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

• US 20140213635A1
• Filed: 04/08/2014
• Published: 07/31/2014
• Est. Priority Date: 03/21/2003
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Van DEUTEKOM, Judith Christina Theodora
• Granted Patent: US 10,113,165 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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. A method for inducing the skipping of exon 53 of the human dystrophin pre-mRNA in a subject with Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD), or a cell derived from the subject, said method comprising providing an oligonucleotide of 15 to 80 nucleotides in length to said subject or said cell, said oligonucleotide comprising a nucleotide sequence which is complementary to a target sequence of exon 53 of the human dystrophin pre-mRNA, wherein said target sequence comprises a nucleotide sequence that is complementary to CUGUUGCCUCCGGUUCUG (SEQ ID NO:

29), wherein said oligonucleotide induces skipping of said exon in the subject or the cell and wherein mRNA produced from skipping exon 53 of the dystrophin pre-mRNA encodes a functional dystrophin protein or a dystrophin protein of a Becker subject.

3. The method of claim 1, wherein the cell is a muscle cell.

4. The method of claim 1, wherein the oligonucleotide comprises a modification.

5. The method of claim 4, wherein the modification is selected from the group consisting of a morpholine ring, a 2′

-O-methyl ribose moiety, a phosphorothioate internucleoside linkage, a phosphorodiamidate internucleoside linkage, a peptide nucleic acid, and a locked nucleic acid.

6. The method of claim 5, wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage.

7. The method of claim 6, wherein the oligonucleotide is a 2′

-O-methyl phosphorothioate oligonucleotide.

8. The method of claim 5, wherein the oligonucleotide comprises a phosphorodiamidate internucleoside linkage.

9. The method of claim 8, wherein each internucleoside linkage of the oligonucleotide is a phosphorodiamidate internucleoside linkage.

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

11. The method of claim 1, wherein the bases of said oligonucleotide consist of DNA bases or RNA bases.

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

13. The method of claim 1, wherein exon 53 skipping is detected by RT-PCR and/or sequence analysis.

14. The method of claim 12, wherein dystrophin expression at the muscle cell membrane is detected by immunohistochemical and/or western blot analysis.

15. The method of claim 1, wherein said oligonucleotide length is selected from the group consisting of 18 to 80 nucleotides, less than 50 nucleotides and less than 80 nucleotides.

2. A method for treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in a subject by inducing the skipping of exon 53 of the human dystrophin pre-mRNA, said method comprising providing an oligonucleotide of 15 to 80 nucleotides in length, said oligonucleotide comprising a nucleotide sequence which is complementary to a target sequence of exon 53 of the human dystrophin pre-mRNA, wherein said target sequence comprises a nucleotide sequence that is complementary to CUGUUGCCUCCGGUUCUG (SEQ ID NO:

29), and wherein said oligonucleotide induces skipping of said exon in the subject.

16. The method of claim 2, wherein said oligonucleotide length is selected from the group consisting of 18 to 80 nucleotides, less than 50 nucleotides and less than 80 nucleotides.

19. A pharmaceutical composition comprising the antisense oligonucleotide of claim 16.

17. An isolated antisense oligonucleotide comprising:

a) a sequence of 15 to 80 nucleotides comprising at least 15 consecutive nucleotides of CUGUUGCCUCCGGUUCUG (SEQ ID NO;

     29); and

b) at least one modification selected from the group consisting of 2′

-O-methyl, 2′

-O-methyl-phosphorothioate, a morpholine ring, a phosphorodiamidate linkage, a peptide nucleic acid and a locked nucleic acid;

wherein the oligonucleotide specifically hybridizes to an exon 53 target region of human dystrophin DNA or pre-mRNA inducing exon 53 skipping.

18. The oligonucleotide of claim 17, wherein said oligonucleotide length is selected from the group consisting of:

18 to 80 nucleotides, less than 50 nucleotides and less than 80 nucleotides.

20. An expression vector encoding a transcript comprising an oligonucleotide, wherein the oligonucleotide is 15 to 80 nucleotides in length, said oligonucleotide comprising a nucleotide sequence which is complementary to a target sequence of exon 53 of the human dystrophin pre-mRNA, wherein said target sequence comprises a nucleotide sequence that is complementary to CUGUUGCCUCCGGUUCUG (SEQ ID NO:

29).

21. A gene delivery vehicle comprising the expression vector of claim 20, wherein the vehicle is a viral vector.