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 51 of the dystrophin gene. The invention also relates to methods of inducing exon 51 skipping using the oligonucleotides.

1 . An isolated antisense oligonucleotide 16 to 50 nucleotides in length, wherein a part of said oligonucleotide is complementary to a strand of a double-stranded closed structure of the dystrophin exon 51 pre-mRNA and another part of said oligonucleotide is complementary to a single stranded open structure of the dystrophin exon 51 pre-mRNA, wherein said exon 51 pre-mRNA assumes a secondary structure within said exon comprising said open and closed structures, wherein said single stranded open structure is contiguous with said strand of said closed structure, wherein said oligonucleotide is capable of inducing exon 51 skipping, said oligonucleotide comprising a modification. 
     
     
         2 . The oligonucleotide of  claim 1 , wherein said single stranded open structure is contiguous with both strands of said double-stranded closed structure. 
     
     
         3 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is fully complementary to said open and closed structures. 
     
     
         4 . The oligonucleotide of  claim 1 , wherein said open and closed structures comprise a consecutive part of between 16 and 50 nucleotides of said exon and said oligonucleotide is complementary to said consecutive part. 
     
     
         5 . The oligonucleotide of  claim 4 , wherein oligonucleotide is fully complementary to said consecutive part. 
     
     
         6 . 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. 
     
     
         7 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is a 2′-O-methyl oligonucleotide. 
     
     
         8 . The oligonucleotide of  claim 7 , wherein said oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide. 
     
     
         9 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is morpholino oligonucleotide. 
     
     
         10 . The oligonucleotide of  claim 9 , wherein said oligonucleotide is a morpholino phosphorodiamidate oligonucleotide. 
     
     
         11 . The oligonucleotide of  claim 1 , wherein oligonucleotide is capable of inducing exon 51 skipping in a human myotube in culture. 
     
     
         12 . The oligonucleotide of  claim 11 , wherein said human myotube is from a Duchenne Muscular Dystrophy (DMD) patient. 
     
     
         13 . The oligonucleotide of  claim 12 , wherein said oligonucleotide is capable of inducing exon 51 skipping of the human dystrophin pre-mRNA upon transfection of said oligonucleotide into human myotubes in culture so as to induce dystrophin expression. 
     
     
         14 . The oligonucleotide of  claim 13 , wherein said transfection comprises introducing at least 100nM of said oligonucleotide into human myotubes so as to induce dystrophin expression. 
     
     
         15 . The oligonucleotide of  claim 14 , wherein said exon 51 skipping is detected by RT-PCR and/or sequence analysis. 
     
     
         16 . The oligonucleotide of  claim 14 , wherein said dystrophin expression is detected at least 16 hours after transfection by immunohistochemical and/or western blot analysis. 
     
     
         17 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is an RNA oligonucleotide. 
     
     
         18 . The oligonucleotide of  claim 1 , said oligonucleotide comprising 20 to 50 nucleotides in length. 
     
     
         19 . The oligonucleotide of  claim 1 , comprising less than 50 nucleotides in length. 
     
     
         20 . The oligonucleotide of  claim 1 , wherein said oligonucleotide comprises a greater number of purine residues than pyrimidine residues. 
     
     
         21 . The oligonucleotide of  claim 1  wherein said oligonucleotide does not bind to a splice donor and/or a splice acceptor sequence of said exon. 
     
     
         22 . A pharmaceutical composition comprising the oligonucleotide of  claim 1  and a pharmaceutically acceptable carrier.