EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY

• US 20140315977A1
• Filed: 03/14/2014
• Published: 10/23/2014
• Est. Priority Date: 03/14/2013
• Status: Abandoned Application

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: BESTWICK, Richard K., FRANK, Diane Elizabeth
• Application Number:
  US14/213,641
• Publication Number:
  US 20140315977A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3535 Nitrogen
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon 53 skipping are described.

Claims

1. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 10 consecutive nucleotides of a nucleotide sequence selected from the group consisting of:

SEQ ID NOs;

1, 9, 11, and 15-18, wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the human dystrophin gene and induces exon 53 skipping, and wherein thymine bases are optionally uracil bases.

2. The antisense oligonucleotide of claim 1, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:

1, 9, 11, and 15-18.

3. The antisense oligonucleotide of claim 1, comprising SEQ ID NO:

16.

4. The antisense oligonucleotide of claim 1, consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs:

1, 9, 11, and 15-18.

5. The antisense oligonucleotide of claim 1, wherein the oligonucleotide does not activate RNase H.

6. The antisense oligonucleotide of claim 1, comprising a non-natural backbone.

7. The antisense oligonucleotide of claim 1, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

8. The antisense oligonucleotide of claim 7, wherein the non-natural moieties are morpholinos.

9. The antisense oligonucleotide of claim 1, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

10. The antisense oligonucleotide of claim 9, wherein the non-natural inter-nucleotide linkages are modified phosphates.

11. The antisense oligonucleotide of claim 1, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

12. The antisense oligonucleotide of claim 11, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

13. The antisense oligonucleotide of claim 12, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates, or phosphoroamidates.

14. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is a 2′

-O-methyl-oligoribonucleotide.

15. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is a peptide nucleic acid.

16. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

17. The antisense oligonucleotide of claim 16, wherein the oligonucleotide is conjugated to an arginine-rich cell penetrating peptide.

18. The antisense oligonucleotide of claim 16, wherein the oligonucleotide is chemically linked to a polyethylene glycol moiety.

36. An expression vector comprising the antisense oligonucleotide of claim 1.

37. The expression vector of claim 36, wherein the expression vector is a modified retrovirus or a non-retroviral vector.

38. The expression vector of claim 37, wherein the non-retroviral vector is an adeno-associated virus (AAV).

39. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 1, and a saline solution that includes a phosphate buffer.

40. A method of treating Duchenne muscular dystrophy, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of claim 39.

41. Use of an antisense molecule according to claim 1 for the manufacture of a medicament for treating muscular dystrophy.

42. An antisense molecule according to claim 1 for use in antisense molecule based therapy.

43. A kit comprising at least one antisense molecule according to claim 1, a suitable carrier, and instructions for use.

19. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 10 consecutive nucleotides complementary to an exon 53 target region of the human dystrophin gene designated as an annealing site selected from the group consisting of:

H53A(+33+60), H53A(+31+55), H53A(+22+46), H53A(+30+57), H53A (+30+56), H53A(+30+55) and H53A(+33+57), wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 53 skipping.

20. The antisense oligonucleotide of claim 19, comprising 25 to 28 nucleotides in length.

21. The antisense oligonucleotide of claim 19, wherein thymine bases are uracil bases.

22. The antisense oligonucleotide of claim 19, wherein the oligonucleotide does not activate RNase H.

23. The antisense oligonucleotide of claim 20, comprising a non-natural backbone.

24. The antisense oligonucleotide of claim 19, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

25. The antisense oligonucleotide of claim 24, wherein the non-natural moieties are morpholinos.

26. The antisense oligonucleotide of claim 19, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

27. The antisense oligonucleotide of claim 26, wherein the non-natural inter-nucleotide linkages are modified phosphates.

28. The antisense oligonucleotide of claim 19, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

29. The antisense oligonucleotide of claim 28, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

30. The antisense oligonucleotide of claim 29, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates, or phosphoroamidates.

31. The antisense oligonucleotide of claim 19, wherein the oligonucleotide is a 2′

-O-methyl-oligoribonucleotide.

32. The antisense oligonucleotide of claim 19, wherein the oligonucleotide is a peptide nucleic acid.

33. The antisense oligonucleotide of claim 19, wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

34. The antisense oligonucleotide of claim 33, wherein the oligonucleotide is conjugated to an arginine-rich cell penetrating peptide.

35. The antisense oligonucleotide of claim 33, wherein the oligonucleotide is chemically linked to a polyethylene glycol moiety.