Antisense oligonucleotides for inducing exon skipping and methods of use thereof
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.
1. An isolated antisense oligonucleotide that is specifically hybridizable to an exon 53 target region of the Dystrophin gene designated as annealing site H53A (+23+47), wherein the antisense oligonucleotide consists of the sequence identified as SEQ ID NO: 195, wherein the uracil bases are optionally thymine bases.
2. The antisense oligonucleotide of claim 1 , wherein the uracil bases are thymine bases.
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.
11. The antisense oligonucleotide of claim 10 , wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.
12. The antisense oligonucleotide of claim 1 , wherein the oligonucleotide is a 2′-O-methyl-oligoribonucleotide.
13. The antisense oligonucleotide of claim 1 , wherein the oligonucleotide is a peptide nucleic acid.
14. 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.
15. The antisense oligonucleotide of claim 14 , wherein the oligonucleotide is chemically linked to a polyethylene glycol molecule.
16. A pharmaceutical composition, comprising an antisense oligonucleotide of claim 1 and a saline solution that includes a phosphate buffer.
17. The antisense oligonucleotide of claim 8 , wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.
18. The antisense oligonucleotide of claim 17 , wherein the modified phosphates are phosphoramidates.
19. The antisense oligonucleotide of claim 17 , wherein the modified phosphates are phosphoromornpholidates.
20. A method of inducing exon-skipping of a dystrophin exon 53, comprising delivering an antisense oligonucleotide that is specifically hybridizable to an exon 53 target region of the dystrophin gene designated as annealing site H53A (+23+47), wherein the antisense oligonucleotide consists of the sequence identified as SEQ ID NO: 195, wherein the uracil bases are optionally thymine bases, thereby inducing exon-skipping of the dystrophin exon.
21. The method of claim 20 , 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 11 , wherein the patient has muscular dystrophy.
24. The method of claim 23 , wherein the muscular dystrophy is Duchenne Muscular Dystrophy.
25. The method of claim 20 , wherein the uracil bases are thymine bases.