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1. (WO2017072590) MATÉRIAUX ET MÉTHODES POUR TRAITER LA DYSTROPHIE MUSCULAIRE DE DUCHENNE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

Claims

What is claimed is:

1 . A method for editing a dystrophin gene in a human cell by genome editing, the method comprising the step of: introducing into the human cell one or more

deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene that results in a permanent deletion, insertion, or replacement of one or more exons or aberrant intronic splice acceptor or donor sites within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity.

2. The method of claim 1 , wherein the human cell is a muscle cell or muscle precursor cell.

3. An ex vivo method for treating a patient with Duchenne Muscular Dystrophy (DMD), the method comprising the steps of:

i) creating a DMD patient specific induced pluripotent stem cell (iPSC);

ii) editing within or near a dystrophin gene of the iPSC;

iii) differentiating the genome-edited iPSC into a Pax7+ muscle progenitor cell; and

iv) implanting the Pax7+ muscle progenitor cell into the patient.

4. The method of claim 3, wherein the creating step comprises:

a) isolating a somatic cell from the patient; and

b) introducing a set of pluripotency-associated genes into the somatic cell to induce the somatic cell to become a pluripotent stem cell.

5. The method of claim 4, wherein the somatic cell is a fibroblast.

6. The method of claim 4, wherein the set of pluripotency-associated genes is one or more of the genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG and cMYC.

7. The method of any one of claims 3-6, wherein the editing step comprises introducing into the iPSC one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene that results in a permanent deletion, insertion, or

replacement of one or more exons or aberrant intronic splice acceptor or donor sites within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity.

8. The method of any one of claims 3-7, wherein the differentiating step comprises one or more of the following to differentiate the genome-edited iPSC into a Pax7+ muscle progenitor cell: contacting the genome-edited iPSC with specific media formulations, including small molecule drugs; transgene overexpression; or serum withdrawal.

9. The method of any one of claims 3-8, wherein the implanting step comprises implanting the Pax7+ muscle progenitor cell into the patient by local injection into the desired muscle.

10. An in vivo method for treating a patient with Duchenne Muscular Dystrophy (DMD), the method comprising the step of editing a dystrophin gene in a cell of the patient.

1 1 . The method of claim 10, wherein the editing step comprises introducing into the cell of the patient one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene that results in a permanent deletion, insertion, or replacement of one or more exons or aberrant intronic splice acceptor or donor sites within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity.

12. The method of claim 1 1 , wherein the cell is a muscle cell or muscle precursor cell.

13. The method of any one of claims 1 , 7 or 1 1 , wherein the one or more DNA endonucleases is a Cas1 , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Casl OO, Csy1 , Csy2, Csy3, Cse1 , Cse2, Csd , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Crn , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, a codon-optimized thereof, or modified version thereof, and combinations thereof.

14. The method of claim 13, wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more DNA endonucleases.

15. The method of claim 13, wherein the method comprises introducing into the cell one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

16. The method of any one of claims 14 or 15, wherein the one or more

polynucleotides or one or more RNAs is one or more modified polynucleotides or one or more modified RNAs.

17. The method of claim 13, wherein the one or more DNA endonuclease is one or more proteins or polypeptides.

18. The method of any one of the preceding claims, wherein the method further comprises introducing into the cell one or more guide ribonucleic acids (gRNAs).

19. The method of claim 18, wherein the one or more gRNAs are single-molecule guide RNA (sgRNAs).

20. The method of claims 18 or 19, wherein the one or more gRNAs or one or more sgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

21 . The method of any one of claims 18-20, wherein the one or more DNA

endonucleases is pre-complexed with one or more gRNAs or one or more sgRNAs.

22. The method of any one of the preceding claims, wherein the method further comprises introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene or cDNA.

23. The method of claim 22, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes at least a part of exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

24. The method of claim 22, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions,

synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

25. The method of any one of claims 22-24, wherein the donor template is a single or double stranded polynucleotide.

26. The method of any one of claims 1 , 7 or 1 1 , wherein the method further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs), and wherein the one or more DNA endonucleases is one or more Cas9 or Cpf1

endonucleases that effect a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first SSB or DSB break at a 5' locus and the second SSB or DSB break at a 3' locus, that results in a permanent deletion or replacement of one or more exons or aberrant intronic splice acceptor or donor sites between the 5' locus and the 3' locus within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity.

