Oligonucleotide comprising an inosine for treating DMD
The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the use of said oligonucleotide for preventing or treating DMD or BMD.
1 . An isolated antisense oligonucleotide 8 to 50 nucleotides in length comprising a base sequence which is complementary to a consecutive part comprising 8 nucleotides of an exon of a dystrophin pre-mRNA exon, wherein said oligonucleotide comprises at least one inosine base and is functional to induce skipping of said exon.
2 . The oligonucleotide according to claim 1 , wherein said consecutive part comprises 13 to 50 nucleotides.
3 . The oligonucleotide according to claim 2 , wherein said consecutive part comprises 14 to 25 nucleotides.
4 . The oligonucleotide according to claim 1 , wherein said exon is selected from the group consisting of 51, 45, 53, 44, 46, 52, 50, 43 and 55.
5 . The oligonucleotide according to claim 1 , wherein said oligonucleotide comprises RNA bases.
6 . The oligonucleotide of claim 1 , wherein said oligonucleotide comprises a modified base, and/or a modified sugar moiety and/or a modified internucleoside linkage.
7 . The oligonucleotide according to claim 6 , wherein said oligonucleotide comprises a modified backbone.
8 . The oligonucleotide according to claim 7 , wherein said 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.
9 . The oligonucleotide according to claim 6 , wherein said oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid (PNA), and/or locked nucleic acid (LNA).
10 . The oligonucleotide according to claim 6 , wherein said oligonucleotide comprises a phosphorothioate internucleoside linkage and a 2′-O-methyl substituted ribose moiety.
11 . The oligonucleotide according to claim 6 , wherein said modified internucleoside linkage is a phosphorothioate moiety, said modified sugar moiety is a 2′-O-methyl substituted ribose and wherein said oligonucleotide comprises the sequence 5′- UUUGCCICUGCCCAAUGCCAUCCUG -3′ (SEQ ID NO:557).
12 . The oligonucleotide according to claim 1 , wherein said oligonucleotide comprises the base sequence 5′-UUUGCCICUGCCCAAUGCCAUCCUG-3′ (SEQ ID NO:557).
13 . The isolated antisense oligonucleotide of claim 12 , wherein said oligonucleotide comprises a modified base, and/or a modified sugar moiety and/or a modified internucleoside linkage.
14 . An isolated antisense oligonucleotide comprising or consisting of a base or a nucleotide sequence selected from the group consisting of: SEQ ID NO: 2-473, 539-556 and 558-576, wherein said oligonucleotide comprises at least one inosine base.
15 . The oligonucleotide of claim 14 , wherein said oligonucleotide comprises RNA bases.
16 . The oligonucleotide of claim 14 , wherein said oligonucleotide comprises a modified base, and/or a modified sugar moiety and/or a modified internucleoside linkage.
17 . An isolated antisense oligonucleotide, comprising a base sequence consisting of the base sequence 5′- UUUGCCICUGCCCAAUGCCAUCCUG -3′ (SEQ ID NO:557), wherein the oligonucleotide consists of phosphorothioate internucleoside linkages and 2′-O-methyl substituted ribose moieties.
18 . An isolated antisense oligonucleotide of 13 to 50 nucleotides in length, said oligonucleotide comprising a sequence which is fully complementary to a consecutive part comprising 8 nucleotides of exon 45 of the human dystrophin pre-mRNA, wherein said consecutive part is fully complementary to a portion of the base sequence 5′- UUUGCCICUGCCCAAUGCCAUCCUG -3′ (SEQ ID NO: 557).
19 . The isolated oligonucleotide of claim 18 , said consecutive part comprising 13 nucleotides of exon 45.
20 . The isolated oligonucleotide of claim 18 , said consecutive part comprising 25 nucleotides of exon 45.
21 . The oligonucleotide of claim 18 , wherein said oligonucleotide comprises a modified base, and/or a modified sugar moiety, and/or a modified internucleoside linkage.
22 . The oligonucleotide claim 21 , wherein said modified sugar moiety is mono- or di-substituted at the 2′, 3′ and/or 5′ position.
23 . The oligonucleotide of claim 21 , wherein said modified internucleoside linkage is a phosphorothioate moiety and said modified sugar moiety is a 2′-O-substituted ribose.
24 . The oligonucleotide of claim 23 , wherein said modified sugar moiety is a 2′-O-methyl ribose.
25 . The oligonucleotide of claim 24 , wherein each sugar moiety is a 2′-O-methyl ribose and each internucleoside linkage is a phosphorothioate.
26 . The oligonucleotide of claim 18 , wherein said oligonucleotide comprises RNA bases.
27 . A method for inducing skipping of an exon of human dystrophin pre-mRNA in a muscle cell, the method comprising contacting said cell with an oligonucleotide of claim 1 for a time and under conditions which permit exon skipping.
28 . A method for inducing skipping of an exon of human dystrophin pre-mRNA in a human subject, the method comprising administering an oligonucleotide of claim 1 to said subject in an amount and for a time which is effective to induce exon skipping.
29 . A method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual, the method comprising administering to said individual an oligonucleotide of claim 1 , wherein said oligonucleotide induces skipping of an exon of a dystrophin pre-mRNA.
30 . A method for inducing and/or promoting skipping of exon 43, 44, 45, 46, 50, 51, 52 or 53 of the dystrophin pre-mRNA in a patient, the method comprising administering an oligonucleotide of claim 4 to said patient.