Antisense oligonucleotides for inducing exon skipping and methods of use thereof

• US 8,455,635 B2
• Filed: 10/11/2011
• 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:
  US13/270,937
• Publication Number:
  US 20120029059A1
• Time in Patent Office: 602 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 20-50 nucleotides in length comprising at least 17 consecutive nucleotides of SEQ ID NO:

175, wherein the oligonucleotide specifically hybridizes to an exon 50 target region of the Dystrophin gene and has ability to skip exon 50, and wherein the uracil bases are optionally thymine bases.

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

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

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

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

6. The antisense oligonucleotide of claim 5, wherein the non-natural moieties are morpholinos.

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

8. The antisense oligonucleotide of claim 7, wherein the non-natural inter-nucleotide linkages are modified phosphates.

9. 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.

10. The antisense oligonucleotide of claim 9, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

11. The antisense oligonucleotide of claim 10, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

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

-0-methyl-oligoribonucleotide.

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

14. 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.

15. The antisense oligonucleotide of claim 14, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

16. The antisense oligonucleotide of claim 8, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

17. The antisense oligonucleotide of claim 16, wherein the modified phosphates are phosphoramidates.

18. The antisense oligonucleotide of claim 16, wherein the modified phosphates are phosphoromorpholidates.

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

175.

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

21. The antisense oligonucleotide of claim 1, consisting of SEQ ID NO:

175.

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

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

42. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 19 and a saline solution that includes a phosphate buffer.

43. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 21 and a saline solution that includes a phosphate buffer.

44. A method of inducing exon-skipping of dystrophin exon 50, comprising administering the pharmaceutical composition of claim 40 to a subject.

45. The method of claim 44, wherein said subject is a patient with muscular dystrophy.

46. The method of claim 45, wherein said muscular dystrophy is Duchenne Muscular Dystrophy.

23. An isolated antisense oligonucleotide of 20-50 nucleotides comprising at least 17consecutive nucleotides complementary to an exon 50 target region of the Dystrophin gene designated as annealing site H50D(+06-18), wherein the oligonucleotide specifically hybridizes the exon 50 target site inducing exon 50 skipping.

24. The antisense oligonucleotide of claim 23, wherein the oligonucleotide does not activate RNase H.

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

26. The antisense oligonucleotide of claim 23, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

27. The antisense oligonucleotide of claim 26, wherein the non-natural moieties are morpholinos.

28. The antisense oligonucleotide of claim 23, wherein 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 inter-nucleotide linkages are modified phosphates.

30. The antisense oligonucleotide of claim 23, 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.

31. The antisense oligonucleotide of claim 30, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.

32. The antisense oligonucleotide of claim 31, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

33. The antisense oligonucleotide of claim 23, wherein the oligonucleotide is a 2′

-0-methyl-oligoribonucleotide.

34. The antisense oligonucleotide of claim 23, wherein the oligonucleotide is a peptide nucleic acid.

35. The antisense oligonucleotide of claim 23, 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.

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

37. The antisense oligonucleotide of claim 31, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

38. The antisense oligonucleotide of claim 37, wherein the modified phosphates are phosphoramidates.

39. The antisense oligonucleotide of claim 37, wherein the modified phosphates are phosphoromorpholidates.

41. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 23 and a saline solution that includes a phosphate buffer.

47. A method of inducing exon-skipping of dystrophin exon 50, comprising administering the pharmaceutical composition of claim 41 to a subject.