Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means

• US 9,139,828 B2
• Filed: 11/11/2010
• Issued: 09/22/2015
• Est. Priority Date: 05/14/2008
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.
• Original Assignee: Academisch Ziekenhuis Leiden, Prosensa Technologies B.V.
• Inventors: Platenburg, Gerard Johannes, de Kimpe, Josephus Johannes, van Deutekom, Judith Christina Theodora, van Ommen, Garrit-Jan Boudewijn, Aartsma-Rus, Annemieke
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US12/992,218
• Publication Number:
  US 20120059042A1
• Time in Patent Office: 1,776 Days
• Field of Search: None
• 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 21/04 for myasthenia gravis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

The invention relates to oligonucleotides that bind to and induce skipping of exon 44 in the human dystrophin pre-mRNA, and methods of use.

Claims

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.

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.

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).