Compositions and methods for correcting limb girdle muscular dystrophy type 2C using exon skipping

• US 10,273,483 B2
• Filed: 04/07/2016
• Issued: 04/30/2019
• Est. Priority Date: 04/08/2015
• Status: Active Grant

• Current Assignee: Northwestern University, University of Chicago
• Original Assignee: Northwestern University, University of Chicago
• Inventors: McNally, Elizabeth, Wyatt, Eugene
• Primary Examiner(s): Gibbs, Terra C
• Application Number:
  US15/564,681
• Publication Number:
  US 20180080030A1
• Time in Patent Office: 1,118 Days
• 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 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

The invention is directed to one or more antisense polynucleotides and their use in pharmaceutical compositions in a strategy to induce exon skipping in the γ-sarcoglycan gene in patients suffering from Limb-Girdle Muscular Dystrophy-2C (LGM-D2C) or in patients at risk of such a disease. The invention also provides methods of preventing or treating muscular dystrophy, e.g., LGMD2C, by exon skipping in the gamma sarcoglycan gene using antisense polynucleotides. Accordingly, in some aspects the invention provides an isolated antisense oligonucleotide, wherein the oligonucleotide specifically hybridizes to an exon target region of a γ-sarcoglycan RNA. In another aspect, the invention provides a method of inducing exon-skipping of a gamma sarcoglycan RNA, comprising delivering an antisense oligonucleotide or a composition to a cell.

Claims

1. A modified antisense oligonucleotide (AON) selected from the group consisting of oligonucleotides having a sequence as set out in SEQ ID NOs:

4-9, 11-12, 14-29, and 31-34.

2. The antisense oligonucleotide of claim 1, wherein the oligonucleotide cannot form an RNase H substrate.

3. The antisense oligonucleotide of claim 1, comprising a modified oligonucleotide backbone.

4. The antisense oligonucleotide of claim 3, wherein the modified oligonucleotide backbone comprises a modified moiety substituted for the sugar.

5. The antisense oligonucleotide of claim 4, wherein the modified moiety is a Morpholino.

6. The antisense oligonucleotide of claim 3, wherein the modified oligonucleotide backbone comprises at least one modified internucleotide linkage.

7. The antisense oligonucleotide of claim 6, wherein the modified internucleotide linkage is a tricyclo-DNA (tc-DNA) modification.

8. The antisense oligonucleotide of claim 6, wherein the modified internucleotide linkage comprises a modified phosphate.

9. The antisense oligonucleotide of claim 8, wherein the modified phosphate is selected from the group consisting of a methyl phosphonate, a methyl phosphorothioate, a phosphoromorpholidate, a phosphoropiperazidate and a phosphoroamidate.

10. The antisense oligonucleotide of claim 3, wherein the oligonucleotide is a 2′

-O-methyl-oligoribonucleotide.

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

12. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is chemically linked to one or more 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 chemically linked to a polyethylene glycol molecule.

14. The antisense oligonucleotide of claim 12 wherein the conjugate is a peptide that enhances cellular uptake.

15. The antisense oligonucleotide of claim 14 wherein the peptide is selected from the group consisting of a nuclear localization signal (NLS), HIV-1 TAT protein, a peptide comprising an integrin binding domain, oligolysine, adenovirus fiber protein and a peptide comprising a receptor-mediated endocytosis (RME) domain.

16. A method of inducing exon-skipping of a gamma sarcoglycan RNA, comprising delivering to a cell the antisense oligonucleotide of claim 1, thereby inducing exon-skipping of the gamma sarcoglycan RNA.

17. The method of claim 16, wherein the cell is a human muscle cell.

18. A kit comprising the modified antisense oligonucleotide of claim 1, optionally in a container, and a package insert, package label, instructions or other labeling.

19. The kit of claim 18, further comprising an additional modified oligonucleotide, wherein the additional modified oligonucleotide specifically hybridizes to an exon in a gamma sarcoglycan RNA.

20. The composition of claim 1, further comprising a physiologically compatible buffer.

21. The method of claim 16, wherein the cell is a human muscle cell.

22. The method of claim 21, wherein the human muscle cell is in a patient.

23. The method of claim 22, wherein the patient has muscular dystrophy.

24. The method of claim 23, wherein the muscular dystrophy is Limb Girdle Muscular Dystrophy type 2C (LGMD2C).