Oligonucleotide comprising an inosine for treating DMD

• US 20150203849A1
• Filed: 04/03/2015
• Published: 07/23/2015
• Est. Priority Date: 04/24/2009
• Status: Abandoned Application

• Current Assignee: BioMarin Technologies B.V.
• Original Assignee: Prosensa Technologies B.V.
• Inventors: van Deutekom, Judith Christina Theodora, de Kimpe, Josephus Johannes, Platenburg, Gerard Johannes
• Application Number:
  US14/678,517
• Publication Number:
  US 20150203849A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

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.

Claims

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.

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.

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.