MEANS AND METHOD FOR INDUCING EXON-SKIPPING

• US 20090269755A1
• Filed: 03/18/2007
• Published: 10/29/2009
• Est. Priority Date: 05/19/2006
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Aartsma-Rus, Annemieke, Van Deutekom, Judith Christina Theodora, Van Ommen, Garrit-Jan Boudewijn
• Granted Patent: US 8,361,979 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 48/0016 wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells
• 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 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

In the present invention means and method are provided for optimising exon-skipping using exon-internal AON. We show that skipping efficiencies are improved by targeting putative splicing regulatory sequences (ESEs) within an exon. Such double targeting may be particularly useful for exons with which efficient skipping was difficult to obtain prior to the invention.

Claims

1. A method for determining whether the efficiency, with which an exon is excluded from a mature mRNA produced from an exon containing precursor of said mRNA produced in a cell, can be increased

said method comprising providing said cell with a first anti-sense oligonucleotide (AON) that can hybridise to an exon internal part of said exon, and determining whether said efficiency is increased upon providing said cell with a second AON that can hybridise to another exon internal part of said exon.

2. A method according to claim 1, wherein the hybridisation site of said first AON and the hybridisation site of said second AON exhibit less than 5 nucleotides overlap.

3. A method according to claim 1, wherein the hybridisation site of said first AON on said exon RNA overlaps a predicted exonic splicing enhancer (ESE) site on said exon RNA.

4. A method according to claim 1, wherein a hybridisation site of said second AON on RNA of said exon RNA overlaps a predicted ESE site on said exon RNA.

5. A method according to claim 1, wherein said exon comprises at least two independent ESE sites.

8. A set of AON which includes at least a first and second AON wherein said second AON enhances the efficiency of excluding an exon from an mRNA precursor as determinable by the method of claim 1.

9. A set of AON according to claim 8, comprising at least one AON of table 1 or table 2.

13. A method to enhance efficiency of exclusion an exon from a mature mRNA produced from an exon-containing precursor of said mRNA produced in a cell, which method comprises administering to said cell at least a first AON that can hybridise to an internal part of said exon and a second AON that can hybridise to another exon internal part of said exon wherein said second AON has been determined to enhance the efficiency of said exclusion by the method of claim 1.

6-7. -7. (canceled)

10. An AON of table 2.

11. A cell that produces an exon containing precursor of an mRNA, wherein said cell comprises a first AON that can hybridise to an exon internal part of said exon, and a second AON that can hybridise to another exon internal part of said exon.

12. A method to enhance efficiency of exclusion an exon from a mature mRNA produced from an exon-containing precursor of said mRNA produced in a cell, which method comprises administering to said cell at least a first AON that can hybridise to an internal part of said exon and a second AON that can hybridise to another exon internal part of said exon.