Multiple exon skipping compositions for DMD

• US 8,865,883 B2
• Filed: 03/14/2013
• Issued: 10/21/2014
• Est. Priority Date: 10/24/2008
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Sazani, Peter, Kole, Ryszard
• Primary Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US13/830,253
• Publication Number:
  US 20130190390A1
• Time in Patent Office: 586 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: A61P 21/04 for myasthenia gravis
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Provided are antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping, and methods of use thereof to treat muscular dystrophy.

Claims

1. An isolated antisense oligonucleotide of 20 to 35 nucleotides in length comprising at least 17 contiguous nucleotides of a nucleotide sequence set forth as SEQ ID NO:

2 wherein the oligonucleotide is capable of binding to human dystrophin pre-mRNA to induce exon 44 skipping, and wherein the oligonucleotide comprises a modification to resist degradation of an oligonucleotide;

RNA heteroduplex by RNase H, and thymine bases (T) are optionally uracil bases (U).

2. The antisense oligonucleotide of claim 1, which is substantially uncharged.

3. The antisense oligonucleotide of claim 2, which comprises morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

4. The antisense oligonucleotide of claim 2, which comprises morpholino subunits linked by substantially uncharged phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

5. The antisense oligonucleotide of claim 4, wherein 3-35% of the linkages are positively charged.

6. The antisense oligonucleotide of claim 3, wherein the intersubunit linkages are uncharged and interspersed with linkages that are positively charged at physiological pH, wherein the total number of positively charged linkages is between 2 and no more than half of the total number of linkages.

7. The antisense oligonucleotide of claim 4, wherein the intersubunit linkages are uncharged and interspersed with linkages that are positively charged at physiological pH, wherein the total number of positively charged linkages is between 2 and no more than half of the total number of linkages.

8. The antisense oligonucleotide of claim 1, comprising morpholino subunits and phosphorodiamidate intersubunit linkages.

9. The antisense oligonucleotide of claim 3, wherein at least one and up to 50% of the intersubunit linkages are modified with a pendent cationic group.

10. The antisense oligonucleotide of claim 4, wherein at least one and up to 50% of the intersubunit linkages are modified with a pendent cationic group.

11. The antisense oligonucleotide of claim 1, which is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.

12. The antisense oligonucleotide of claim 11, which is conjugated to a polyamine.

13. The antisense oligonucleotide of claim 11, which is chemically linked to polyethylene glycol.

14. The antisense oligonucleotide of claim 11, which is conjugated to an arginine-rich peptide.

15. The antisense oligonucleotide of claim 1 which specifically hybridizes to a target region in exon 44 and the oligonucleotide sequence consists of SEQ ID NO:

2.

16. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 1, and a pharmaceutically acceptable carrier.

17. A method of treating muscular dystrophy, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the muscular dystrophy is Duchenne'"'"'s muscular dystrophy (DMD or Becker'"'"'s muscular dystrophy (BMD).

19. The antisense oligonucleotide of claim 1, having uracil bases (U) in place of thymine bases (T).

20. The antisense oligonucleotide of claim 1, comprising a 5-methylcytosine base.

21. The antisense oligonucleotide of claim 14, wherein the arginine-rich peptide comprises a sequence selected from SEQ ID NOs:

570-578.