Methods of Inducing Exon Skipping

Abstract

Methods for inducing skipping of exons, including exon 51 of the dystrophin gene. Oligonucleotides are used for inducing exon skipping and 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 method for treating Duchenne Muscular Dystrophy, said method comprising:
 administering to a DMD patient a composition including:
 means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and 
 a backbone covalently associated with said means for inducing. 
   
     
     
         2 . A method according to  claim 1 , wherein the backbone comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate. 
     
     
         3 . A method 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. 
     
     
         4 . A method according to  claim 3 , 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. 
     
     
         5 . A method according to  claim 3 , 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. 
     
     
         6 . A method according to  claim 2 , wherein the composition includes an antisense oligonucleotide that is twenty bases in length. 
     
     
         7 . A method according to  claim 2 , wherein the composition includes an antisense oligonucleotide that is twenty-three bases in length. 
     
     
         8 . A method according to  claim 2 , wherein the composition includes an antisense oligonucleotide that is fifty bases in length. 
     
     
         9 . A method for treating Duchenne Muscular Dystrophy, said method comprising:
 administering to a DMD patient an oligonucleotide including:
 means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and 
 means for providing the oligonucleotide with resistance to RNaseH. 
   
     
     
         10 . A method 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 . A method 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 . A method 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 . A method 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 . A method according to  claim 10 , wherein the oligonucleotide is twenty bases in length. 
     
     
         15 . A method according to  claim 10 , wherein the oligonucleotide is twenty-three bases in length. 
     
     
         16 . A method according to  claim 10 , wherein the oligonucleotide is fifty bases in length. 
     
     
         17 . A method for inducing the skipping of exon 51 of the human dystrophin pre-mRNA, said method comprising:
 providing a composition including:
 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; and 
 a backbone covalently associated with said means for binding. 
   
     
     
         18 . A method according to  claim 17 , wherein the backbone comprises a chemical modification selected from the group consisting morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate. 
     
     
         19 . A method according to  claim 18 , wherein said composition is operative in a human muscle cell. 
     
     
         20 . A method according to  claim 17 , wherein the backbone is selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′-O-methyl phosphorothioate backbone. 
     
     
         21 . A method according to  claim 20 , wherein said composition is operative in a human muscle cell. 
     
     
         22 . A method for inducing the skipping of exon 51 of the human dystrophin pre-mRNA, said method comprising:
 providing a composition including 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.   
     
     
         23 . A method according to  claim 22 , wherein said oligonucleotide further comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′-O-methyl phosphorothioate. 
     
     
         24 . A method according to  claim 22 , wherein said oligonucleotide is selected from the group consisting of a morpholino phosphorodiamidate and a 2′-O-methyl phosphorothioate. 
     
     
         25 . A method according to  claim 22 , wherein said composition is operative in a human muscle cell.