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 55 of the dystrophin gene. The invention also relates to methods of inducing exon 55 skipping using the oligonucleotides.

1 - 3 . (canceled) 
     
     
         4 . An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, wherein the oligonucleotide binds to said exon 55 and induces skipping of said exon. 
     
     
         5 . An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, and wherein the oligonucleotide binds to exon 55, interferes with the structure of said exon and induces skipping of said exon. 
     
     
         6 . An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, and wherein the oligonucleotide binds to said exon, masks said exon from the splicing apparatus and induces skipping of said exon. 
     
     
         7 . The oligonucleotide of  claim 4 , wherein the oligonucleotide interferes with one or more components of the splicing machinery so that the exon is no longer recognized by the splicing machinery. 
     
     
         8 . The oligonucleotide of  claim 7 , wherein the one or more components of the splicing machinery is an SR protein. 
     
     
         9 . The oligonucleotide of  claim 4 , which is fully complementary to exon 55 of the human dystrophin pre-mRNA. 
     
     
         10 . The oligonucleotide of  claim 4 , wherein the oligonucleotide comprises a modification. 
     
     
         11 . The oligonucleotide of  claim 10 , wherein the oligonucleotide comprises a 2′-O-methyl-phosphorothioate oligoribonucleotide modification. 
     
     
         12 . The oligonucleotide of  claim 10 , wherein the oligonucleotide consists of a 2′-O-methyl oligonucleotide or a 2′-O-methyl-phosphorothioate oligonucleotide. 
     
     
         13 . The oligonucleotide of  claim 10 , wherein the oligonucleotide comprises a morpholino ring. 
     
     
         14 . The oligonucleotide of  claim 13 , wherein the oligonucleotide comprises a morpholino ring and a phosphorodiamidate linkage. 
     
     
         15 . The oligonucleotide of  claim 10 , wherein the oligonucleotide comprises a peptide nucleic acid and/or locked nucleic acid oligomer. 
     
     
         16 . The oligonucleotide of  claim 10 , wherein the modification comprises a base substitution of a U by a T. 
     
     
         17 . The oligonucleotide of  claim 4 , wherein the oligonucleotide induces skipping of exon 55 of the human dystrophin pre-mRNA and dystrophin expression at the myotube membrane upon transfection of primary human myotubes with at least 100 nM of said oligonucleotide and incubation for at least 16 hours. 
     
     
         18 . The oligonucleotide of  claim 17 , wherein skipping of exon 55 is detected by RT-PCR and sequence analysis. 
     
     
         19 . The oligonucleotide of  claim 17 , wherein dystrophin expression at the myotube is detected by immunohistochemical or western blot analysis. 
     
     
         20 . An expression vector encoding the oligonucleotide of  claim 4 . 
     
     
         21 . A gene delivery vehicle comprising the expression vector of  claim 20 . 
     
     
         22 . The gene delivery vehicle of  claim 21 , wherein the vehicle is a viral vector. 
     
     
         23 . A method of altering recognition of exon 55 in a human dystrophin pre-mRNA, by the splicing machinery, said method comprising:
 combining the pre-mRNA with the oligonucleotide of  claim 4  under conditions wherein recognition of the exon by the splicing machinery is altered;   and altering recognition of exon 55 in the pre-mRNA, wherein alteration of the recognition of exon 55 by the splicing machinery is detected by RT-PCR and sequence analysis.   
     
     
         24 . A method of inducing skipping of exon 55 of the human dystrophin pre-mRNA, said method comprising:
 combining the pre-mRNA with the oligonucleotide of  claim 4  under conditions wherein skipping of exon 55 can occur;   and inducing the skipping of exon 55, wherein skipping of exon 55 is detected by RT-PCR and sequence analysis.