Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means
The invention relates to oligonucleotides that bind to and induce skipping of exon 44 in the human dystrophin pre-mRNA, and methods of use.
1. An isolated antisense oligonucleotide consisting of 7, 8, 9, or 10 nucleotides and comprising the base sequence of 5′-UCAGCUUCUGUUAGCCACUG-3′ (SEQ ID NO: 5), said oligonucleotide comprising a modification.
2. The oligonucleotide according to claim 1 , consisting of 20 nucleotides having the base sequence of 5′-UCAGCUUCUGUUAGCCACUG-3′ (SEQ ID NO: 5).
3. The oligonucleotide according to claim 1 , wherein said nucleotides comprise one or more DNA bases.
4. The oligonucleotide according to claim 1 , wherein said modification is selected from the group consisting of: a modified base, a modified sugar moiety, and a modified internucleoside linkage.
5. The oligonucleotide of claim 1 , having a modified backbone.
6. The oligonucleotide of claim 4 , comprising a ribose moiety that is mono- or di-substituted at the 2′, 3′ and/or 5′ position.
7. The oligonucleotide according to claim 6 , comprising a 2′-O substituted ribose and a phosphorothioate modified internucleoside linkage.
8. The antisense oligonucleotide of claim 6 , comprising a 2′-O-methyl ribose and a phosphorothioate modified internucleoside linkage.
9. The oligonucleotide according to claim 1 , comprising a phosphorothioate modified internucleoside linkage ribose, wherein ribose moieties are each 2′-O-methyl substituted.
10. An isolated antisense oligonucleotide comprising a phosphorothioate backbone consisting of 20 nucleotides having the base sequence of 5′-UCAGCUUCUGUUAGCCACUG-3′ (SEQ ID NO: 5), wherein each of the sugar moieties is 2′-O-methyl substituted.
11. A viral-based vector comprising an expression cassette for expression of the oligonucleotide consisting of 7, 8, 9 or 10 nucleotides and comprising the base sequence of 5′-UCAGCUUCUGUUAGCCACUG-3′ (SEQ ID NO: 5).
12. A pharmaceutical composition comprising the oligonucleotide of claim 1 or the vector of claim 11 and a pharmaceutically acceptable carrier.
13. A method for inducing skipping of exon 44 of pre-mRNA of the dystrophin gene in a DMD or BMD patient, wherein the genome of said patient comprises a mutation in the DMD gene selected from the group consisting of: one of a deletion of exon 03-43, exon 05-43, exon 06-43, exon 10-43, exon 13-43, exon 14-43, exon 17-43, exon 19-43, exon 28-43, exon 30-43, exon 31-43, exon 33-43, exon 34-43, exon 35-43, exon 36-43, exon 37-43, exon 38-43, exon 40-43, exon 41-43, exon 42-43, exon 43-45, exon 45-54 and exon 45-68, a duplication of exon 44, a deletion of exon 43, and a deletion of exon 45, a nonsense point mutation in exon 43, and a nonsense point mutation in exon 45 of the dystrophin pre-mRNA, wherein the method comprises administering to said patient an amount of the oligonucleotide of claim 1 , the vector of claim 11 or the pharmaceutical composition of claim 12 effective to induce the skipping of said exon 44.
14. A method for inducing skipping of exon 44 of the pre-mRNA of the dystrophin gene in a cell comprising providing the cell with the oligonucleotide of claim 1 , the vector of claim 11 or the pharmaceutical composition of claim 12 .
15. The oligonucleotide according to claim 1 , wherein said oligonucleotide induces skipping of exon 44 of the dystrophin pre-mRNA.