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

• US 20150218559A1
• Filed: 04/16/2015
• Published: 08/06/2015
• 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, Van Ommen, Garrit-Jan Boudewijn, Aartsma-Rus, Annemieke, den DUNNEN, Johannes Theodorus
• Application Number:
  US14/688,871
• Publication Number:
  US 20150218559A1
• 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 of 15 to 80 nucleotides in length wherein said oligonucleotide is capable of binding to a sequence internal to exon 55 of the human dystrophin pre-mRNA and inducing skipping of said exon, said oligonucleotide comprising a modification.

2. The oligonucleotide of claim 1, wherein said oligonucleotide is 16 to 50 nucleotides in length.

3. The oligonucleotide of claim 2, said oligonucleotide being less than 50 nucleotides in length.

4. The oligonucleotide of claim 3, wherein said oligonucleotide is 16 to 24 nucleotides in length.

5. The oligonucleotide of claim 2, wherein said oligonucleotide is complementary to said sequence internal to exon 55 of the human dystrophin pre-mRNA.

6. The oligonucleotide of claim 5, wherein said oligonucleotide is fully complementary to said sequence internal to exon 55 of the human dystrophin pre-mRNA.

7. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a 2′

-O-methyl-phosphorothioate oligonucleotide modification.

8. The oligonucleotide of claim 1, wherein the oligonucleotide consists of a 2′

-O-methyl oligonucleotide or a 2″

-O-methyl-phosphorothioate oligonucleotide.

9. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a morpholine ring.

10. The oligonucleotide of claim 9, wherein the oligonucleotide further comprises a phosphorodiamidate linkage.

11. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a peptide nucleic acid and/or locked nucleic acid.

12. The oligonucleotide of claim 1, wherein said oligonucleotide is capable of binding said sequence internal to exon 55 of the human dystrophin pre-mRNA and inducing skipping of said exon in primary human myotubes upon transfection of at least 100 nM of said oligonucleotide to said myotubes and incubation of transfected myotubes for at least 16 hours.

13. The oligonucleotide of claim 12, wherein skipping is detectable by RT-PCR and nucleic acid sequencing.

18. The oligonucleotide of claim 5, wherein said sequence internal to exon 55 comprises is predicted by the m-Fold RNA structure-modeling program to assume both a double-stranded stretch and a single-stranded stretch of nucleotides, said double-stranded stretch comprising two single-stranded nucleic acid stretches of exon 55 that form a duplex.

19. A method for inducing skipping of exon 55 of the human dystrophin pre-mRNA in a muscle cell, the method comprising contacting said cell with an oligonucleotide of claim 1 for a time and under conditions which permit induction of exon 55 skipping.

20. A method for inducing skipping of exon 55 of the human dystrophin pre-mRNA in a human subject, the method comprising administering an oligonucleotide of claim 1 to said subject in an amount and for a time which permits induction of exon 55 skipping.

14. An expression vector encoding an oligonucleotide of 15 to 80 nucleotides wherein said oligonucleotide is capable of binding to a sequence internal to exon 55 of the human dystrophin pre-mRNA and inducing skipping of said exon.

15. A gene delivery vehicle comprising the expression vector of claim 14.

16. An antisense oligonucleotide of 15 to 80 nucleotides in length, wherein said oligonucleotide is complementary to a consecutive part of between 16 and 50 nucleotides of an sequence internal to exon 55 of the human dystrophin pre-mRNA and said oligonucleotide is capable of inducing skipping of exon 55 in a muscle cell, said oligonucleotide comprising a modification.

17. The antisense oligonucleotide of claim 16, wherein said oligonucleotide is between 16 and 50 nucleotides in length.