ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF

• US 20110263686A1
• Filed: 06/24/2011
• Published: 10/27/2011
• Est. Priority Date: 06/28/2004
• Status: Abandoned Application

• Current Assignee: University of Western Australia
• Original Assignee: University of Western Australia
• Inventors: MCCLOREY, GRAHAM, FLETCHER, SUE, WILTON, STEPHEN DONALD
• Application Number:
  US13/168,857
• Publication Number:
  US 20110263686A1
• Time in Patent Office: Days
• US Class Current: 514/44.00A
• 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

1-14. -14. (canceled)

15. An isolated antisense oligonucleotide of 10 to 50 nucleotides in length comprising a sequence that is specifically hybridizable to an exon 45 target region of the Dystrophin gene designated as annealing site H45A(−

6+20), H45A(+91+110), H45A(+125+151), H45D(+16−

04), H45A(+71+90) or a combination thereof.

16. The antisense oligonucleotide of claim 15, comprising SEQ ID NO:

207, SEQ ID NO;

208, SEQ ID NO;

209, SEQ ID NO;

210 or SEQ ID NO;

211 wherein the uracil bases are optionally thymine bases.

17. The antisense oligonucleotide of claim 16, wherein the uracil bases are thymine bases.

18. The antisense oligonucleotide of claim 15, which is 20 to 31 nucleotides in length.

19. The antisense oligonucleotide of claim 15, which is 25 to 31 nucleotides in length.

20. The antisense oligonucleotide of claim 15, wherein the oligonucleotide does not activate RNase H.

21. The antisense oligonucleotide of claim 15, comprising a non-natural backbone.

22. The antisense oligonucleotide of claim 21, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.

23. The antisense oligonucleotide of claim 22, wherein the non-natural moieties are morpholinos.

24. The antisense oligonucleotide of claim 15, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

25. The antisense oligonucleotide of claim 24, wherein the non-natural inter-nucleotide linkages are modified phosphates.

26. The antisense oligonucleotide of claim 25, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.

27. The antisense oligonucleotide of claim 26, wherein the modified phosphates are phosphoramidates.

28. The antisense oligonucleotide of claim 26, wherein the modified phosphates are phosphoromorpholidates.

29. The antisense oligonucleotide of claim 15, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the internucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.

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

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

32. The antisense oligonucleotide of claim 31, wherein the modified phosphates are phosphoramidates.

33. The antisense oligonucleotide of claim 31, wherein the modified phosphates are phosphoromorpholidates.

34. The antisense oligonucleotide of claim 15, wherein the oligonucleotide is a 2′

-O-methyl-oligoribonucleotide.

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

36. The antisense oligonucleotide of claim 15, 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.

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

38. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide consists of the sequence identified as SEQ ID NO:

207, SEQ ID NO;

208, SEQ ID NO;

209, SEQ ID NO;

210 or SEQ ID NO;

211, wherein the uracil bases are optionally thymine bases.

39. The antisense oligonucleotide of claim 38, wherein the uracil bases are thymine bases.

40. A pharmaceutical composition, comprising the antisense oligonucleotide of claim 15 and a pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 40, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline.

42. A method of inducing exon-skipping of dystrophin exon 45 comprising delivering an antisense oligonucleotide of claim 15 to a cell, thereby inducing exon-skipping of the dystrophin exon.

43. The method of claim 42, wherein the cell is a human muscle cell.

44. The method of claim 43, wherein the human muscle cell is in a patient.

45. The method of claim 44, wherein the patient has muscular dystrophy.

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

47. A pharmaceutically acceptable salt of the antisense oligonucleotide of claim 15.