Antisense oligonucleotides for inducing exon skipping and methods of use thereof

• US 8,455,634 B2
• Filed: 06/24/2011
• Issued: 06/04/2013
• Est. Priority Date: 06/28/2004
• Status: Active Grant

• Current Assignee: University of Western Australia
• Original Assignee: University of Western Australia
• Inventors: Wilton, Stephen Donald, Fletcher, Sue, McClorey, Graham
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US13/168,863
• Publication Number:
  US 20120041050A1
• Time in Patent Office: 711 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• 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 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

An antisense molecule capable of binding to a selected target site to induce exon skipping in the dystrophin gene, as set forth in SEQ ID NO: 1 to 202.

Claims

1. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 17 consecutive nucleotides complementary to an exon 52 target region of the Dystrophin gene designated as annealing site H52A(+17+37), wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 52 skipping, and wherein uracil bases in the antisense oligonucleotide are optionally thymine bases.

2. The antisense oligonucleotide of claim 1 comprising 20-31 nucleotides in length.

3. The antisense oligonucleotide of claim 1, wherein the uracil bases are thymine bases.

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

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

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

7. The antisense oligonucleotide of claim 6, wherein the non-natural moieties are morpholinos.

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

9. The antisense oligonucleotide of claim 8, wherein the non-natural inter-nucleotide linkages are modified phosphates.

10. 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.

11. The antisense oligonucleotide of claim 10, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

12. The antisense oligonucleotide of claim 11, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

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

-O-methyl-oligoribonucleotide.

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

15. 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.

16. The antisense oligonucleotide of claim 15, wherein the oligonucleotide is conjugated to polyamine.

17. The antisense oligonucleotide of claim 15, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

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

37. A method of inducing exon-skipping of a dystrophin exon 52, comprising administering to a subject a pharmaceutical composition of claim 36.

38. The method of claim 37, wherein the subject is a human subject.

39. The method of claim 38, wherein the human subject has muscular dystrophy.

40. The method of claim 39, wherein the muscular dystrophy is Duchenne muscular dystrophy.

42. The antisense oligonucleotide of claim 7, comprising SEQ ID NO:

188, wherein the uracil bases are thymine bases.

44. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 42, and a saline solution that includes a phosphate buffer.

18. An isolated antisense of 20 to 50 nucleotides in length comprising at least 17 consecutive nucleotides of SEQ ID NO:

188, wherein the oligonucleotide specifically hybridizes to an exon 52 target region of the Dystrophin gene inducing exon 52 skipping, and wherein the uracil bases are optionally thymine bases.

19. The antisense oligonucleotide of claim 18 comprising SEQ ID NO:

188.

20. The antisense oligonucleotide of claim 18 consisting of SEQ ID NO:

188.

21. The antisense oligonucleotide of claim 18 comprising 20-31 nucleotides in length.

22. The antisense oligonucleotide of claim 18, wherein the oligonucleotide does not activate RNase H.

23. The antisense oligonucleotide of claim 18, comprising a non-natural backbone.

24. The antisense oligonucleotide of claim 18, 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 18, 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 18, 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 18, wherein the oligonucleotide is a 2′

-O-methyl-oligoribonucleotide.

32. The antisense oligonucleotide of claim 18, wherein the oligonucleotide is a peptide nucleic acid.

33. The antisense oligonucleotide of claim 18, 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 a polyamine.

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

41. The antisense oligonucleotide of claim 18, wherein the uracil bases are thymine bases.

43. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 18, and a saline solution that includes a phosphate buffer.

45. A method of inducing exon-skipping of a dystrophin exon 52, comprising administering to a subject a pharmaceutical composition of claim 43.

46. The method of claim 45, wherein the subject is a human subject.

47. The method of claim 46, wherein the human subject has muscular dystrophy.

48. The method of claim 47, wherein the muscular dystrophy is Duchenne muscular dystrophy.