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 isolated antisense oligonucleotide of 15 to 80 nucleotides comprising at least 15 bases of the sequence cuguugccuccgguucug (SEQ ID NO: 29), wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. 
     
     
         2 . An isolated antisense oligonucleotide of 15 to 80 nucleotides comprising at least 15 bases of the sequence cuguugccuccgguucug (SEQ ID NO: 29), wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorodiamidate internucleoside linkage. 
     
     
         3 . An isolated antisense oligonucleotide of 18 to 80 nucleotides comprising at least the base sequence cuguugccuccgguucug (SEQ ID NO: 29), wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. 
     
     
         4 . An isolated antisense oligonucleotide of 18 to 80 nucleotides comprising at least the base sequence cuguugccuccgguucug (SEQ ID NO: 29), wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorodiamidate internucleoside linkage. 
     
     
         5 . The oligonucleotide of  claim 1  or  3 , wherein the oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide. 
     
     
         6 . The oligonucleotide of  claim 1  or  2 , wherein the oligonucleotide is 18 nucleotides and comprises the base sequence cuguugccuccgguucug (SEQ ID NO: 29), wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA. 
     
     
         7 . 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 exon 53, and wherein h53AON1 (cuguugccuccgguucug) (SEQ ID NO: 29) binds to said exon-internal sequence of exon 53 pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. 
     
     
         8 . 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 exon 53, and wherein h53AON1 (cuguugccuccgguucug) (SEQ ID NO: 29) binds to said exon-internal sequence of exon 53 pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorodiamidate internucleoside linkage. 
     
     
         9 . The oligonucleotide of  claim 7 , wherein the oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide. 
     
     
         10 . The oligonucleotide of  claim 7  or  8 , 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. 
     
     
         11 . The oligonucleotide of  claim 1  or  2 , wherein the oligonucleotide is complementary to exon 53 of the human dystrophin pre-mRNA. 
     
     
         12 . An isolated 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 the human dystrophin pre-mRNA and induces skipping of exon 53, and wherein h53AON1 (cuguugccuccgguucug) (SEQ ID NO: 29) binds to said exon-internal sequence of exon 53 pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. 
     
     
         13 . An isolated 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 the human dystrophin pre-mRNA and induces skipping of exon 53, and wherein h53AON1 (cuguugccuccgguucug) (SEQ ID NO: 29) binds to said exon-internal sequence of exon 53 pre-mRNA, wherein each internucleoside linkage of the oligonucleotide is a phosphorodiamidate internucleoside linkage. 
     
     
         14 . The oligonucleotide of  claim 12 , wherein the oligonucleotide is a 2′-O-methyl phosphorothioate oligonucleotide. 
     
     
         15 . The oligonucleotide of  claim 1 ,  2 ,  12  or  13  wherein the 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. 
     
     
         16 . The antisense oligonucleotide of  claim 12 , wherein exon 53 skipping is detected by RT-PCR and/or sequence analysis. 
     
     
         17 . The oligonucleotide of  claim 12 , wherein dystrophin expression in the muscle cell is detected by immunohistochemical and/or western blot analysis. 
     
     
         18 . The oligonucleotide of  claim 7 ,  8 ,  12  or  13 , wherein the bases of the nucleotides of said oligonucleotide consist of DNA bases or consist of RNA bases. 
     
     
         19 . The oligonucleotide of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  12  or  13 , said oligonucleotide consisting of RNA. 
     
     
         20 . The oligonucleotide of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  12  or  13 , said oligonucleotide being less than 50 nucleotides in length. 
     
     
         21 . The oligonucleotide of  claim 1 ,  2 ,  3 , or  4 , said oligonucleotide being less than 80 nucleotides in length. 
     
     
         22 . The oligonucleotide of  claim 7 ,  8 ,  12  or  13 , wherein said oligonucleotide is capable of binding without mismatches to said exon-internal sequence. 
     
     
         23 . The oligonucleotide of  claim 1 ,  2 ,  3  or  4 , wherein said oligonucleotide does not bind to a splice donor and/or a splice