Induction of exon skipping in eukaryotic cells

• US 20080209581A1
• Filed: 10/31/2007
• Published: 08/28/2008
• Est. Priority Date: 09/21/2000
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: den Dunnen, Johannes Theodorus, van Ommen, Garrit-Jan Boudewijn, van Deutekom, Judith Christina, T.
• Application Number:
  US11/982,285
• Publication Number:
  US 20080209581A1
• Time in Patent Office: Days
• US Class Current: 800/13
• 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

Described is 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 pre-mRNA in a cell capable of performing a splicing operation to reduce the production of an undesired protein in the cell, the method comprising:

contacting the pre-mRNA in the cell with an antisense-oligonucleotide capable of specifically inhibiting an exon inclusion signal of at least one exon in the pre-mRNA, wherein the antisense-oligonucleotide is directed against the interior of the at least one exon and contains between 14-40 nucleotides.

2. The method according to claim 1, wherein the mRNA encodes a functional protein.

3. The method according to claim 1, wherein the undesired protein comprises two or more domains, wherein at least one of the domains is encoded by the mRNA as a result of skipping of at least part of an exon in the pre-mRNA.

4. The method according to claim 1, wherein the contacting results in activation of a cryptic splice site in a contacted exon.

6. The method according to claim 1, wherein the exon inclusion signal comprises an exon recognition sequence.

7. The method according to claim 1, wherein the exon inclusion signal is present in an exon comprising a strong splice donor/acceptor pair.

8. The method according to claim 1, wherein the translation results in a mutant or normal dystrophin protein.

9. The method according to claim 8, wherein the mutant dystrophin protein is equivalent to a dystrophin protein of a Becker Muscular Dystrophy patient.

10. The method according to claim 9, wherein the antisense-oligonucleotide contains between 15-25 nucleotides.

11. The method according to claim 1, further comprising providing the cell with another antisense-oligonucleotide capable of inhibiting an exon inclusion signal present in another exon of the pre-mRNA.

20. The method according to claim 1, wherein the undesired protein in the wild-type has at least two functional domains generated from distinct parts of the primary amino acid sequence.

21. The method according to claim 1, wherein the undesired protein is an aberrant protein.

22. The method according to claim 21, wherein the aberrant protein is an oncoprotein or viral protein.

23. The method according to claim 1, wherein the undesired protein is involved in a genetic disease or genetic predisposition to disease.

24. The method according to claim 1, wherein the undesired protein is involved in breast cancer, colon cancer, tuberous sclerosis, neurofibromatosis, hemophilia A or congenital hypothyroidism.

5. 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, the method comprising:

providing the cell with an antisense-oligonucleotide capable of specifically inhibiting an exon inclusion signal of at least one of the exons, wherein the antisense-oligonucleotide is directed against the interior of the at least one exon and contains between 14-40 nucleotides, the method further comprising allowing translation of mRNA produced from splicing of the pre-mRNA.

12. A method for determining whether a nucleic acid, having complementarity to a part of an exon, is capable of specifically inhibiting an exon inclusion signal of the exon, the method comprising:

providing a cell having a pre-mRNA containing the exon, with the nucleic acid,

culturing the cell to allow the formation of an mRNA from the pre-mRNA, and

determining whether the exon is absent from the mRNA.

13. The method according to claim 12, further comprising determining in vitro the relative binding affinity of the nucleic acid to an RNA molecule comprising the exon.

14. A nucleic acid obtainable by the method according to claim 12.

15. A nucleic acid delivery vehicle comprising a nucleic acid according to claim 14, or the complement thereof.

16. A nucleic acid delivery vehicle capable of expressing the nucleic acid of claim 14.

17. A non-human animal provided with the nucleic acid of claim 14.

18. The non-human animal of claim 17, further comprising a nucleic acid encoding a human protein.

19. The non-human animal of claim 18, further comprising a silencing mutation in the gene encoding an animal homologue of the human protein.