MODULATION OF EXON RECOGNITION IN PRE-MRNA BY INTERFERING WITH THE SECONDARY RNA STRUCTURE

• US 20130302806A1
• Filed: 12/18/2012
• Published: 11/14/2013
• Est. Priority Date: 03/21/2003
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Van Deutekom, Judith Christina Theodora
• Application Number:
  US13/718,666
• Publication Number:
  US 20130302806A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 48/0016 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 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• 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 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• 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 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/33 : Alteration of splicing
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 33/6887 : from muscle, cartilage or connective tissue

Abstract

The invention relates to oligonucleotides for inducing skipping of exon 55 of the dystrophin gene. The invention also relates to methods of inducing exon 55 skipping using the oligonucleotides.

Claims

1-3. -3. (canceled)

4. An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, wherein the oligonucleotide binds to said exon 55 and induces skipping of said exon.

7. The oligonucleotide of claim 4, wherein the oligonucleotide interferes with one or more components of the splicing machinery so that the exon is no longer recognized by the splicing machinery.

8. The oligonucleotide of claim 7, wherein the one or more components of the splicing machinery is an SR protein.

9. The oligonucleotide of claim 4, which is fully complementary to exon 55 of the human dystrophin pre-mRNA.

10. The oligonucleotide of claim 4, wherein the oligonucleotide comprises a modification.

11. The oligonucleotide of claim 10, wherein the oligonucleotide comprises a 2′

-O-methyl-phosphorothioate oligoribonucleotide modification.

12. The oligonucleotide of claim 10, wherein the oligonucleotide consists of a 2′

-O-methyl oligonucleotide or a 2′

-O-methyl-phosphorothioate oligonucleotide.

13. The oligonucleotide of claim 10, wherein the oligonucleotide comprises a morpholino ring.

14. The oligonucleotide of claim 13, wherein the oligonucleotide comprises a morpholino ring and a phosphorodiamidate linkage.

15. The oligonucleotide of claim 10, wherein the oligonucleotide comprises a peptide nucleic acid and/or locked nucleic acid oligomer.

16. The oligonucleotide of claim 10, wherein the modification comprises a base substitution of a U by a T.

17. The oligonucleotide of claim 4, wherein the oligonucleotide induces skipping of exon 55 of the human dystrophin pre-mRNA and dystrophin expression at the myotube membrane upon transfection of primary human myotubes with at least 100 nM of said oligonucleotide and incubation for at least 16 hours.

18. The oligonucleotide of claim 17, wherein skipping of exon 55 is detected by RT-PCR and sequence analysis.

19. The oligonucleotide of claim 17, wherein dystrophin expression at the myotube is detected by immunohistochemical or western blot analysis.

20. An expression vector encoding the oligonucleotide of claim 4.

21. A gene delivery vehicle comprising the expression vector of claim 20.

22. The gene delivery vehicle of claim 21, wherein the vehicle is a viral vector.

23. A method of altering recognition of exon 55 in a human dystrophin pre-mRNA, by the splicing machinery, said method comprising:

combining the pre-mRNA with the oligonucleotide of claim 4 under conditions wherein recognition of the exon by the splicing machinery is altered;

and altering recognition of exon 55 in the pre-mRNA, wherein alteration of the recognition of exon 55 by the splicing machinery is detected by RT-PCR and sequence analysis.

24. A method of inducing skipping of exon 55 of the human dystrophin pre-mRNA, said method comprising:

combining the pre-mRNA with the oligonucleotide of claim 4 under conditions wherein skipping of exon 55 can occur;

and inducing the skipping of exon 55, wherein skipping of exon 55 is detected by RT-PCR and sequence analysis.

5. An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, and wherein the oligonucleotide binds to exon 55, interferes with the structure of said exon and induces skipping of said exon.

6. An isolated antisense oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is complementary to exon 55 of the human dystrophin pre-mRNA, and wherein the oligonucleotide binds to said exon, masks said exon from the splicing apparatus and induces skipping of said exon.