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

• US 20160194636A1
• Filed: 02/18/2016
• Published: 07/07/2016
• Est. Priority Date: 03/21/2003
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Original Assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Inventors: Van DEUTEKOM, Judith Christina Theodora, Van OMMEN, Garrit-Jan Boudewijn, AARTSMA-RUS, Annemieke, den DUNNEN, Johannes Theodorus
• Application Number:
  US15/047,233
• Publication Number:
  US 20160194636A1
• 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 53 of the dystrophin gene. The invention also relates to methods of inducing exon 53 skipping using the oligonucleotides.

Claims

1. An isolated antisense oligonucleotide 15 to 80 nucleotides in length, wherein said oligonucleotide induces exon 53 skipping in the human dystrophin pre-mRNA in a muscle cell, said oligonucleotide comprising a modification.

2. The oligonucleotide of claim 1, wherein said oligonucleotide is 18 to 80 nucleotides in length.

3. The oligonucleotide of claim 1, wherein said oligonucleotide is complementary to exon 53 of the human dystrophin pre-mRNA.

7. The oligonucleotide of claim 1, wherein said muscle cell is from a DMD patient.

8. 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.

9. The oligonucleotide of claim 1, wherein said modification consists of 2′

-O-methyl or 2′

-O-methyl phosphorothioate.

10. The oligonucleotide of claim 1, wherein said modification comprises a morpholine ring and a phosphorodiamidate linkage.

11. The oligonucleotide of claim 1, which is a morpholino phosphorodiamidate oligoribonucleotide.

12. The oligonucleotide of claim 1, wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate linkage.

13. The oligonucleotide of claim 1, wherein said oligonucleotide induces exon 53 skipping of the human dystrophin pre-mRNA and induces dystrophin expression in the muscle cell upon transfection of human muscle cells with at least 100 nM of said oligonucleotide and incubation for at least 16 hours.

14. The oligonucleotide of claim 13, wherein exon 53 skipping is detected by RT-PCR and/or sequence analysis.

15. The oligonucleotide of claim 13, wherein dystrophin expression in said muscle cell is detected by immunohistochemical and/or western blot analysis.

16. The oligonucleotide of claim 1, wherein the bases of the nucleotides of said oligonucleotide consist of DNA bases or consist of RNA bases.

17. The oligonucleotide of claim 1, said oligonucleotide consisting of RNA.

18. The oligonucleotide of claim 1, said oligonucleotide being less than 50 nucleotides in length.

19. The oligonucleotide of claim 1, said oligonucleotide being less than 80 nucleotides in length.

21. The oligonucleotide of claim 1, wherein said oligonucleotide binds to a purine rich sequence.

22. The oligonucleotide of claim 1, wherein said oligonucleotide binds to an ERS or a SR protein binding site.

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

24. The oligonucleotide of claim 1, wherein at least a part of said oligonucleotide is complementary to a region of the dystrophin exon 53 pre-mRNA that assumes a structure that is hybridized to another part of said pre-mRNA (closed structure) and wherein at least a part of said oligonucleotide is complementary to a region of a pre-mRNA of an exon that is not hybridized (open structure).

25. A pharmaceutical composition comprising the oligonucleotide of claim 1.

26. A method for inducing the skipping exon 53 of the human dystrophin pre-mRNA in a subject with Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD), or a cell of said subject, said method comprising providing the oligonucleotide of claim 1, to said subject or said cell, wherein said oligonucleotide induces skipping of said exon in said subject or said cell and wherein mRNA produced from skipping exon 53 of the dystrophin pre-mRNA encodes a functional dystrophin protein.

27. A method for treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in a subject by inducing skipping of exon 53 of the human dystrophin pre-mRNA, said method comprising providing the oligonucleotide of claim 1, wherein said oligonucleotide induces skipping of said exon in said subject.

4. An isolated antisense oligonucleotide of 18 to 50 nucleotides in length, wherein said oligonucleotide binds to an exon-internal sequence of exon 53 of the human dystrophin pre-mRNA and induces skipping of said exon in a muscle cell, said oligonucleotide comprising a modification.

5. The oligonucleotide of claim 4, wherein said exon-internal sequence comprises a consecutive part of between 16 and 50 nucleotides of said exon and said oligonucleotide is complementary to said consecutive part.

20. The oligonucleotide of claim 4, wherein said oligonucleotide is capable of binding without mismatches to said exon-internal sequence.

6. An antisense oligonucleotide of 18 to 50 nucleotides in length, wherein said oligonucleotide is complementary to a consecutive part of between 16 and 50 nucleotides of an exon-internal sequence of exon 53 of said human dystrophin pre-mRNA and induces skipping of exon 53 in a muscle cell.