protein related to micro-dystrophin

Based on germline genomics analysis of sequence conservation and genotype-phenotype correlations among BMD/DMD patients, we modeled in silico a spectrum of AAV-encodable miniaturized dystrophin-related proteins that maintained a combination of the homeodomain of calcineurin, the first three and the last three spectrin-like repeats, and the WW, EF hand and ZZ domains. To preserve amino acid side chain interactions between the interspiral loops of adjacent spectrin-like repeats, we focused on only a subset of μ dystrophin-related proteins with the first or last three repeats remaining intact. To minimize immunogenicity, we considered only those μ dystrophin-related proteins that could be produced by a single combination of internal deletions and C-terminal truncations. Although the spectrin-like repeats are homologous to the consensus sequence, the divergence is such that no splicing can be found between the identical decapeptides in any mammalian dystrophin-related protein. Therefore, we used the hidden markov model of the sequence as performed on hmmer janelia org/search/hmmscan online to define and annotate spectrin-like triple helix repeat boundaries in the full-length canine dystrophin-related protein sequence (3456 aa, XP — 005615306). We used transgenes encoding proteins matching canine and human proteins in neonatal mice, where antigen-specific Tolerance was readily induced by intraperitoneal injection of AAV45, but only in canine form in neonatal and older dogs, where Tolerance is expected to require earlier prenatal exposure to isogenic native proteins during Immune ontogeny (Davey, m. g. et al, Induction of Immune Tolerance to Foreign Protein via addition-Associated Viral Vector Gene, Gene Transfer in Mid-stage magnetic sheet, PLoS year 12, e0171132, (2017)). The mu dystrophin-related protein transgene was designed to contain the actin binding domain, triple helix repeats 1-3 and 22, a disordered proline rich region approximating what was previously identified as the "hinge" 2, and the C-terminal WW, EF hand and ZZ domains, thereby generating recombinant proteins designed to match the canine and human dystrophin-related protein sequences except for a single splice site at the deletion junction, relative to that of the deletion junction The previously reported transgenes minimize potential immunogenicity in dystrophin deficient dogs and eventually humans (Wang, B. et al, Adeno-assisted virus vector Carrying human minor genes affecting expression in mdx mouse model. Proc Natl Acad Sci U A97, 13714. medi.13719. (2000); Harper, S. Q. et al, modulation flexibility of expression: markers for gene therapy of Duchen molecular dynamics. Nat Med.8, 253. 261 (2002); Grorevic, P. et al, System of expression vectors for expression therapy, 78. medium 2006; G. 2004; Grorevic, P. et al, System of expression vectors genes 789, Nat. 10. expression vector 120, 2. 10. et al, g, L, et al, Microutherin delivery through rAAV6, microorganisms function in a steroid kinetic/neurotrophin-specific microorganism. Mol Ther 16, 1539-. The coding sequences selected for use in our study were selected as the highest expression candidates in the pool of cDNAs optimized and synthesized by competitive biotechnology companies (GeneArt and DNA 2.0). By being at mdxExpression was determined by immunofluorescence staining and western blot following electroporation of 50 μ g of DNA in the anterior tibialis muscle of mice. Synthetic coding sequences selected for further use were found to drive expression of about 30-fold in vitro and in vivo assays of wild-type canine cDNA sequences encoding recombinant proteins of the same primary structure. A significant difference between the optimally synthesized cDNA and the wild-type is the level of codon bias, whereas only the optimized synthesized cDNA closely matches the extreme bias of mammalian myosin heavy chain (e.g., 154 CTG leucine, 0 TTA leucine). The synthetic canine mu dystrophin-related protein cDNA was subcloned into AAV2 expression vector cassettes driven by the 833 bp fragment of the CMV immediate early enhancer/promoter or the synthetic promoter spc5-12 (CMV and SP, respectively). As described previously, AAV9 vector was generated and purified in HEK 293 cells by the University of Pennsylvania preclinical vector core (University of Pennsylvania preclinical vector core) using triple transfection (Vandenberghe, L.H. et al, effective se) methodType-dependent release of functional vector into the culture medium induced-associated virus manufacturing Hum Gene Ther 21, 1251-1257, doi:10.1089/hum.2010.107 (2010)). In merging for merging with 2 × 10 ¹¹AAV9 mu dystrophin-related protein vg injected intomdxThe quality, purity and endotoxin level of the vector preparation were determined in the mouse anterior tibialis muscle (Lock, M.et al, Analysis of particulate content of recombinant acquired viral serogroups 8 vectors by ion-exchange chromatography. Hum Gene therapeutics, 23, 56-64, doi:10.1089/hgtb.2011.217 [ pii: pi.]。

c. Animal-in general

All Animal protocols in mice and dogs were approved by the Animal Care and Use Committee of University of a & M (a & M University) and pennsylvania University.

d. Murine model vector administration

Mouse strains C57BL/10SnJ andmdxfrom Jackson laboratory (Bar Harbor, ME). The study included 23C 57BL/10SnJ mice and 30mdxMice, all injected at 9 ± 2 days of age. Before receiving intraperitoneal injections of AAV9 μ dystrophin-related protein or Phosphate Buffered Saline (PBS), individual litters were subjected to toe tattoo using the Aramis Micro tattoo kit (Ketchum Manufacturing Inc, canada) and randomly assigned to different dose groups. The investigator was blind during all injections and tissue harvesting. Based on the protocol, C57BL/10SnJ and mdxPups were injected with 50-250 μ l of PBS as a negative control or AAV9 μ dystrophin-related protein diluted in PBS. Each mouse was weighed prior to injection. After vehicle administration, all mice were returned to their litter (litter) and isolated after weaning.

