ANTISENSE OLIGONUCLEOTIDE DIRECTED REMOVAL OF PROTEOLYTIC CLEAVAGE SITES FROM PROTEINS

• US 20130198877A1
• Filed: 08/04/2011
• Published: 08/01/2013
• Est. Priority Date: 08/05/2010
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Original Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Inventors: Aartsma-Rus, Annemieke, Van Ommen, Garrit-Jan Boudewijn, van Roon-Mom, Wilhelmina M. C., Evers, Melvin Maurice, Pepers, Barry Antonius
• Granted Patent: US 9,611,471 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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: C12Y 304/19012 Ubiquitinyl hydrolase 1 (3.4.19.12)

Abstract

Described are means and methods for removing a proteolytic cleavage site from a protein, the method comprising providing a cell that expresses pre-mRNA encoding the protein with an anti-sense oligonucleotide that induces skipping of the exonic sequence that encodes the proteolytic cleavage site, and allowing translation of mRNA produced from the pre-mRNA.

Claims

1. A method for removing a proteolytic cleavage site from a protein, the method comprising:

providing a cell that expresses pre-mRNA encoding said protein with an anti-sense oligonucleotide that induces skipping of an exonic sequence that encodes said proteolytic cleavage site, and

allowing translation of mRNA produced from said pre-mRNA.

2. A method according to claim 1, wherein said protein is involved in a neurodegenerative disorder.

3. The method according to claim 1, wherein said proteolytic cleavage site comprises a caspase cleavage site or a secretase cleavage site.

4. The method according to claim 1, wherein said protein is a protein that is normally expressed in an animal'"'"'s brain.

5. The method according to claim 1, wherein the gene encoding said protein is a mutant gene that encodes a trinucleotide repeat expansion when compared to the gene from a normal individual.

6. A method according to claim 5, wherein the mutant gene is the causative gene in a polyglutamine disorder.

7. The method according to claim 6, wherein said mutant gene is the huntingtin gene.

8. The method according to claim 1, wherein said anti-sense oligonucleotide is directed toward the interior of the exon encoding said proteolytic cleavage site.

9. The method according to claim 1, wherein said exon is skipped in its entirety.

10. A method of removing a proteolytic cleavage site from a protein in a cell, the method comprising:

utilizing exon-skipping in a cell to remove the proteolytic cleavage site from the protein.

12. The method according to claim 10, wherein said protein is involved in a neurodegenerative disorder.

11. A method of removing a proteolytic cleavage site in a protein, the method comprising:

utilizing an anti-sense oligonucleotide that induces skipping of an exon that encodes a proteolytic cleavage site in the protein, so as to remove the proteolytic cleavage site from said protein in a cell that produces pre-mRNA encoding said protein.

13. An oligonucleotide of between 14-40 nucleotides that induces skipping of an exon that encodes a proteolytic cleavage site in a protein.

14. A method of treating a disease, the method comprising:

utilizing the oligonucleotide of claim 13 in the treatment of a disease that is associated with a proteolytic cleavage product of said protein.

15. The oligonucleotide of claim 13, wherein said oligonucleotide binds to the pre-mRNA of said protein to form a double-stranded nucleic acid complex and wherein said oligonucleotide is chemically modified to render said double-stranded nucleic acid complex RNAse H resistant.

18. A non-human animal comprising the oligonucleotide of claim 13.

19. A non-human animal according to claim 18, that comprises a mutant gene that encodes a trinucleotide repeat expansion when compared to the gene of a normal individual.

16. A method for altering the proteolytic processing of a protein that comprises a proteolytic cleavage site, the method comprising:

providing a cell that produces a pre-mRNA that codes for said protein with an anti-sense oligonucleotide that is specific for said pre-mRNA; and

that prevents inclusion of the code for said proteolytic cleavage site into mature mRNA produced from said pre-mRNA, and

allowing translation of said mRNA to produce the protein of which the proteolytic processing is altered.

17. The method of claim 16, wherein said protein is involved in a neurodegenerative disorder.

20. A method for removing a proteolytic cleavage site from a protein involved in Huntington'"'"'s disease, the method comprising:

providing a cell that expresses pre-mRNA encoding the protein with an anti-sense oligonucleotide directed toward the interior of the exon encoding the proteolytic cleavage site that induces skipping of an exonic sequence that encodes the proteolytic cleavage site so that the exon is skipped in its entirety, and

allowing translation of mRNA produced from the pre-mRNA,

wherein the gene encoding the protein is a mutant gene that encodes a trinucleotide repeat expansion in comparison to the gene of a non-Huntington'"'"'s individual.