27. The method of claim 26, wherein one gRNA creates a pair of SSBs or DSBs.

28. The method of claim 26, wherein one gRNA comprises a spacer sequence that is complementary to either the 5' locus, the 3' locus, or a segment between the 5' locus and 3' locus.

29. The method of claim 26, wherein the method comprises a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence that is

complementary to a segment of the 5' locus and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3' locus.

30. The method of any one of claims 26-29, wherein the one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).

31 . The method of any one of claims 26-30, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

32. The method of any one of claims 26-31 , wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs or one or more sgRNAs.

33. The method of any one of claims 26-32, wherein there is a deletion of the chromosomal DNA between the 5' locus and the 3' locus.

34. The method of any one of claims 26-33, wherein the deletion is a single exon deletion.

35. The method of claim 34, wherein the single exon deletion is a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, or exon 53.

36. The method of any one of claims 34-35, wherein the 5' locus is proximal to a 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, and exon 53.

37. The method of any one of claims 34-36, wherein the 3' locus is proximal to a 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, and exon 53.

38. The method of any one of claims 34-37, wherein the 5' locus is proximal to a 5' boundary and the 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, and exon 53.

39. The method of any one of claims 36-38, wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron.

40. The method of any one of claims 26-33, wherein the deletion is a multi-exon deletion.

41 . The method of claim 40, wherein the multi-exon deletion is a deletion of exons 45-53 or exons 45-55.

42. The method of any one of claims 40-41 , wherein the 5' locus is proximal to a 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

43. The method of any one of claims 40-42, wherein the 3' locus is proximal to a 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

44. The method of any one of claims 40-43, wherein the 5' locus is proximal to a 5' boundary and a 3' locus is proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

45. The method of any one of claims 42-44, wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron.

46. The method of any one of claims 26-32, wherein there is a replacement of the chromosomal DNA between the 5' locus and the 3' locus.

47. The method of any one of claims 26-32 or 46, wherein the replacement is a single exon replacement.

48. The method of any one of claims 26-32 or 46-47, wherein the single exon replacement is a replacement of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, or exon 70.

49. The method of any one of claims 47-48, wherein the 5' locus is proximal to a 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, or exon 70.

50. The method of any one of claims 47-49, wherein the 3' locus is proximal to a 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, or exon 70.

51 . The method of any one of claims 47-50, wherein the 5' locus is proximal to a 5' boundary and a 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, or exon 70.

52. The method of any one of claims 49-51 , wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron or neighboring exon.

53. The method of any one of claims 26-32 or 46, wherein the replacement is a multi-exon replacement.

54. The method of claim 53, wherein the multi-exon replacement is a replacement of exons 45-53 or exons 45-55.

55. The method of any one of claims 53-54, wherein the 5' locus is proximal to a 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

56. The method of any one of claims 53-55, wherein the 3' locus is proximal to a 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

57. The method of any one of claims 53-56, wherein the 5' locus is proximal to a 5' boundary and a 3' locus is proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

58. The method of any one of claims 55-57, wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron.

59. The method of any one of claims 46-58, wherein the method further comprises introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene or cDNA, and the replacement is by homology directed repair (HDR).

60. The method of claim 59, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes at least a part of exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

61 . The method of claim 59, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

62. The method of any one of claims 1 , 7 or 1 1 , wherein the method further comprises introducing into the cell one guide ribonucleic acid (gRNA) and a

polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene, and wherein the one or more DNA endonucleases is one or more Cas9 or Cpf1

endonucleases that effect one single-strand break (SSB) or double-strand break (DSB) at a locus within or near the dystrophin gene that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA at the locus that results in permanent insertion or correction of one or more exons or aberrant intronic splice acceptor or donor sites within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus.

