METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA

Abstract

The invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing an isolate muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. The invention further relates to such molecule used in the method.

What is claimed is: 
     
         1 . An antisense oligonucleotide of 25-29 nucleotides in length, comprising the base sequence of 5′-UUUGCCGCUGCCCAAUGCCAUCCUG-3′ (SEQ ID: NO: 3) or a DNA oligonucleotide thereof, said oligonucleotide comprising a modification and is complementary along its entire length to a sequence within exon 45 of a human dystrophin pre-mRNA, said oligonucleotide being capable of inducing skipping of exon 45 by at least 50%. 
     
     
         2 . The antisense oligonucleotide of  claim 1 , said oligonucleotide consisting of said base sequence or consisting of the DNA sequence thereof. 
     
     
         3 . The antisense oligonucleotide of  claim 1  wherein said modification comprises a modified sugar, a modified base and/or a modified a modified internucleoside linkage. 
     
     
         4 . The antisense oligonucleotide of  claim 1  wherein said oligonucleotide comprises a phosphorothioate internucleoside linkage and a 2′-O-alkyl substituted ribose moiety. 
     
     
         5 . The antisense oligonucleotide of  claim 3 , wherein the modified sugar moiety is selected from the group consisting of: a ribose that is mono- or di-substituted at the 2′, 3′, and/or 5′ position. 
     
     
         6 . The antisense oligonucleotide of  claim 5 , wherein the ribose is a 2′-O-substituted ribose. 
     
     
         7 . The antisense oligonucleotide of  claim 6 , wherein the ribose is a 2′-O-methyl ribose. 
     
     
         8 . The oligonucleotide of  claim 3 , wherein all internucleoside linkages of said oligonucleotide are modified. 
     
     
         9 . The oligonucleotide of  claim 8 , said internucleoside linkages comprising phosphorothioate. 
     
     
         10 . The oligonucleotide of  claim 8 , said internucleoside linkages comprising a phosphorodiamidate morpholino oligomer (PMO). 
     
     
         11 . The oligonucleotide of  claim 1  said modification comprising a peptide nucleic acid, and/or locked nucleic acid. 
     
     
         12 . The oligonucleotide of  claim 8 , wherein said linkages are selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone. 
     
     
         13 . The oligonucleotide of  claim 1 , wherein said oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). 
     
     
         14 . A method for directing splicing of a Duchenne Muscular Dystrophy (DMD) gene pre-mRNA in muscle cells of a DMD patient to direct exclusion of exon 45 during splicing of said DMD gene pre-mRNA to a DMD gene mRNA, the method comprising:
 providing said cells with an isolated antisense RNA or DNA oligonucleotide 25-29 nucleotides in length whose sequence is fully complementary to exon 45 of a DMD gene pre-mRNA, said RNA oligonucleotide comprising the base sequence 5′-UUUGCCGCUGCCCAAUGCCAUCCUG-3′ (SEQ ID: NO: 3) and said DNA oligonucleotide comprising the DNA base sequence thereof, said oligonucleotide comprising a modification, such that said antisense oligonucleotide functions to produce an in-frame DMD gene mRNA lacking exon 45, thereby directing exclusion of exon 45 during splicing.   
     
     
         15 . The method of  claim 14 , wherein said mRNA encodes a functional shortened dystrophin protein. 
     
     
         16 . The method of  claim 15 , wherein expression of said shortened dystrophin protein is detected at the muscle cell membrane by immunohistochemical and/or western blot analysis 
     
     
         17 . The method of  claim 14 , wherein said RNA or DNA oligonucleotide consists of the nucleotide base sequence 5′-UUUGCCGCUGCCCAAUGCCAUCCUG 3′ (SEQ ID NO: 3) or the DNA sequence thereof, respectively. 
     
     
         19 . The method of  claim 14 , wherein said modification is selected from the group consisting of: a modified base, a modified sugar, a non-natural internucleoside linkage, and a modified backbone. 
     
     
         20 . The method of  claim 19 , wherein the modified backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone. 
     
     
         21 . The method of  claim 14 , wherein said oligonucleotide is a phosphorothioate oligonucleotide. 
     
     
         22 . The method of  claim 14 , wherein said oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide. 
     
     
         23 . The method of  claim 14 , wherein said oligonucleotide is a phosphorodiamidate oligonucleotideage. 
     
     
         24 . The method of  claim 14 , wherein said modification is selected from a group consisting of 2′-O-Methyl phosphorothioate, phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid, and locked nucleic acid.