e. Murine model tissue acquisition and storage

According to institutional policy, at about 8 weeks of age,mdxand C57BL/10SnJ mice experience CO₂And (6) performing euthanasia. Based on the display in AAV9 in mice<100-fold lower off-target Gene tablesTo study, harvest heart, tibialis anterior, gastrocnemius, quadriceps, triceps, abdomen, diaphragm, engage muscle and liver, and further process; others are stored but do not utilize Zincarelli, C.et al, Analysis of AAV serotyps 1-9 mediated gene expression and tropism in semiconductor system injection. Mol Ther 16, 1073-1080 (2008)). Designated histological Tissue samples were placed in OCT (Tissue-Tek) containing embedded molds (Richard-alan Scientific) and snap frozen in liquid nitrogen cooled isopentane. Additional designated biological tissue samples were placed in tissue containers and flash frozen in liquid nitrogen. All samples were stored at-80 ℃. Frozen sections of 5-7 μm thickness were cut on a-25 ℃ cryostat (Microm HM550, Thermo Scientific, usa) and mounted on slides (Superfrost Plus, Fisher Scientific, usa).

f. Basic histology-hematoxylin and eosin staining (H & E) and Alizarin Red Staining (ARS)

The 7 μm thick cross-sections were air-dried at room temperature for 15 minutes. Slides were then stained with Harris hematoxylin dye for 2.5 minutes, rinsed in distilled water, immersed in 0.1% acetic acid for 15 seconds, followed by repeated rinsing in tap water for 4 minutes and counterstaining with 1% eosin for 1 minute. As a final step, the slides were dehydrated 3 times in ethanol for 2 minutes each. Representative non-overlapping High Power Fields (HPFs) were taken for scoring (data is now displayed). Alizarin red staining was also performed on 7 μm thick cross sections. After fixation with 10% buffered formalin phosphate for 10 minutes, sections were washed with PBS for 3 × 5 minutes and incubated with alizarin red dye for 15 minutes at room temperature. After the procedure, the slides were washed 3 times in ethanol and mounted by cytoseal 60 (Thermo Scientific).

g. Morphometric analysis

Three groups of mice, C57BL/Sn10J (control) andmdxthey were randomized to injection with PBS or AAV9 μ dystrophin-related protein. H & E for 4-5 muscles from each of tibialis anterior, gastrocnemius, quadriceps, triceps, engage and abdominal muscles Randomly selected areas were stained and screened for quantification of centrally nucleated muscle fibers using light microscopy. Since the region at the tendon junctions (myofibers) is abundant in centrally nucleated muscle fibers in both mdx and control, they were excluded from the measurement. A total of 11,649 fibers were evaluated.

h. Immunofluorescence staining procedure: double staining of N-terminal and C-terminal dystrophin-related proteins

Sections from all muscle samples were processed for dystrophin-related protein immunostaining by using both N-terminal polyclonal (anti-full-length and recombinant dystrophin-related proteins) and C-terminal monoclonal (anti-full-length dystrophin-related protein) antibodies. After an initial incubation of 20 minutes in a 1% solution of Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) diluted in 0.01M PBS (Roche Diagnostics GmbH, Mannheim, Germany), the samples were rinsed 3 times for 5 minutes (3X 5 minutes) each in PBS. The sections were then incubated in 5% normal donkey serum for 15 minutes followed by incubation with an antibody to the N-terminal dystrophin-related protein (N-19, sc-7460, goat polyclonal IgG, Santa Cruz, Calif., U.S.A., dilution 1: 50) for 60 minutes at 37 ℃. After a second cycle of 3 × 5 min PBS washes, the slides were incubated with 5% normal donkey serum for 15 min at room temperature. The prepared sections were then incubated in donkey anti-goat IgG-FITC (sc-2024, Santa Cruz, Calif., U.S.A., dilution 1: 300) for 30 minutes at 37 ℃. After a third PBS wash for 3X 5 minutes, sections were first incubated with 10% normal goat serum (Invitrogen, Scotland, UK) for 15 minutes and then with C-terminal dystrophin-related protein antibodies (MANCHO 7, mouse monoclonal IgG2a, Santa Cruz, CA, USA, dilution 1: 25) for 60 minutes at 37 ℃. After washing in PBS for 3X 5 minutes and incubation with 10% normal goat serum, the sections were incubated in goat anti-mouse IgG2a-Alexa Fluor 594 (A-21140, Life Technologies, USA, Dilute 1: 300) for 30 minutes at 37 ℃. The sections were washed again in PBS for 3X 5 minutes and mounted in either Vectashield mounting medium (H-1000) (Vector Laboratories, CA, USA) or mounting medium with DAPI (H-1500) (Vector Laboratories). Photographs were taken with a Leica DM6000B microscope (Leica, germany).

i. Immunofluorescence staining procedure: dual immunofluorescent staining for gamma-tropomyosin/laminin, MuRF-1/laminin, and MyHC-embryo/laminin

The staining procedure for these proteins followed the same protocol as previously described (Song, Y. et al, Effects on related proteins, after which unsolvated tissue staining and externalization. PloS one 7, e52230, doi:10.1371/journal. pane. 0052230 (2012)). Rabbit anti-gamma-tropomyosin (NBP 1-59744, Novus Biologicals, Littleton, CO) and MURF1 (NBP 1-31207, Novus Biologicals, Littleton, CO) polyclonal antibodies were used at a dilution of 1:50 in PBS with Bovine Serum Albumin (BSA). MyHC-embryonic monoclonal antibody (F1.652) (development studios, Hybridoma Bank, Iowa, USA) was used at a dilution of 1:50 to 1:100 in PBS. Laminin chicken polyclonal antibodies (ab 14055-50, Abcam, Cambridge, MA, USA) at a dilution of 1:500-1:1000 were used along with a second goat anti-chicken IgY (TR) antibody (ab 7116, Abcam, Cambridge, MA, USA, dilution 1: 300) to identify muscle fibers. MYH16 rabbit polyclonal antibody peptide sequences were generated using human canine sequences of the "loop 2" region of MYH 16. Peptide sequence:

j. TUNEL assay

Sections were initially fixed in 10% buffered formalin phosphate (Fisher Scientific, usa) for 20 minutes. The fragmented DNA was then subjected to in situ nick end labeling using the TACS 2 TdT fluorescein apoptosis detection kit (Trevigen, Gaithersburg, MD, usa) as described by the manufacturer's instructions.

k. Serum Creatine Kinase (CK) assay

Serum was collected via venipuncture of the inframandibular vein using a 5mm Animal Lancet (golden Animal Lancet, Braintree Scientific, Inc, Braintree, MA). A total of 150 μ L were collected in heparinized blood collection tubes (Terumo, Cat: TMLH). 30 minutes after blood draw, mice were carefully monitored to observe potential adverse stress signs. CK levels were determined by the Clinical Pathology Laboratory (Clinical Pathology Laboratory) at Matthey J. Ryan Veterinary Hospital, university of Pa.

