Induction of exon skipping in eukaryotic cells

• US 20030235845A1
• Filed: 03/21/2003
• Published: 12/25/2003
• Est. Priority Date: 09/21/2000
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: den Dunnen, Johannes Theodorus, van Ommen, Garrit-Jan Boudewijn, van Deutekom, Judith Christina, T.
• Granted Patent: US 7,973,015 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 pre-mRNA in a system capable of performing a splicing operation comprising:

contacting said pre-mRNA in said system with an agent capable of specifically inhibiting an exon inclusion signal of at least one exon in said pre-mRNA, and

allowing splicing of said pre-mRNA.

2. The method according to claim 1, further comprising allowing translation of an mRNA produced from splicing of said pre-mRNA.

3. The method according to claim 1 or claim 2, wherein said mRNA encodes a functional protein.

4. The method according to any one of claims 1-3, wherein said functional protein comprises at least two domains, wherein at least one of said domains is encoded by said mRNA as a result of skipping of at least part of an exon in said pre-mRNA.

5. The method according to any one of claims 1-4, wherein said contacting results in activation of a cryptic splice site in a contacted exon.

7. The method according to any one of claims 1-6, wherein said exon inclusion signal comprises an exon recognition sequence.

8. The method according to any one of claims 1-6, wherein said exon inclusion signal is present in an exon comprising a strong splice donor/acceptor pair.

9. The method according to any one of claims 1-8, wherein said translation results in a mutant dystrophin protein or a normal dystrophin protein.

10. The method according to claim 9, wherein said mutant dystrophin protein is equivalent to a dystrophin protein of a Becker patient.

11. The method according to claim 9 or claim 10, wherein said exon inclusion signal is present in exon number 2, 8, 43, 44, 45, 46, 50, 51, 52, or 53.

12. The method according to any one of claims 1-11, wherein said agent comprises a nucleic acid or a functional equivalent thereof.

13. The method according to claim 12, wherein said nucleic-acid contains between 15-25 nucleotides or a functional equivalent thereof.

14. The method according to any one of claims 1-13, further comprising providing said cell with another agent capable of inhibiting an exon inclusion signal present in another exon of said pre-mRNA.

6. A method for at least in part decreasing the production of an aberrant protein in a cell, said cell comprising pre-mRNA comprising exons coding for said aberrant protein, the method comprising:

providing said cell with an agent capable of specifically inhibiting an exon inclusion signal of at least one of said exons, and

allowing translation of mRNA produced from splicing of said pre-mRNA.

15. A method for determining whether a nucleic acid or a functional equivalent thereof is capable of specifically inhibiting an exon inclusion signal of an exon, said nucleic acid or said functional equivalent thereof comprising complementarity to a part of said exon, the method comprising:

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

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

determining whether said exon is absent from said mRNA.

16. The method according to claim 15 further comprising determining in vitro the relative binding affinity of said nucleic acid or said functional equivalent thereof to an RNA molecule comprising said exon.

17. A nucleic acid or a functional equivalent thereof obtainable by the method according to claim 15 or claim 16.

18. A nucleic acid delivery vehicle comprising the nucleic acid or the functional equivalent thereof of claim 17, or the complement thereof.

19. A nucleic acid delivery vehicle capable of expressing the nucleic acid or the functional equivalent thereof of claim 17.

20. A method for treating a subject with an inherited disease or a predisposition to a disease, comprising administering to the subject the nucleic acid or the functional equivalent thereof of claim 17.

21. A method for treating a subject with an inherited disease or a predisposition to a disease, comprising administering to the subject the nucleic acid delivery vehicle of claim 18.

22. A method for treating a subject with an inherited disease or a predisposition to a disease, comprising administering to the subject the nucleic acid delivery vehicle of claim 19.

24. A non-human animal provided with the nucleic acid or the functional equivalent thereof of claim 17.

25. The non-human animal according to claim 24, further comprising a nucleic acid encoding a human protein or a functional equivalent thereof.

26. The non-human animal according to claim 25, further comprising a silencing mutation in a gene encoding an animal homologue of said human protein.

27. A pharmaceutical composition useful in treating a subject with Duchenne muscular dystrophy, comprising the nucleic acid or the functional equivalent thereof of claim 17.

28. A pharmaceutical composition useful in treating a subject with Duchenne muscular dystrophy, comprising the nucleic acid delivery vehicle of claim 18.

29. A pharmaceutical composition useful in treating a subject with Duchenne muscular dystrophy, comprising the nucleic acid delivery vehicle of claim 19.

23. A method for treating a subject with an inherited disease or a predisposition to a disease, comprising administering to the subject a nucleic acid or a functional equivalent thereof, the nucleic acid or the functional equivalent thereof comprising an exon inclusion signal inhibiting quality.

30. A pharmaceutical composition useful in treating a subject with Duchenne muscular dystrophy, comprising a nucleic acid or a functional equivalent thereof, the nucleic acid or the functional equivalent thereof comprising an exon inclusion signal inhibiting quality.