Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof

• US 20120022145A1
• Filed: 10/11/2011
• Published: 01/26/2012
• 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
• Granted Patent: US 8,455,636 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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 comprising AACUGUUGCC (base positions 6-15 of SEQ ID NO:

193), wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the Dystrophin, wherein said uracil bases are optionally thymidine bases.

16. The isolated antisense oligonucleotide of claim 15, wherein said oligonucleotide has the ability to skip exon 53 at about 600 nM concentration

17. The isolated antisense oligonucleotide of claim 15, wherein said oligonucleotide is 10-50 nucleotides in length.

18. The isolated antisense oligonucleotide of claim 15, wherein said oligonucleotide is 17-30 nucleotides in length.

19. The isolated antisense oligonucleotide of claim 15, wherein said oligonucleotide induces exon skipping when tested in an assay comprising the steps:

a. preparing myoblasts obtained from differentiated human muscle biopsy cultures;

b. introducing the antisense oligonucleotide of claim 15 into the myoblasts;

c. culturing the myoblasts of (b) for at least 24 hours;

d. extracting RNA;

e. conducting reverse-transcriptase polymerase chain reaction; and

f. analyzing the targeted regions of the dystrophin pre-mRNA for induced exonic rearrangements.

20. The oligonucleotide of claim 15, further comprising an expression vector.

21. The oligonucleotide of claim 20, wherein said expression vector is a modified retrovirus or a non-retroviral vector.

22. The oligonucleotide of claim 21, wherein said non-retroviral vector is an adeno-associated virus (AAV).

23. The oligonucleotide of claim 15, wherein said oligonucleotide is 100% complementary to the target region.

24. The oligonucleotide of claim 15, where said oligonucleotide has been modified to minimize or prevent cleavage by RNaseH.

25. A method of inducing exon-skipping of dystrophin exon 53, comprising introducing a nucleic acid molecule into a cell by way of an expression vector, wherein the nucleic acid molecule is an isolated antisense oligonucleotide comprising AACUGUUGCC (base positions 6-15 of SEQ ID NO:

193), wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the Dystrophin gene, wherein said uracil bases are optionally thymidine bases.

26. The method of claim 25, wherein said oligonucleotide has the ability to skip exon 53 at about 600 nM concentration.

27. The method of claim 25, wherein said expression vector is a modified retrovirus or a non-retroviral vector.

28. The method of claim 27, wherein said non-retroviral vector is an adeno-associated virus (AAV).

29. The method of claim 25, wherein the cell is a human muscle cell.

30. The method of claim 29, wherein said human muscle cell is in a patient.

31. The method of claim 30, wherein said patient has muscular dystrophy.

32. The method of claim 31, wherein said muscular dystrophy is Duchenne Muscular Dystrophy.

33. The method of claim 25, wherein said oligonucleotide is 100% complementary to the target region.

34. The method of claim 25, wherein said nucleic acid molecule and expression vector are delivered to the cell in a colloidal dispersion system.

35. An isolated antisense oligonucleotide comprising CCUUGGUUUC (base positions 7-16 of SEQ ID NO:

194) wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the Dystrophin, wherein said uracil bases are optionally thymidine bases.

36. A method of inducing exon-skipping of dystrophin exon 53, comprising introducing a nucleic acid molecule into a cell by way of an expression vector, wherein the nucleic acid molecule is an isolated antisense oligonucleotide comprising CCUUGGUUUC (base positions 7-16 of SEQ ID NO:

194) wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the Dystrophin gene, wherein said uracil bases are optionally thymidine bases.

37. A method of treating Duchenne muscular dystrophy, comprising administering to a patient in need thereof an effective amount of an isolated antisense oligonucleotide comprising AACUGUUGCC (base positions 6-15 of SEQ ID NO:

193), wherein the uracil bases are optionally thymidine bases.

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

39. The method of claim 37, wherein said antisense oligonucleotide is administered as a colloidal dispersion system.

40. The method of claim 39, further comprising administering a steroid to said patient.

41. A method of treating Duchenne muscular dystrophy, comprising administering to a patient in need thereof an effective amount of an isolated antisense oligonucleotide comprising AACUGUUGCC (base positions 6-15 of SEQ ID NO:

193), wherein the oligonucleotide specifically hybridizes to an exon 53 target region of a Dystrophin gene and induces exon-skipping of dystrophin exon 53, and wherein the uracil bases are optionally thymidine bases.

42. The method of claim 41, wherein the patient is a human.

43. The method of claim 41, wherein said antisense oligonucleotide is administered as a colloidal dispersion system.

44. The method of claim 43, further comprising administering a steroid to said patient.