Ex vivo evaluation of EDL muscle contraction properties

Ex vivo evaluation was performed by the Muscle Physiology evaluation center of the university of pennsylvania (Muscle Physiology evaluation Core). Using an Aurora Mouse 1200A system equipped with Dynamic Muscle Control v.5.3 software, at 2 months of age from mdxPhysiological properties were quantified on freshly isolated EDL muscle from mice, including isometric twitch force, isometric tonic force (isocratic force) and force decline following ECCs. All of these mice have undergone in vivo grip testing 24 hours prior to euthanasia ex vivo testing. EDL muscle was maintained in constantly oxygenated ringer's solution (100 mM NaCl, 4.7 mM KCl, 3.4 mM CaCl) at 24 deg.C₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄25 mM HEPES and 5.5 mM D-glucose). The twitch stimulation protocol applied was a single stimulation with a duration of 0.2 ms. For measuring tonic maximal force production, the same stimulation was repeated at a frequency of 120 Hz for 500 ms. Five minutes were allowed between two tonic contractions to ensure muscle recovery. Muscle length was adjusted to obtain the maximum twitch response and measured between the outermost visible tips of the tendon junctions and recorded as the optimal length (L0). By dividing muscle mass by the muscle density factor (1.06 g/cm)³) Muscle cross-sectional area (CSA) of EDL muscle was calculated as the product of muscle L0 and fiber length coefficient (0.45 for EDL). The specific force is determined by normalizing the force against the CSA.

After testing the isometric properties of the EDL, the muscle was stretched L starting from a repeated 500 ms isometric contraction followed by the application of the maximum tonic stimulus ₀A series of five eccentric contractions (ECCs, once every five minutes) was applied for 10% of the cycles. The reported absolute force of each ECC corresponds to the ECC during a long period of timeThe peak force of (c).

Vertical movement and grip Strength test

Mice were carefully placed in open field cages and their baseline vertical activity was measured for five minutes. The mice were then returned to their original cages and allowed to rest for three minutes. An axial Force Sensor was used to measure Force (Vernier LabPro & Vernier Dual-Range Force Sensor + -10N, Beaverton Oregon) while data was collected using the accompanying software (Logger Lite version 1.8.1). All experiments were performed by the same experimenter in a blind manner. To reduce the chance of bias and ensure the robustness of the blind experiment, we used the method as described (Song, Y. et al, Suite of clinical innovative functional analysis to address thermal efficacy and disease mechanism in the dynamic mdx mouse. J Appl Physiol 122, 593-.

n. dog model

Two groups of dogs were used in our experiments. The first group was housed in the group at university of A & M and was assigned to the university of Pennsylvania. The study included five dogs with GRMD and four age-matched littermates, including one wild-type and three carrier females, which served as a control group. All dystrophic dogs were identified by elevated serum Creatine Phosphokinase (CPK) levels and genotyped by PCR assays. All pups were randomly assigned to treatment groups and the investigators were kept blind during clinical and histological evaluation. Young animals are 1.0 × 10 at 6-10 days old ¹³vg/kg and 1.0X10^13.5 Dose of vg/kg AAV9 μ dystrophin-related proteins were injected via the external jugular vein route. Two pups were injected with a low dose of AAV9 μ dystrophin-related protein, two were injected with a high dose, and the remaining one was injected with saline only. Each dog was weighed daily for the first 6 weeks and weekly thereafter.

The second group of dogs included two GRMD dogs of about 7.5 weeks of age. Three days prior to vehicle administration, dogs were scheduled for a 1mg/kg oral prednisolone regimen for 25 days. The same method was used to obtain 1.25X 10¹⁴ vg/kg and 5.0X 10¹³Two different single injections of vg/kg AAV9 μ dystrophin related proteins. Dogs were randomly assigned to doses and researchers remained blinded during clinical and histological evaluation.

Deficient null German Short hair Boeing dogs (GSHPMD) were housed and placed in Texas A&University of M (Texas A)&M University). Two seven-year-old dogs with GSHPMD Ned and Grinch weighed 21 kg and 24.2 kg, respectively. Each dog received 5 times to a total equivalent dose of 1.0x10 in their tibialis anterior compartment¹²Intramuscular injection of vg/kg of AAV9- μ dystrophin (right) and AAV9- μ dystrophin related protein (left). All five injection sites were tattooed, allowing us to pinpoint injection sites for a muscle biopsy 4 weeks after injection. Peripheral blood was collected before injection, 2, 4, 6, 8 weeks after injection to collect Peripheral Blood Mononuclear Cells (PBMCs).

Canine tissue acquisition and storage

The first group of GRMD dogs underwent needle biopsies of the cranium sartorius, lateral femoral muscle, and triceps muscle approximately 6 weeks after vehicle injection. The samples were stored with a set of blinding codes to prevent bias in the analysis and interpretation process. Biopsies were obtained through a spring-loaded 14-gauge needle trocar, thereby significantly minimizing post-operative pain in the animals. Muscle biopsies were then snap frozen in liquid nitrogen-cooled isopentane, embedded in OCT media (Sakuru, usa), and stored at-80 ℃. After tissue analysis, the blinded code was destroyed by individuals unrelated to the original author of the study. One month after vehicle exposure, a second group of injected dogs underwent an open-sight (open) muscle biopsy of the same muscle. Seven weeks after vehicle administration, the dogs were euthanized and harvested tissue was cryopreserved in the same manner.

