Antisense oligonucleotides for inducing exon skipping and methods of use thereof

Abstract

Antisense molecules capable of binding to a selected target site in the dystrophin gene to induce exon skipping are described.

1 .- 20 . (canceled) 
     
     
         21 . An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 20 nucleotides of SEQ ID NO:193, wherein at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base and uracil bases are optionally thymine bases, and wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the human dystrophin gene inducing exon 53 skipping. 
     
     
         22 . The antisense oligonucleotide of  claim 21 , wherein the pyrimidine base is cytosine. 
     
     
         23 . The antisense oligonucleotide of  claim 21 , wherein the pyrimidine base is uracil. 
     
     
         24 . The antisense oligonucleotide of  claim 21 , wherein the pyrimidine base is thymine. 
     
     
         25 . The antisense oligonucleotide of  claim 21 , wherein the 5-substituted pyrimidine base is selected from the group consisting of 5-propynyluracil and 5-propynylcytosine. 
     
     
         26 . The antisense oligonucleotide of  claim 21 , wherein the 5-substituted pyrimidine base is 5-methylcytosine. 
     
     
         27 . The antisense oligonucleotide of  claim 21  comprising 20-31 nucleotides in length. 
     
     
         28 . The antisense oligonucleotide of  claim 21  comprising a non-natural backbone. 
     
     
         29 . The antisense oligonucleotide of  claim 28 , wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties. 
     
     
         30 . The antisense oligonucleotide of  claim 29 , wherein the non-natural moieties are morpholinos and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages. 
     
     
         31 . The antisense oligonucleotide of  claim 30 , wherein the non-natural inter-nucleotide linkages are modified phosphates. 
     
     
         32 . The antisense oligonucleotide of  claim 31 , wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates. 
     
     
         33 . The antisense oligonucleotide of  claim 21 , wherein the oligonucleotide is a 2′-β-methyl-oligoribonucleotide. 
     
     
         34 . The antisense oligonucleotide of  claim 21 , 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. 
     
     
         35 . The antisense oligonucleotide of  claim 34 , wherein the oligonucleotide is conjugated to a polyamine. 
     
     
         36 . The antisense oligonucleotide of  claim 34 , wherein the oligonucleotide is chemically linked to a polyethylene glycol chain. 
     
     
         37 . An isolated antisense oligonucleotide of 20 to 50 nucleotides in length comprising at least 20 nucleotides of SEQ ID NO:193, wherein at least one purine base of the oligonucleotide comprises an N-2, N-6 substituted purine base and uracil bases are optionally thymine bases, and wherein the oligonucleotide specifically hybridizes to an exon 53 target region of the human dystrophin gene inducing exon 53 skipping. 
     
     
         38 . The antisense oligonucleotide of  claim 37  comprising 20-31 nucleotides in length. 
     
     
         39 . The antisense oligonucleotide of  claim 37  comprising a non-natural backbone. 
     
     
         40 . The antisense oligonucleotide of  claim 39 , wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties. 
     
     
         41 . The antisense oligonucleotide of  claim 40 , wherein the non-natural moieties are morpholinos and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages. 
     
     
         42 . The antisense oligonucleotide of  claim 41 , wherein the non-natural inter-nucleotide linkages are modified phosphates. 
     
     
         43 . The antisense oligonucleotide of  claim 42 , wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates. 
     
     
         44 . The antisense oligonucleotide of  claim 37 , wherein the oligonucleotide is a 2′-β-methyl-oligoribonucleotide. 
     
     
         45 . The antisense oligonucleotide of  claim 37 , 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. 
     
     
         46 . The antisense oligonucleotide of  claim 45 , wherein the oligonucleotide is conjugated to a polyamine. 
     
     
         47 . The antisense oligonucleotide of  claim 45 , wherein the oligonucleotide is chemically linked to a polyethylene glycol chain. 
     
     
         48 . A composition comprising the antisense oligonucleotide of  claim 21  and a pharmaceutically acceptable carrier. 
     
     
         49 . A composition comprising the antisense oligonucleotide of  claim 37  and a pharmaceutically acceptable carrier.