63. The method of any one of claims 1 , 7, or 1 1 , wherein the method further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs) and a polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene, and wherein the one or more DNA endonucleases is one or more Cas9 or Cpf1 endonucleases that effect a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first at a 5' locus and the second at a 3' locus, within or near the dystrophin gene that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent insertion or correction of one or more exons or aberrant intronic splice acceptor or donor sites between the 5' locus and the 3' locus within or near the dystrophin gene and results in restoration of the dystrophin reading frame and restoration of the dystrophin protein activity.

64. The method of claim 63, wherein one gRNA creates a pair of SSBs or DSBs.

65. The method of claim 63, wherein one gRNA comprises a spacer sequence that is complementary to either the 5' locus, the 3' locus, or a segment between the 5' locus and the 3' locus.

66. The method of claim 63, wherein the method comprises a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence that is

complementary to a segment of the 5' locus and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3' locus.

67. The method of claim 62 or 63, wherein the one or more gRNAs are one or more single-molecule guide RNA (sgRNAs).

68. The method of any one of claims 62-63 or 67, wherein the one or more gRNAs or one or more sgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

69. The method of any one of claims 62-63 or 67-68, wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs or one or more sgRNAs.

70. The method of any one of claims 62-69, wherein the insertion is a single exon insertion.

71 . The method of claim 70, wherein the single exon insertion is an insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, or exon 70.

72. The method of any one of claims 70-71 , wherein the locus, 5' locus or 3' locus is proximal to a boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51 , exon 52, exon 53, and exon 70.

73. The method of claim 72, wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron or neighboring exon.

74. The method of any one of claims 62-69, wherein the insertion is a multi- exon insertion.

75. The method of claim 74, wherein the multi-exon insertion is an insertion of exons 45-53 or exons 45-55.

76. The method of any one of claims 74-75, wherein the locus, 5' locus or 3' locus is proximal to a boundary of multiple-exons selected from the group consisting of exons 45-53 or exons 45-55.

77. The method of any one of claims 76, wherein proximal to the boundary of the exon includes the surrounding splice donors and acceptors of the neighboring intron.

78. The method of claim 62 or 63, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes at least a part of exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

79. The method of claim 62 or 63, wherein the at least a portion of the wild-type dystrophin gene or cDNA includes exon 1 , exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 1 1 , exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21 , exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31 , exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 , exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51 , exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61 , exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71 , exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intronic regions, synthetic intronic regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

80. The method of any one of claims 62-79, wherein the insertion is by homology directed repair (HDR).

81 . The method of any one of claims 62-80, wherein the donor template is a single or double stranded polynucleotide.

82. The method of any one of claims 26-81 , wherein the Cas9 or Cpfl mRNA, gRNA, and donor template are each formulated into separate lipid nanoparticles or all co-formulated into a lipid nanoparticle.

83. The method of any one of claims 26-81 , wherein the Cas9 or Cpfl mRNA is formulated into a lipid nanoparticle, and both the gRNA and donor template are delivered to the cell by an adeno-associated virus (AAV) vector.

84. The method of any one of claims 26-81 , wherein the Cas9 or Cpfl mRNA is formulated into a lipid nanoparticle, and the gRNA is delivered to the cell by

electroporation and donor template is delivered to the cell by an adeno-associated virus (AAV) vector.

85. The method of any one of the preceding claims, wherein the dystrophin gene is located on Chromosome X: 31 , 1 17,228-33,344,609 (Genome Reference Consortium -GRCh38/hg38).

86. One or more guide ribonucleic acids (gRNAs) for editing a dystrophin gene in a cell from a patient with Duchenne Muscular Dystrophy, the one or more gRNAs comprising a spacer sequence selected from the group consisting of the nucleic acid sequences in SEQ ID NOs: 1 - 1 ,410,472 of the Sequence Listing.

87. The one or more gRNAs of claim 86, wherein the one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).

88. The one or more gRNAs or sgRNAs of claims 86 or 87, wherein the one or more gRNAs or one or more sgRNAs is one or more modified gRNAs or one or more modified sgRNAs.