Histological analysis of dogs

Transversely cut 7 μm serial sections were used for bright field microscopy analysis and Immunofluorescence (IF) staining to examine micro-dystrophin-related protein expression and retrieval of sarcoleminoprotectans. Muscle sections were stained with H & E for bright field microscopy and mounted with permount. For IF staining, sections were blocked in 5% donkey serum in PBS for 45 minutes, followed by incubation at 37 ℃ for 60 minutes using a 1:350 dilution of polyclonal goat anti-dystrophin related protein antibody (N-19, sc-7460, Santa Cruz, USA) and a 1:250 dilution of monoclonal gamma-troponin antibody (ab 55683, Abcam, USA). Sections were then rinsed 3 times in PBS and incubated for 45 minutes in 1:1000 dilution Alexa488 donkey anti-goat secondary antibody or Alexa540 donkey anti-mouse secondary antibody. Slides were washed twice with PBS for 5 minutes followed by a single wash with water and mounted with fade resistant mounting media containing DAPI (H-1500, Vector Labs). Images were captured using an Olympus B-65 fluorescence microscope at the same settings and processed in the same manner by setting limits and gains all over to avoid any inconsistencies in the IF images. The minimum ferett diameter and coefficient of variation were calculated according to TREAT _ NMD protocol DMD _ m.1.2.001 updated as 1 month 28 days 2014.

Immunoblot analysis

Immunoblot analysis was performed by loading 20-40 μ g/lane of whole cells or whole muscle lysate onto 10% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membranes. The micro-dystrophin related proteins were detected by goat polyclonal antibodies against the N-terminal epitope at a dilution of 1:500 (N-19, sc-7460, Santa Cruz, USA) and a secondary antibody at a dilution of 1:5000 with horse radish peroxidase conjugated donkey anti-goat antibody (Sigma-Aldrich). Protein detection and quantification was performed using the Odyssey infrared imaging system (LI-COR). Gamma-myomembrane proteoglycans were detected by a mouse monoclonal antibody (Vector Labs VP-G803) and donkey, anti-mouse HRP conjugated secondary antibody (Santa Cruz Biotechnology).

Detection of neutralizing anti-AAV antibodies

To assess the humoral immune response against AAV 9 capsid protein, sera were collected via peripheral veins on the day of birth, then at weeks 4 and 8. HEK 293 cells in 200 μ L DMEM with 10% fetal bovine serum at 10⁵The density of individual cells/well was seeded in 48-well plates. Placing the cells inIncubated at 37 ℃ for 3-4 hours and allowed to adhere to the wells.

AAV 9-Green Fluorescent Protein (GFP) vector (1X 10)⁸Individual particles) were incubated with mouse serum serially diluted with PBS for 2 hours at 4 ℃ in a total volume of 25 μ L. The mixture was then added to the cells in a final volume of 200 μ l, which contained 4 x 10⁶AAV9 particles were incubated at 37 ℃ for 24 or 48 hours. Cells expressing GFP were counted under a fluorescent microscope. The highest dilution was used to calculate neutralizing antibody titers, with 50% less percentage of GFP-positive cells than serum-free controls.

Evaluation of the reactivity of T-cells to capsid-derived peptides

Peripheral Blood T cell responses to novel AAV capsid antigens were quantified by IFN- γ ELISpot assay (Mingozzi, F., et al, AAV-1-mediated gene transfer to viral muscles in humans in vivo dependent activity of capsule-specific T cells, Blood 114, 2077-2086 (2009)). Briefly, Peripheral Blood Mononuclear Cells (PBMCs) were isolated on a Ficoll hypaque gradient and cultured with synthetic peptides (20 amino acids in length, overlapping 10 residues) spanning the VP1 capsid protein. To identify individual peptides in the pool that elicit IFN- γ activity, each peptide was present in two cross-mapped sub-pools. After incubation at 37 ℃ for 36 hours, IFN-. gamma.SFU were counted. Less than 10 SFU/well (enhanced GFP) was observed with peptides from the control pool. When SFU exceeds 50/10 in the repeat well ⁶The response was considered positive with PBMC.

Statistical analysis

Estimation based on one of the most sensitive and most widely used histological assays availablemdxInformative group sizes of AAV vector injections in mice: proportion of central nucleated muscle fibers from mice at 8 weeks of age necropsy. To maximize the statistical power of the number of animals used, we used the method described by Aarts et al (Aarts, E. et al, A solubility to dependency: using multilevel analysis to acacommod ate new data. Nat Neurosci, 2014.17 (4): p. 491-6) to accommodate multiple High Power Fields (HPFs) in miceThe dependence of (c). A mixed effect model, taking an exchangeable correlation structure into account for the grouping in mice, was used for comparing the three groups defined by genotype and treatment. Estimates of intra-cluster correlation (ICC) from these models show low values: (<10%), suggesting a relatively highly effective sample size, despite the necessarily small number of mice per group (at least four). Other random effect parameters calculated in the analysis include: variance between groups, σ² _u(ii) a Variance within the group, σ² _e(ii) a And effective sample amount, n _eff。

To characterize the distribution of the minimum feret diameters in wild-type, treated and untreated malnourished dogs, points representing individual measurements from representative HPFs were plotted. Because of the small number of dogs, the analysis is completely descriptive.

All analyses except the proportion of centrally nucleated muscle fibers are provided as mean ± s.d. Statistical analysis was processed using Prism 7 software (GraphPad). Statistical significance of p-values is shown in the figure: p < 0.05; p < 0.001; p < 0.0001; n.s. was not significant.

Example 3 systemic Gene therapy for DMD Using AAV-mediated delivery of Nano-dystrophin-related proteins and Nano-dystrophin

A. Human nano-dystrophin-related protein design

The main role of the rod domain is the longitudinal transmission of force, and the abnormal length of dystrophins reflects the evolutionary heritage of the protein. As a result of this understanding, we have identified approaches that address the important limitations of other approaches and designed new transgenes to achieve levels of force transduction and muscle protection indistinguishable from wild-type.

