Induction of exon skipping in eukaryotic cells

• US 7,973,015 B2
• Filed: 03/21/2003
• Issued: 07/05/2011
• Est. Priority Date: 09/21/2000
• Status: Expired due to Fees

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: van Deutekom, Judith C. T., den Dunnen, Johannes T., van Ommen, Garrit-Jan B.
• Primary Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US10/395,031
• Publication Number:
  US 20030235845A1
• Time in Patent Office: 3,028 Days
• Field of Search: None
• US Class Current: 514/44.00R
• 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-called 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. A method for directing splicing of a dystrophin pre-mRNA in a first cell, the method comprising:

contacting said dystrophin pre-mRNA in said first cell with a first antisense oligonucleotide having from 14 to 40 nucleotides, and being complementary to a portion of a first exon of the dystrophin pre-mRNA, said first exon selected from the group consisting of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52 and 53 of a dystrophin gene, and wherein said first antisense oligonucleotide is able to bind to said first exon and mask said first exon from said first cell'"'"'s splicing apparatus,

providing said first cell with a second antisense oligonucleotide, said second antisense oligonucleotide having from 14 to 40 nucleotides, and being complementary to a portion of a second exon of said dystrophin pre-mRNA wherein said second exon is different from said first exon, wherein said second antisense oligonucleotide is able to bind to said second exon and mask the second exon from the first cell'"'"'s splicing apparatus,

splicing said dystrophin pre-mRNA, and

translating an mRNA produced from splicing of said dystrophin pre-mRNA to form a final mRNA excluding said first exon and said second exon, wherein said translation of said mRNA results in a mutant dystrophin protein having a Becker mutant'"'"'s functionality,

wherein said first antisense oligonucleotide is obtained by;

providing a second cell with said first oligonucleotide, the cell having a pre-mRNA containing said first exon,

culturing said second cell to form mRNA in said second cell from said pre-mRNA in said second cell, and

determining whether said first exon is absent from the thus formed mRNA in said second cell,

wherein said second antisense oligonucleotide is obtained by;

providing a third cell or said second cell with said second oligonucleotide, said third cell or said second cell having a pre-mRNA containing said second exon,

culturing said third cell or said second cell to form mRNA in said third cell or said second cell from said pre-mRNA in said third cell or said second cell, and determining whether said second exon is absent from the thus formed mRNA in said third cell or said second cell, and

wherein the method of determining whether said first exon or said second exon is absent from the thus formed mRNA comprises RT-PCR and sequence analysis.

2. The method according to claim 1, wherein said first and/or second antisense oligonucleotide is a 2′

O-methyl phosphorothioate oligoribonucleotide or a peptide nucleic acid.

3. The method according to claim 1, wherein said first exon is exon 45 of dystrophin and wherein said second exon is exon 51 of dystrophin.

4. The method according to claim 1, wherein said first and/or said second antisense oligonucleotide have from 14 to 25 nucleotides.