Modulation of exon recognition in pre-mrna by interfering with the secondary rna structure

Abstract

The invention relates to oligonucleotides for inducing skipping of exon 53 of the dystrophin gene. The invention also relates to methods of inducing exon 53 skipping using the oligonucleotides.

We claim: 
     
         1 . An isolated antisense oligonucleotide 15 to 80 nucleotides in length, wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA in a muscle cell, said oligonucleotide comprising a modification. 
     
     
         2 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is 18 to 80 nucleotides in length. 
     
     
         3 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is complementary to exon 53 of the human dystrophin pre-mRNA. 
     
     
         4 . An isolated antisense oligonucleotide of 18 to 50 nucleotides in length, wherein said oligonucleotide binds to an exon-internal sequence of exon 53 of the human dystrophin pre-mRNA and induces skipping of said exon in a muscle cell, said oligonucleotide comprising a modification. 
     
     
         5 . The oligonucleotide of  claim 4 , wherein said exon-internal sequence comprises a consecutive part of between 16 and 50 nucleotides of said exon and said oligonucleotide is complementary to said consecutive part. 
     
     
         6 . An antisense oligonucleotide of 18 to 50 nucleotides in length, wherein said oligonucleotide is complementary to a consecutive part of between 16 and 50 nucleotides of an exon-internal sequence of exon 53 of said human dystrophin pre-mRNA and induces skipping of exon 53 in a muscle cell. 
     
     
         7 . The oligonucleotide of  claim 1 , wherein said muscle cell is from a DMD patient. 
     
     
         8 . The oligonucleotide of  claim 1 , wherein said modification is selected from the group consisting of: 2′-O-methyl, 2′-O-methyl-phosphorothioate, a morpholine ring, a phosphorodiamidate linkage, a modification to increase resistance to RNAseH, a peptide nucleic acid and a locked nucleic acid. 
     
     
         9 . The oligonucleotide of  claim 1 , wherein said modification consists of 2′-O-methyl or 2′-O-methyl phosphorothioate. 
     
     
         10 . The oligonucleotide of  claim 1 , wherein said modification comprises a morpholine ring and a phosphorodiamidate linkage. 
     
     
         11 . The oligonucleotide of  claim 1 , which is a morpholino phosphorodiamidate oligoribonucleotide. 
     
     
         12 . The oligonucleotide of  claim 1 , wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate linkage. 
     
     
         13 . The oligonucleotide of  claim 1 , wherein said oligonucleotide induces exon 53 skipping of the human dystrophin pre-mRNA and induces dystrophin expression in the muscle cell upon transfection of human muscle cells with at least 100 nM of said oligonucleotide and incubation for at least 16 hours. 
     
     
         14 . The oligonucleotide of  claim 13 , wherein exon 53 skipping is detected by RT-PCR and/or sequence analysis. 
     
     
         15 . The oligonucleotide of  claim 13 , wherein dystrophin expression in said muscle cell is detected by immunohistochemical and/or western blot analysis. 
     
     
         16 . The oligonucleotide of  claim 1 , wherein the bases of the nucleotides of said oligonucleotide consist of DNA bases or consist of RNA bases. 
     
     
         17 . The oligonucleotide of  claim 1 , said oligonucleotide consisting of RNA. 
     
     
         18 . The oligonucleotide of  claim 1 , said oligonucleotide being less than 50 nucleotides in length. 
     
     
         19 . The oligonucleotide of  claim 1 , said oligonucleotide being less than 80 nucleotides in length. 
     
     
         20 . The oligonucleotide of  claim 4 , wherein said oligonucleotide is capable of binding without mismatches to said exon-internal sequence. 
     
     
         21 . The oligonucleotide of  claim 1 , wherein said oligonucleotide binds to a purine rich sequence. 
     
     
         22 . The oligonucleotide of  claim 1 , wherein said oligonucleotide binds to an ERS or a SR protein binding site. 
     
     
         23 . The oligonucleotide of  claim 1 , wherein said oligonucleotide does not bind to a splice donor and/or a splice acceptor sequence of said exon. 
     
     
         24 . The oligonucleotide of  claim 1 , wherein at least a part of said oligonucleotide is complementary to a region of the dystrophin exon 53 pre-mRNA that assumes a structure that is hybridized to another part of said pre-mRNA (closed structure) and wherein at least a part of said oligonucleotide is complementary to a region of a pre-mRNA of an exon that is not hybridized (open structure). 
     
     
         25 . A pharmaceutical composition comprising the oligonucleotide of  claim 1 . 
     
     
         26 . A method for inducing the skipping exon 53 of the human dystrophin pre-mRNA in a subject with Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD), or a cell of said subject, said method comprising providing the oligonucleotide of  claim 1 , to said subject or said cell, wherein said oligonucleotide induces skipping of said exon in said subject or said cell and wherein mRNA produced from skipping exon 53 of the dystrophin pre-mRNA encodes a functional dystrophin protein. 
     
     
         27 . A method for treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in a subject by inducing skipping of exon 53 of the human dystrophin pre-mRNA, said method comprising providing the oligonucleotide of  claim 1 , wherein said oligonucleotide induces skipping of said exon in said subject.