Our sequence analysis of orthologues and paralogues of dystrophin genes and transcripts from widely sampled metazoan taxa led us to hypothesize that BMD in patients with deletions or duplications of conserved reading frames is caused by local destabilization of the rod domain during mechanical stress. The hypothesis is supported by crystallographic data from dystrophin and other proteins containing triple-helical repeat domains, bioinformatic analysis of evolutionary coupling between amino acids in adjacent triple-helical repeats, and unpublished data from our studies of miniaturized recombinant proteins in mechanically stressed muscle. None of the recombinant mini-dystrophins (mini-dystrophins) reported to date has the potential to avoid the weakest link at the internal deletion site, since all design methods used model triple helical repeats as interchangeable modular units. Despite its improved phenotype in animal models giving profound prints, even micro-dystrophin-related proteins (micro-Utrophin) risk this limitation. To address this possibility and further shorten the transgene, we have developed a model for triple-spliced "nano-dystrophin-related proteins" (figure 4). We "cut" and "bind" the rods of dystrophin and dystrophin-related proteins across the most structurally conserved three-dimensional planes of two non-adjacent triple-helical repeats to maintain all evolutionarily coupled amino acid side chain interactions at the load-bearing loop-to-loop interfaces with less conservation between adjacent repeats. This is modeled in figure 4 using a domino metaphor. In the next section, this will be described in more detail using a graphical method from molecular modelling. Potential improvements over micro-dystrophin related proteins were evaluated with respect to triple spliced nano-dystrophin related proteins by sequential testing in dystrophin deficient mice (intermediate) and rats (better pathologic genetic models of DMD fibrosis, degeneration and weakness, but requiring 10x more vector) to identify therapeutic transgenes.

B. Regulatable production of AAV for systemic delivery in DMD

AAV9 and Anc80 vectors, which have been utilized to date in our preclinical studies in dystrophic mice and dogs, have been prepared in human HEK 293 cells by applying a triple plasmid transfection method. The technique relies on anchorage dependent cell growth in rich media, whereas the input plasmid is propagated on a large scale in bacterial strains prior to transfection, so that the final product retains a potentially immunogenic bacterial DNA methylation pattern. AAV vector discovery and production using a large number of capsids and platforms was performed, including anchorage-dependent and suspension-dependent HEK 293 cells and baculovirus-infected Sf9 insect cells.

C. Animal model

Rigorous, blind testing of AAV μ -to n-dystrophin-related protein systemic therapy was investigated using primary, secondary, and exploratory endpoints reporting the design and execution of future clinical trials. Two primary endpoints were selected: the performance in our published comprehensive physiological assay Suite of the mdx mice (mixed limb force vertical activity test, see the graph from (Song, Y. et al, Suite of clinical Relevitative Functional Assays to Address Therapeutic Efficacy and Disease Mechanism in the Dystrophe mdx mouse, p. jap 007762016), and quantitative immunoassay of the N-terminal 25 kDa fragment of titin (Robertson, A.S. et al, pharmaceutical expression in vitro assay of tertiary necrosis by using two polypeptide fragments) and the results of the quantitative immunoassay of the N-terminal 25 kDa fragment of titin (map 12, 12. et al, the latter map is based on the systemic dynamics of necrosis of muscle fragments and clinical experiments, see FIG. 12. No. 12. the latter map is based on the systemic dynamics of necrosis of muscle tissues and diseases, found in the Biodynamic range of the muscle proteins found in the A.S. 12, see FIG. 12. No. 3. A. shows, and is based on the role of the adiponectin isoform in myofibril formation (example 1). The main objective of the proposed aim is to quantitatively characterize the extent and persistence of phenotypic improvement in a cost-effective model that reproduces the main pathological features of DMD. The study in mdx mice has systematically expanded to dystrophin deficient rats because the latter model more closely resembles the hallmark pathological myodegeneration and fibrosis seen in DMD, with an easily quantifiable progressive muscle weakness and cardiomyopathy index Robertson, a.s. et al, a.s. with a simultaneous elevation in the urinary amino terminal tissue and by two muscle dysfunction in the respiratory tissue and in the dystrophin defect nerves. See Petrof, B.J. et al, Dystrophin technologies, the sarcolema from structures depleted reduced muscle connection, Proc Natl Acad Sci, 1993.90: p.3710-14; neuroguscu disc, 2017.27 (7): p, 635-; (iii) Laccher, T. et al, chromatography of kinetic specificity rates a new model for Duchenne molecular dynamics. PLoS One, 2014.9 (10): p.e 110371; nakamura, K. et al, Generation of a molecular dynamics model rates with a CRISPR/Cas system. Sci Rep, 2014.4: p. 5635; stedman, H.H. et al, The mdx mouse diaphragm processes The generative changes of Duchenne molecular dynamics, Nature, 1991.352 (6335): p.536-9; shrager, J.B. et al, The mdx mouse and mdx diaphragma indications for The pathogenesis of Duchenne Musculus dystrophy, in neurous Development and Disease, A.M. Kelly and H.M. Blau, eds. 1992, Raven Press, Ltd.: New York. p.317. 328; krupnick, A.S. et al, instrumentation loads not access computer systems in mdx mouse diaphragm, amplification for regenerating therapy J Appl Physiol, 2003.94 (2) p.411-9; and Song, Y, et al, Suite of clinical reuse functional assays to address therapeutic effects and disease mechanisms in the dynamic mdx mouse, J Appl Physiol, 2017.122 (3): p.593-. Several canine disease models (Cooper, B.J. et al, The homology of The double recess of The cultured in X-linked mucosal dynamics of domains. Nature, 1988.334 (6178): p.154-6; Smith, B.F. et al, Molecular basis of cancer type selectivity. J Biol. Chem. 1996.271 (33): p.200704; Bridges, C.R. et al, Global cardiac-specific expression using cardiac substrate with cardiac analysis of cardiac isolation, Ann thor. Surg, 2002.73 (2002.73; p.1939-46; Arrowa, V.R. large et al, Molecular analysis of cardiac substrate with Molecular analysis of cardiac isolation. Ann. Thoror. Surg., p.9-46; AAV, V.R. large et al, Molecular analysis of viral expression, AAV 2. D.A. P.A. P.M. P.154-6, 2010.115 (23) p.4678-88; mead, A.F. et al, Diapthragm remodelling and machinery reactivity in a Canine model of Duchenne molecular dynamics. J Appl physical (1985), 2014.116 (7) p.807-15; and Su, L.T. et al, in the first clinical basic test and background test of gene therapy using AAV vectors in human muscular dystrophy, the general scale-independent gene transfer to structured mouse after delivery of vector Circulation, 2005.112 (12): p.1780-8) can be used, as well as hamster models of LGMD (e.g., histological assays in FIGS. 14A-14G, from Greelish et al, Nature Medicine, (Greelish, J.P. et al, Stable recovery of the synthetic complex in dynamic mouse tissue with high stability a structural additional viral vector Nat, 1999.5 (4): p.439-43): stedman et al, Human Gene therapy, Stedman, H, et al, Phase I clinical trial therapy for slide gird transformer alpha-, beta-, gamma-, or delta-saturated Gene delivery with intra-vascular implants of amino-associated vectors Hum Gene therapy, 2000.11 (5): p.777-90).

