INDUCTION OF EXON SKIPPING IN EUKARYOTIC CELLS

• US 20150322434A1
• Filed: 05/14/2015
• Published: 11/12/2015
• Est. Priority Date: 09/21/2000
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: van Deutekom, Judith Christina Theodora
• Application Number:
  US14/712,753
• Publication Number:
  US 20150322434A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 15/86 Viral vectors
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2320/33 : Alteration of splicing
• CPC Class Codes: C12N 2750/14142 : virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Abstract

The present invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, by inducing so-call exon skipping in the cell. Exon-skipping results in mature mRNA that does not contain the skipped exon, which leads to an altered product of the exon codes for amino acids. Exon skipping is performed by providing a cell with an agent capable of specifically inhibiting an exon inclusion signal, for instance, an exon recognition sequence, of the exon. The exon inclusion signal can be interfered with by a nucleic acid comprising complementarity to a part of the exon. The nucleic acid, which is also herewith provided, can be used for the preparation of a medicament, for instance, for the treatment of an inherited disease.

Claims

1. An isolated antisense oligonucleotide 17 to 40 nucleotides in length comprising a nucleotide sequence capable of binding to a purine-rich sequence of exon 51 of the human dystrophin pre-mRNA and inducing skipping of exon 51 of said pre-mRNA in a human muscle cell, wherein hAON#23:

5′

-TGGCATTTCTAGTTTGG-3′

(SEQ ID NO;

     18) binds to said purine-rich sequence and causes skipping of exon 51 of said pre-mRNA in a human muscle cell, said antisense oligonucleotide comprising a modification for increasing its resistance to an endonuclease in a cell.

2. The oligonucleotide of claim 1, wherein the endonuclease is RNAse.

3. The oligonucleotide of claim 1, which is a Peptide Nucleic Acid (PNA).

4. The oligonucleotide of claim 1, wherein each T has been replaced by a U.

7. A nucleic acid delivery vehicle comprising a transcription unit expressing the antisense oligonucleotide of claim 1.

8. A composition comprising the oligonucleotide of claim 1.

9. A pharmaceutical formulation comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

10. A method for directing splicing of a dystrophin pre-mRNA in a cell by inducing the skipping of exon 51 of the dystrophin pre-mRNA to produce a dystrophin protein comprising:

providing the cell with an antisense oligonucleotide of claim 1 and wherein said oligonucleotide induces skipping of exon 51 from said dystrophin pre-mRNA in said cell.

5. The oligonucleotide of claim 5, wherein said oligonucleotide is a 2′

-O-methyl oligoribonucleotide.

6. The oligonucleotide of claim 5, which is a 2′

-O-methyl phosphorothioate oligoribonucleotide.