MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD

• US 20150152415A1
• Filed: 10/24/2014
• Published: 06/04/2015
• Est. Priority Date: 10/24/2008
• Status: Abandoned Application

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: SAZANI, Peter, KOLE, Ryszard
• Application Number:
  US14/523,610
• Publication Number:
  US 20150152415A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Provided are antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping, and methods of use thereof to treat muscular dystrophy.

Claims

1. A composition for use in producing skipping of exon 44 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

1, 3-20, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 44, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

2. The composition of claim 1, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

8, 11 and 12.

3. The composition of claim 1, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

4. A composition for use in producing skipping of exon 45 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

21-76 and 612 to 624, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 45, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

5. The composition of claim 4, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

27, 29, 34 and 39.

6. The composition of claim 5, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

29 and 34.

7. The composition of claim 4, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

8. A composition for use in producing skipping of exon 46 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

77-125, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 46, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

9. The composition of claim 8, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

77-105.

10. The composition of claim 9, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

82, 84-87, 90, 96, 98, 99 and 101.

11. The composition of claim 8, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

12. A composition for use in producing skipping of exon 47 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

126-169, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 47, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

13. The composition of claim 12, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

126-149.

14. The composition of claim 13, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

126, 128-130, 132, 144 and 146-149.

15. The composition of claim 12, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

16. A composition for use in producing skipping of exon 48 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

170-224 and 634, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 48, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

17. The composition of claim 16, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

170-201 and 634.

18. The composition of claim 17, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

176, 178, 181-183, 194 and 198-201.

19. The composition of claim 16, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

20. A composition for use in producing skipping of exon 49 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

225-266, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 49, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

21. The composition of claim 20, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

225-248.

22. The composition of claim 21, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

227, 229, 234, 236, 237 and 244-248.

23. The composition of claim 20, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

24. A composition for use in producing skipping of exon 50 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

267-286 and 288-308, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 50, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

25. The composition of claim 24, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

277, 290 and 291.

26. The composition of claim 25, wherein the compound contains the sequence consisting of SEQ ID NO:

290.

27. The composition of claim 24, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

28. A composition for use in producing skipping of exon 51 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

309-371, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 51, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

29. The composition of claim 28, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

324, 326 and 327.

30. The composition of claim 29, wherein the compound contains the sequence consisting of SEQ ID NO:

327.

31. The composition of claim 28, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

32. A composition for use in producing skipping of exon 52 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

372-415, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 52, a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

33. The composition of claim 32, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

372-397.

34. The composition of claim 33, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

379-382, 384, 390 and 392-395.

35. The composition of claim 32, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

36. A composition for use in producing skipping of exon 53 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

416-475 and 625-633, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 53 a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

37. The composition of claim 36, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

428, 429 and 431.

38. The composition of claim 37, wherein the compound contains a sequence consisting of SEQ ID NO:

429.

39. The composition of claim 36, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

40. A composition for use in producing skipping of exon 54 in the processing of human dystrophin pre-processed mRNA, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

476-519, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 54 a heteroduplex structure between said compound and mRNA having a Trn of at least 45°

C.

41. The composition of claim 40, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

476-499.

42. The composition of claim 41, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

479-482, 484, 489 and 491-493.

43. The composition of claim 40, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

44. A composition for use in producing skipping of exon 55 in the processing of human dystrophin-gene pre-processed mRNA transcript, comprising

a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

520-569 and 635, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 55 a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C.

45. The composition of claim 44, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

520-546 and 635.

46. The composition of claim 45, wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS:

524-528, 537, 539, 540, 542 and 544.

47. The composition of claim 44, wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS:

570-578.

48. A method of treating muscular dystrophy in a subject, comprising administering to the subject an effective amount of a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS;

1, 3 to 286, 288 to 569 and 612 to 635, and capable of forming with the complementary mRNA sequence in a dystrophin-gene exon a heteroduplex structure between said compound and mRNA having a Tm of at least 45°

C., wherein the exon is selected from the group consisting of exons 44-55.

49. The method of claim 48, wherein the muscular dystrophy is Duchenne'"'"'s muscular dystrophy (DMD).

50. The method of claim 48, wherein the muscular dystrophy is Becker muscular dystrophy (BMD).

51. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

1, 3-20, and the exon is exon 44.

52. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

21-76 and 612 to 624, and the exon is exon 45.

53. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

77-125, and the exon is exon 46.

54. The method of claim 48, wherein the sequence selected from the group consisting SEQ ID NOS:

126-169, and the exon is exon 47.

55. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

170-224 and 634, and the exon is exon 48.

56. The method of claim 48, wherein the sequence selected from the group consisting SEQ ID NOS:

225-266, and the exon is exon 49.

57. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

267-286 and 288-308, and the exon is exon 50.

58. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

309-371, and the exon is exon 51.

59. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

372-415, and the exon is exon 52.

60. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

416-475 and 625-633, and the exon is exon 53.

61. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

476-519, and the exon is exon 54.

62. The method of claim 48, wherein the sequence is selected from the group consisting SEQ ID NOS:

520-569 and 635, and the exon is exon 55.

63. The method of claim 48, wherein the sequence comprises SEQ ID NO:

290.

64. The method of claim 48, wherein the compound is conjugated to an arginine-rich peptide.

65. The method of claim 64, wherein the arginine-rich peptide comprises a sequence selected from the group consisting of SEQ ID NOS:

570-578.