D. Product profile

Target product profile for AAV μ dystrophin-related proteins or AAVn dystrophin-related proteins.

The table above depicts the target product profile for AAV μ dystrophin-related proteins or AAVn dystrophin-related proteins. Our current understanding of the progressive loss of striated muscle cells and fibro-fat replacement (fibro-fat replacement) in DMD suggests that significant reversal of disease may be limited in older subjects, but that further muscle cell loss may be prevented at any stage after the onset of recombinant mu-or n-dystrophin-related protein expression. Experiments were performed mainly on dystrophin deficient mice and rats to report the expected values for the ideal parameters in DMD patients treated in infancy. Transduction with sufficient AAV μ -or n-dystrophin-related proteins during infancy to normalize sarcoleminopolyn expression throughout growth to skeletal maturation may allow for relatively normal muscle growth and thus an increase in normal maturation in strength. At least four factors may limit the therapeutic benefit in older patients: 1) the extent of pre-treatment irreversible myocyte loss, 2) fibro-fat replacement of muscle impairs vector delivery and the extent of myocyte transduction, 3) a decrease in maturation in endothelial permeability to the vector, which may require forced extravasation from the vascular lumen distal to the tourniquet in older patients, and 4) an expected increase in natural exposure to AAV viruses with increasing patient age, thereby increasing the proportion of memory T cells with both high titers of antibodies to multiple AAV serotypes and peptides derived from conserved AAV capsids. Dose-limiting toxicity may involve the innate immune system and/or liver.

E. Experimental and theoretical basis for mu-or n-dystrophin-related protein substitution for dystrophin deficiency

Dystrophin-related proteins were originally discovered based on their homology to the coding sequence of dystrophin (Love, d.r. et al, An autosomal transcript in skin muscle with homology to dystrophin, Nature, 1989.339 (6219): p.55-8). We have recently reconstructed the evolutionary history of dystrophin and dystrophin-related proteins based on publicly available whole genome sequences from a large number of taxa. Two observations were relevant: 1) before The appearance of striated muscle, The "donor" genes for The rod-like domains of both proteins have at least 21 spectrin-like repeat domains in tandem, well before they are linked to The Dp 71-like domain by partial gene repeats, 2) The individual genes for dystrophin-related proteins and dystrophin are fixed after lineage divergence of The cephalopods along a common ancestor to both amantadine and maxillo vertebrates and before The evolution of oligodendrocytes appears (Putnam, n.h. et al, The opioxus genome and The evolution of The chorddate kayotype, 2008.453 (7198): p.1064-71; and Smith, J.J. et al, Sequencing of the sea laboratory (Petromyzon marinaus) genome languages into a transformed solution, Nat Genet, 2013.45 (4): p.415-21, 421e 1-2). Both proteins retain the binding interface for the transmembrane glycoproteins of the cytoskeletal actin and dystrophin (/ dystrophin-related protein) binding protein complex (D/UAPC). It is well established that full length recombinant derivatives of dystrophin-related proteins can even reverse the severe dystrophic phenotype in mice (Tinsey, J. et al, Expression of full-length human genes Expression in mdx. Nat. Med 1998.4 (12): p. 1441-4; Gilbert, R. et al, Adenoviral-mediated Gene transfer of the dynamic Gene Expression of mdx mice Gene theory 1999.10 (8): p. 1299;. and Odom, G.L. et al, microprotein gradient Gene Expression 6 in microorganisms and tissue Expression 2008.16/1539). The unresolved gaps in our knowledge are the property of selective pressure resulting in the initial tandem repeat of the triple helix repeat of the ancestral protein, and whether the current physiological effect or effects of dystrophin "require" the estimated 6 nm/repeat x 24 repeats or 144nm length of native protein Dp 427. The severe clinical phenotype of some BMD patients with small internal deletions suggests that the length of Dp427 is essential for their role as shock absorber, but repeat-BMD raises questions to this explanation because these patients have longer dystrophins than wild-type. Another explanation was drawn from our reconstruction of the remote evolutionary history of dystrophin (example 1).

We asked a seemingly simple question "which dystrophin and sarcomere appeared firstThe objective was to determine whether the available evidence of genome and inferred proteome from existing species is consistent with a model in which the length of the dystrophin bar increases during the period from the appearance of the sarcomere to the recent evolution of the specialized Fast-contracting muscle most susceptible to acute injury in dystrophin deficient mammals (Webster, C. et al, Fast muscle fiber expressed after in Duchenne muscle Cell, 1988.52 (4): p.503-13; and Petrofs, B.JEt al, adaptions in a yeast blood chain expression and a restriction function in a steroid mouse diaphragma, Am. J. physiol., 1993.265: p. C834-C841). As shown in fig. 15, evidence supports dystrophin prior in time to the remodeling of sarcomeres. Detailed analysis showed that dystrophin is the same length in existing vertebrate and stinging animal (jellyfish) species, with highly conserved gene structures, as shown in fig. 6A to 6C. The length of dystrophin is likely to be reached long before sarcomere appears, even before the disc worms (trichoplazoa), the phylum represented by the simplest free living animal species, diverged. The species uses ciliary dynein (not myosin) as their primary source of kinetic energy and their physical developmental layout features only four cell types, none of which show identifiable sarcomere (Srivastava, m. et al, The trichopulex genome and The Nature of planzoans, Nature, 2008.454 (7207): p. 955-60). Innovative analysis of gene structure provides convincing evidence that the rod-like domain of dystrophin, 80% of the protein length, appears to complement as a whole from a larger ancestral protein (not spectrin, noting the qualitatively different pattern in fig. 6B), with a specific role in cross-linking actin filaments and Microtubules (MACF). The fundamental meaning of the findings is that MACF-like proteins, rather than dystrophins themselves, are the subject of selective pressure that drives the initial lengthening of the common ancestral rod domain of both proteins. The crystal structure of MACF differs from that of spectrin in the potential for longitudinal force transmission, providing an explanation for the maintenance of dystrophin and dystrophin-related protein lengths in a large number of taxa. As shown in fig. 16A and 16B, spectrin and dystroplakins have three helices that fold differently in terms of degree of overlap and number of stable amino acid side chain interactions. In fig. 16A and 16B, we modeled the adjacent triple helical repeats of the human dystrophin-related protein using templates derived from (fig. 16A) human β 2-spectrin (3 EDV) and (fig. 16B) human reticulin (5J 1G). Our model predicts that forces are in vivo across a wide inter-domain Interfacial longitudinal transfer, as required for the transfer of mechanical energy from within the cell to the extracellular matrix without disrupting the sarcolemma. Any major disruption in side chain bonding between adjacent and possibly overlapping triple helix repeats will therefore destabilize dystrophin and dystrophin-related proteins during force transmission.

