ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF

• US 20130274313A1
• Filed: 03/14/2013
• Published: 10/17/2013
• 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
• Granted Patent: US 9,175,286 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. 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:

1-212, wherein the oligonucleotide specifically hybridizes to a target region in an exon of the human dystrophin gene inducing exon skipping, and wherein uracil bases in the antisense oligonucleotide are optionally thymine bases.

2. The antisense oligonucleotide of claim 1, wherein the exon in human dystrophin is selected from the group consisting of exons 3-8, 10-16, 19-40, 42-47, and 50-53.

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, and wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates, or phosphoroamidates.

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

12. 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.

13. The antisense oligonucleotide of claim 12, wherein the oligonucleotide is conjugated to a polyamine.

14. The antisense oligonucleotide of claim 12, wherein the oligonucleotide is chemically linked to a polyethylene glycol chain.

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

17. The expression vector of claim 16, wherein the expression vector is a modified retrovirus or a non-retroviral vector.

18. The expression vector of claim 17, wherein the non-retroviral vector is an adeno-associated virus (AAV).

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

20. A method of treating Duchenne muscular dystrophy, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of claim 19.

15. An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 10 consecutive nucleotides complementary to a target region of an exon in the human dystrophin gene designated as an annealing site selected from the group set forth in Table 1A, wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon skipping.