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.

1 . An antisense oligonucleotide of 16 to 80 nucleotides in length, wherein said oligonucleotide is complementary to a consecutive part of between 16 and 50 nucleotides of an sequence internal to exon 55 of the human dystrophin pre-mRNA and said oligonucleotide is capable of inducing skipping of exon 55 in a muscle cell, said oligonucleotide comprising a modification. 
     
     
         2 . The antisense oligonucleotide of  claim 1 , wherein said oligonucleotide is 16 to 50 nucleotides in length. 
     
     
         3 . The oligonucleotide of  claim 3 , wherein said oligonucleotide is 16 to 24 nucleotides in length. 
     
     
         4 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises a 2′-O-methyl-phosphorothioate oligonucleotide modification. 
     
     
         5 . The oligonucleotide of  claim 1 , wherein the oligonucleotide consists of a 2′-O-methyl oligonucleotide or a 2′-O-methyl-phosphorothioate oligonucleotide. 
     
     
         6 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises a morpholine ring. 
     
     
         7 . The oligonucleotide of  claim 6 , wherein the oligonucleotide further comprises a phosphorodiamidate linkage. 
     
     
         8 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises a peptide nucleic acid and/or locked nucleic acid. 
     
     
         9 . The oligonucleotide of  claim 2 , wherein said oligonucleotide is fully complementary to said consecutive part. 
     
     
         10 . The oligonucleotide of  claim 9 , wherein the oligonucleotide comprises a 2′-O-methyl-phosphorothioate oligonucleotide modification. 
     
     
         11 . The oligonucleotide of  claim 9 , wherein the oligonucleotide consists of a 2′-O-methyl oligonucleotide or a 2′-O-methyl-phosphorothioate oligonucleotide. 
     
     
         12 . The oligonucleotide of  claim 9 , wherein the oligonucleotide comprises a morpholine ring and a phosphorodiamidate linkage. 
     
     
         13 . The oligonucleotide of  claim 9 , wherein the oligonucleotide comprises a peptide nucleic acid and/or locked nucleic acid. 
     
     
         14 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is capable of inducing skipping of said exon in primary human myotubes upon transfection of at least 100 nM of said oligonucleotide to said myotubes and incubation of transfected myotubes for at least 16 hours. 
     
     
         15 . The oligonucleotide of  claim 14 , wherein skipping is detectable by RT-PCR and nucleic acid sequencing. 
     
     
         16 . An expression vector encoding an oligonucleotide according to  claim 1 . 
     
     
         17 . A gene delivery vehicle comprising the expression vector of  claim 16 . 
     
     
         18 . The oligonucleotide of  claim 1 , wherein said consecutive part comprises a sequence which assumes both a double-stranded stretch and a single-stranded stretch of nucleotides, said double-stranded stretch comprising two single-stranded nucleic acid stretches of exon 55 that form a duplex. 
     
     
         19 . A method for inducing skipping of exon 55 of the human dystrophin pre-mRNA in a muscle cell in vitro or in vivo, the method comprising contacting said cell or administering to a human subject comprising said cell an oligonucleotide according to  claim 1  for a time and under conditions which permit induction of exon 55 skipping.