The model is in full agreement with the assertion that the most important feature of the rod domain is strength rather than length, while the abnormal size of the protein can be attributed to its historical heritage as a derivative of the partial gene duplication by a much longer MACF homolog (homolog). Thus, a short recombinant protein specifically designed to optimize the structural integrity of the rod domain at the interface between all repeated sequences should completely complement the mechanical function of the full-length protein, e.g., Dp 427. The simplest approach to this conceptually is to avoid an internal rearrangement that directly juxtaposes incompatible triple helical repeats. The problem is that crystallographic information about the structure exists for only one of the 24 triple helix repeats of dystrophin and that there is low primary structural homology between the repeats except for the tryptophan residues conserved in the centers of helices a and C. We selected recombinant proteins initially focused on having one internal and one C-terminal deletion relative to the full-length dystrophin-related protein and named micro-or μ -dystrophin-related proteins. As shown in figure 17, the constructs juxtapose the unstructured, proline-rich, inter-helical "hinge-2" domain relative to the last triple helix repeat (22 nd) of the full-length dystrophin-related protein. In the most suitable case, the "hinge" may act as an inter-helical spacer (spacer) with the ability to transmit longitudinal forces without an exact match to the sequence of the triple helical repeat 22. This is the rationale for our centralized evaluation of μ -dystrophin-related proteins, as described in detail below and in example 2.

Systematic approach for mu-dystrophin-related protein transgene optimization determined that the use of codon bias of sarcomere myosin heavy chain increased the expression of immunodetectable protein in vitro by 30-fold compared to wild type (lane 5 vs lane 8, fig. 18). In fact, the expression level was so high that it competed effectively with the expression of the co-transfected e-gfp control. To analyze the therapeutic efficacy of the optimized μ U constructs after packaging into AAV9, we performed a series of randomized, blind studies in the mdx mouse model of DMD, as described in example 2.

According to the on-computer chip analysis of MHC binding of minimal T cell peptide epitopes, the μ dystrophin-related proteins have a number of potential immunodominant foreign peptides less than 1/100 compared to all hypothesized μ dystrophins in a dystrophin deficient host. This minimizes the risk of post-treatment autoimmunity and the potential need for long-term immunosuppression in DMD. Our blind study of systemic μ dystrophin-related protein expression in non-immunosuppressed GRMD dogs provides further reassurance by demonstrating the complete absence of peripheral T cell reactivity as determined by interferon γ ELISpot. These studies must be performed at doses of 1/10 (normalized to adult body weight) which are the maximum dose used in mdx mice, and can only be expanded at future dates to assess the maximum tolerated dose in large animal DMD models using adjustable manufacturing techniques.

F. Basic principle and structure of dystrophin, the protein related to nano-dystrophin

Our findings are of great significance in the whole field, since they are equally relevant to all AAV-sized dystrophin candidates being developed for transformation into clinical studies.

We started a series of blind experiments to treat the stability of μ -dystrophin-related proteins in AAV-injected mdx mice. These quantitative studies have provided very favorable data on the level of μ -dystrophin-related protein expression at intermediate time points. However, in a further analysis, we noted an unexpected "extra band" on the western blot after staining with an antibody specific for the N-terminus of the dystrophin-related protein. As summarized in fig. 21, the molecular weight assigned to the novel band completely matched the molecular weight of the 79 kd N-terminal portion of the μ -dystrophin-associated protein. In other words, the findings suggest that 135 kd of the μ -dystrophin related protein is disrupted in the immediate vicinity of the junction between the portion flanking the "hinge 2" deleted for the coding sequence corresponding to the full length-dystrophin related protein and the spectrin-like repeat 22, thereby releasing 79 kd N-terminal "subdomain" as a fragment. In our study, it appears that there appears to be a correlation between the appearance and intensity of the 79 kd band and the maximum recent level of force transduction by muscle. We next subjected the 79 kd region of the additional gel to proteomic analysis by a combination of liquid chromatography and tandem mass spectrometry. This confirms our hypothesis, as shown in fig. 22. The findings compel us to reconsider our assumptions about the interchangeability of spectrin repeats, particularly 4 and 22 when placed directly at the primary structure close to hinge 2. Upon revisiting our findings of bioinformatic analyses of dystrophin, dystrophin-related proteins and titin, it is clear that the poly IgG domain of titin shows strong evidence of interchangeability over evolutionary time, but in the same species comparison we see no evidence of interchangeability of the other two proteins (example 1). This provides a convincing explanation for genotype-phenotype association in selected becker MD cases, especially in severe cases associated with gene duplication, where it was concluded that there is a longer than wild-type dystrophin protein with the weakest link in the transmission of new connective axial forces across two non-adjacent ancestral portions of the rod.

