Methods of Inducing Exon Skipping

Abstract

Oligonucleotides for inducing exon skipping, including exon 51 of the dystrophin gene. Compositions are disclosed for treating Duchenne Muscular Dystrophy. Disclosed structures include: (1) h51AON1 (SEQ ID NO: 27; UCAA GGAA GAUG GCAU UUCU), which is 20 bases long, (2) h51AON2 (SEQ ID NO: 28; CCUC UGUG AUUU UAUA ACUU GAU), which is 23 bases long, and (3) the combination of h51AON2 and h45AON5 linked by 10 uracils (i.e., SEQ ID NO: 28 (CCUC UGUG AUUU UAUA ACUU GAU) linked to SEQ ID NO: 16 (GCCC AAUG CCAU CCUG G) by UUUU UUUU UU), which combination is 50 bases long.

What is claimed is: 
     
         1 . A composition comprising:
 means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and   a backbone covalently associated with said means for inducing, wherein the backbone comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate.   
     
     
         2 . A composition according to  claim 1 , wherein the backbone is selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′-O-methyl phosphorothioate backbone. 
     
     
         3 . A composition according to  claim 2 , wherein said means for inducing skipping includes means for binding to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA. 
     
     
         4 . A composition according to  claim 2 , wherein said means for inducing skipping includes means for binding without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA. 
     
     
         5 . A composition according to  claim 4 , wherein the means for inducing skipping is operative in a human muscle cell. 
     
     
         6 . A composition according to  claim 2 , wherein the composition includes an antisense oligonucleotide that is twenty bases in length. 
     
     
         7 . A composition according to  claim 2 , wherein the composition includes an antisense oligonucleotide that is twenty-three bases in length. 
     
     
         8 . A composition according to  claim 2 , wherein composition includes an antisense oligonucleotide that is fifty bases in length. 
     
     
         9 . An oligonucleotide comprising:
 means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and   means for providing the oligonucleotide with resistance to RNaseH.   
     
     
         10 . An oligonucleotide according to  claim 9 , wherein the means for providing the oligonucleotide with resistance to RNaseH comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate. 
     
     
         11 . An oligonucleotide according to  claim 9 , wherein the means for providing the oligonucleotide with resistance to RNaseH comprises a backbone selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′-O-methyl phosphorothioate backbone. 
     
     
         12 . An oligonucleotide according to  claim 11 , wherein said means for inducing skipping includes means for binding to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA. 
     
     
         13 . An oligonucleotide according to  claim 11 , wherein said means for inducing skipping includes means for binding without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA. 
     
     
         14 . An oligonucleotide according to  claim 13 , wherein the means for inducing skipping is operative in a human muscle cell. 
     
     
         15 . An oligonucleotide according to  claim 11 , wherein the oligonucleotide is twenty bases in length. 
     
     
         16 . An oligonucleotide according to  claim 11 , wherein the oligonucleotide is twenty-three bases in length. 
     
     
         17 . An oligonucleotide according to  claim 11 , wherein the oligonucleotide is fifty bases in length. 
     
     
         18 . An oligonucleotide of between 20 to 50 nucleotides comprising a sequence containing h51AON1 (UCAAG GAAGA UGGCA UUUCU) (SEQ ID NO: 27) or the DNA thereof,
 wherein said oligonucleotide binds without mismatches to the complementary sequence of exon 51 of the human dystrophin pre-mRNA, and   wherein said oligonucleotide further comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate.   
     
     
         19 . An oligonucleotide according to  claim 18 , wherein the oligonucleotide is selected from the group consisting of a morpholino phosphorodiamidate and a 2′-O-methyl phosphorothioate. 
     
     
         20 . An oligonucleotide according to  claim 19 , wherein the means for inducing skipping is operative in a human muscle cell. 
     
     
         21 . A morpholino phosphorodiamidate antisense oligonucleotide of 20 to 50 nucleotides in length, wherein said oligonucleotide binds without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA. 
     
     
         22 . An oligonucleotide according to  claim 21 , wherein the means for inducing skipping is operative in a human muscle cell.