Modulation of Exon Recognition in Pre-mRNA By Interfering with the Secondary RNA Structure

• US 20160355810A1
• Filed: 04/14/2016
• Published: 12/08/2016
• 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:
  US15/098,589
• Publication Number:
  US 20160355810A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 51 of the dystrophin gene. The invention also relates to methods of inducing exon 51 skipping using the oligonucleotides.

Claims

1. An isolated antisense oligonucleotide 16 to 50 nucleotides in length, wherein a part of said oligonucleotide is complementary to a strand of a double-stranded closed structure of the dystrophin exon 51 pre-mRNA and another part of said oligonucleotide is complementary to a single stranded open structure of the dystrophin exon 51 pre-mRNA, wherein said exon 51 pre-mRNA assumes a secondary structure within said exon comprising said open and closed structures, wherein said single stranded open structure is contiguous with said strand of said closed structure, wherein said oligonucleotide is capable of inducing exon 51 skipping, said oligonucleotide comprising a modification.

2. The oligonucleotide of claim 1, wherein said single stranded open structure is contiguous with both strands of said double-stranded closed structure.

3. The oligonucleotide of claim 1, wherein said oligonucleotide is fully complementary to said open and closed structures.

4. The oligonucleotide of claim 1, wherein said open and closed structures comprise a consecutive part of between 16 and 50 nucleotides of said exon and said oligonucleotide is complementary to said consecutive part.

5. The oligonucleotide of claim 4, wherein oligonucleotide is fully complementary to said consecutive part.

6. The oligonucleotide of claim 1, wherein said modification is selected from the group consisting of:

2′

-O-methyl, 2′

-O-methyl-phosphorothioate, a morpholine ring, a phosphorodiamidate linkage, a modification to increase resistance to RNAseH, a peptide nucleic acid and a locked nucleic acid.

7. The oligonucleotide of claim 1, wherein said oligonucleotide is a 2′

-O-methyl oligonucleotide.

8. The oligonucleotide of claim 7, wherein said oligonucleotide is a 2′

-O-methyl phosphorothioate oligonucleotide.

9. The oligonucleotide of claim 1, wherein said oligonucleotide is morpholino oligonucleotide.

10. The oligonucleotide of claim 9, wherein said oligonucleotide is a morpholino phosphorodiamidate oligonucleotide.

11. The oligonucleotide of claim 1, wherein oligonucleotide is capable of inducing exon 51 skipping in a human myotube in culture.

12. The oligonucleotide of claim 11, wherein said human myotube is from a Duchenne Muscular Dystrophy (DMD) patient.

13. The oligonucleotide of claim 12, wherein said oligonucleotide is capable of inducing exon 51 skipping of the human dystrophin pre-mRNA upon transfection of said oligonucleotide into human myotubes in culture so as to induce dystrophin expression.

14. The oligonucleotide of claim 13, wherein said transfection comprises introducing at least 100nM of said oligonucleotide into human myotubes so as to induce dystrophin expression.

15. The oligonucleotide of claim 14, wherein said exon 51 skipping is detected by RT-PCR and/or sequence analysis.

16. The oligonucleotide of claim 14, wherein said dystrophin expression is detected at least 16 hours after transfection by immunohistochemical and/or western blot analysis.

17. The oligonucleotide of claim 1, wherein said oligonucleotide is an RNA oligonucleotide.

18. The oligonucleotide of claim 1, said oligonucleotide comprising 20 to 50 nucleotides in length.

19. The oligonucleotide of claim 1, comprising less than 50 nucleotides in length.

20. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a greater number of purine residues than pyrimidine residues.

21. The oligonucleotide of claim 1 wherein said oligonucleotide does not bind to a splice donor and/or a splice acceptor sequence of said exon.

22. A pharmaceutical composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.