One crystal structure from the MACF/banin family may be beneficial because the structural data for dystrophin and dystrophin-related proteins is limited to the first triple helix repeat (i.e. the most N-terminal end of the rod) and the hinge domain is predicted to be "unstructured". Prior to the determination of the structure, there was the speculation that the SH3 domain between the triple helical repeats also acts as a hinge, allowing the appearance of unstable interface images between two strongly powerful portions of the rod. In contrast, the structure revealed that the SH3 domain forms multiple high affinity contacts with compatible amino acid side chains from the flanking adjacent triple helices, a configuration with the potential to transmit longitudinal forces and resist stretching (fig. 23 shows the 3PEo structure of SR4 & 5 of the plaque domain, shown as space-filling to show SH 3: SR4/5 binding interface). Analysis of the primary structure of several dystrophin/dystrophin-related protein orthologs from stinging animal species (jellyfish) revealed the presence of interposed but HMM recognizable domains, but no unstructured "hinges", further supporting our hypothesis on both the evolutionary origin of the dystrophin rod domains from MACF-like proteins and the magnitude of long-axis force transmission across the wide surface area of the interdomain interface.

Given these structural and functional constraints, we revisited the design of AAV-compatible miniaturized substitutions for Dp427, a full-length dystrophin. We have designed a nano-dystrophin-related protein that exploits unique opportunities to rearrange the rod domains internally with minimal disruption of the inter-domain interface. This is achieved on a computer chip by combining pairs of different triple helices across a conserved axial cross-section, one illustrated at the site of the interacting, highly conserved tryptophan residue that stabilizes the core of the triple helix (fig. 3). We have synthesized codon-optimized cDNAs for the constructs, generated and prepared ITR-flanked transcription cassette vectors for triple transfection in 293 cells in large quantities. Further, we performed blind experiments in whichmdxMice were randomized to receive AAVs encoding "micro-" or "nano-" dystrophin-related proteins. The results show that micro-dystrophin-related proteins are cleaved precisely at the end of the 79kd N-terminal subfragment, whereas no detectable cleavage of nano-dystrophin-related proteins is observed under similar physiological load (figure 28). Both proteins are correctly localized mdxSarcolemma in the muscle of mice. The findings from the experiments indicated that the superior strength of the nano-dystrophin-related protein compared to the micro-dystrophin-related protein more closely approximates the mechanics biology of the dystrophin isoform Dp 427. Additional studies were conducted to evaluate phenotypic improvement in various disease models using the methods used in these examples above. At the same time, a transgene was established based on the reduced vector genome sizeDue to the degree of any improvement in the efficiency of packaging into AAV.

G. Reconstituted progenitor AAV capsids for DMD treatment: anc80, 81, 82

AAV vectors based on naturally occurring capsid serotype 9 achieve striking global biodistribution to striated muscle in dogs and non-human primates. Although the capsid residues involved in binding to selected membrane receptors are well defined for other serotypes, the structural basis for this is poorly understood. AAV8 is a good choice for efficient cardiac gene transfer in dogs, reflecting species differences, since AAV9 provides robust cardiac transduction in primates. Both AAV8 and 9 are associated with neutralizing antibodies in a significant proportion of the adult population. In an effort to circumvent this limitation while maintaining overall biodistribution to the striated muscle, Zinn et al (Zinn, E. et al, In silica Reconstruction of the Viral evolution series a content Gene Therapy vector Cell Rep, 2015.12 (6): p. 1056-68) used a combination of ancestral sequence Reconstruction and In vitro Gene synthesis on a computer chip to prepare AAVs with "new" vector capsids (i.e., capsid variants that are likely to occur naturally but have died long ago). Of these, those labeled Anc80, 81, and 82 represent the most immediate opportunity to replicate or expand the favorable biodistribution of AAV8 and 9 while expanding the eligibility pool from a population of patients with the potential to neutralize antibodies at high titers after past exposure to naturally occurring existing AAVs, as described in detail (Zinn et al, cited above).

We recently packaged our μ dystrophin-related protein genome in the Anc80 capsid and used the resulting vectors to evaluate transduction of related muscles in mdx mice. These studies indicate that, in this sense, Anc80 achieved a similar overall biodistribution as AAV9 in the case of strong transduction of cardiac and skeletal muscle (fig. 24A to 24C).

Data from randomized, blind experiments demonstrated the ability of Anc80 and AAV9 to have similar in vivo infectivity in dystrophic muscle based on the expression levels of therapeutic transgenes.

For in the range of 2.5 × 10¹²Biological distribution of μ dystrophin-associated proteins in a group of mdx mice after systemic administration of these vectors at equivalent dose of vg/mouse, experiments designed to qualitatively compare AAV9 and Anc80 were performed. Representative western blots of various muscles from two mice are shown in fig. 25 for each vector, demonstrating broad and efficient transduction of striated muscle with both vectors.

Example 4 quintuplex sequence mutant dystrophin related proteins and dystrophins

We have designed dystrophin-related and dystrophin recombinant proteins containing an additional triple helix relative to the original "nano" mutant described above. These "quintuplex" mutants have 5 spectrin-like triple helix repeats between the N-terminal calmodulin homeodomain and the C-terminal "WW-EF-ZZ" domain present in the full-length protein. These variants also have improved stability due to the presence of triple splice mutations, and further can be packaged into AAV vectors. Importantly, the development of these mutants illustrates the principles on which the design of four-repeat nano-dystrophin and nano-dystrophin related proteins, including those described herein, can be extended to variants with five helical repeats. SEQ ID NO: 22, amino acid sequences corresponding to the five repeat dystrophin protein are provided, wherein the splicing mutations are formed by joining the helical repeats 1 and 20 of the full length dystrophin protein (fig. 29A and 29B). SEQ ID NO: provided in 21 is an amino acid sequence corresponding to a five repeat dystrophin-related protein wherein the splicing mutation is formed by joining helical repeats 1 and 18 of a full length dystrophin-related protein (fig. 30A and 30B).

(free words of sequence listing)

The following information is provided for sequences containing free text under the numeric identifier <223 >.

All publications, patents, patent applications and sequence listings referred to herein, as well as U.S. provisional patent application No. 62/658,464, filed 2018, 4, 16, are hereby incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Many modifications and variations are within the scope of the above-identified description and are expected to be apparent to those skilled in the art. Such modifications and variations to the compositions and methods, for example, the selection of different coding sequences or the selection or dosage of vectors or immunomodulatory agents, are considered to be within the scope of the appended claims.