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 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.
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.
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 .