Antisense oligonucleotides for inducing exon skipping and methods of use thereof

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.

The claims defining the invention are as follows: 
     
       1. A method of inducing skipping of exon 51 in a dystrophin gene in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier. 
     
     
       2. The method of  claim 1 , wherein the antisense oligonucleotide comprises SEQ ID NO:181. 
     
     
       3. The method of  claim 1 , wherein the antisense oligonucleotide consists of SEQ ID NO:181 and wherein the uracil bases are thymine bases. 
     
     
       4. The method of  claim 1 , wherein the subject is a human. 
     
     
       5. The method of  claim 1 , wherein the antisense oligonucleotide does not activate RNase H. 
     
     
       6. The method of  claim 1 , wherein the antisense oligonucleotide comprises a non-natural backbone. 
     
     
       7. The method of  claim 1 , wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties. 
     
     
       8. The method of  claim 7 , wherein the non-natural moieties are morpholinos. 
     
     
       9. The method of  claim 1 , wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages. 
     
     
       10. The method of  claim 9 , wherein the non-natural inter-nucleotide linkages are modified phosphates. 
     
     
       11. The method 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. 
     
     
       12. The method of  claim 11 , wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates. 
     
     
       13. The method of  claim 12 , wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates. 
     
     
       14. The method of  claim 1 , wherein the oligonucleotide is a  2 ′-O-methyl-oligoribonucleotide. 
     
     
       15. The method of  claim 1 , wherein the oligonucleotide is a peptide nucleic acid. 
     
     
       16. The method 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. 
     
     
       17. The method of  claim 1 , wherein the oligonucleotide is conjugated to a polyamine. 
     
     
       18. The method of  claim 1 , wherein the oligonucleotide is chemically linked to a polyethylene glycol chain. 
     
     
       19. A method of correcting a defective gene for dystrophin in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier. 
     
     
       20. The method of  claim 19  wherein the antisense oligonucleotide comprises SEQ ID NO:181. 
     
     
       21. The method of  claim 19 , wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases. 
     
     
       22. A method of restoring or increasing functional dystrophin protein production in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier. 
     
     
       23. The method of  claim 22 , wherein the antisense oligonucleotide comprises SEQ ID NO:181. 
     
     
       24. The method of  claim 22 , wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases. 
     
     
       25. A method of treating muscular dystrophy associated with a defective gene for dystrophin in a subject comprising administering a pharmaceutical composition comprising an antisense oligonucleotide of 30 to 50 nucleotides in length comprising SEQ ID NO: 181, wherein the uracil bases are optionally thymine bases, and a pharmaceutically acceptable carrier. 
     
     
       26. The method of  claim 25 , wherein the antisense oligonucleotide comprises SEQ ID NO:181. 
     
     
       27. The method of  claim 25 , wherein the antisense oligonucleotide consists of SEQ ID NO:181, and wherein the uracil bases are thymine bases. 
     
     
       28. The method of  claim 25 , wherein the subject is a human and the muscular dystrophy is Becker muscular dystrophy. 
     
     
       29. The method of  claim 25 , wherein the subject is a human and the muscular dystrophy is Duchenne muscular dystrophy. 
     
     
       30. The method of  claim 1 , wherein the pharmaceutical carrier is phosphate buffered saline. 
     
     
       31. The method of  claim 1 , further comprising administering a steroid to the subject. 
     
     
       32. A method of inducing skipping of exon 51 in a dystrophin gene in a human patient comprising administering an injectable solution comprising:
 an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;   and phosphate-buffered saline,   wherein the injectable solution is formulated for intravenous administration.   
     
     
       33. A method of correcting a defective gene for dystrophin in a human patient comprising administering an injectable solution comprising:
 an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;   and phosphate-buffered saline,   wherein the injectable solution is formulated for intravenous administration.   
     
     
       34. A method of restoring or increasing functional dystrophin protein production in a human patient comprising administering an injectable solution comprising:
 an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;   and phosphate-buffered saline,   wherein the injectable solution is formulated for intravenous administration.   
     
     
       35. A method of treating Duchenne muscular dystrophy in a human patient comprising administering an injectable solution comprising:
 an antisense oligonucleotide of 30 nucleotides in length comprising the base sequence 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 181), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain;   and phosphate-buffered saline,   wherein the injectable solution is formulated for intravenous administration.