Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure

• US 20060099616A1
• Filed: 09/21/2005
• Published: 05/11/2006
• Est. Priority Date: 03/21/2003
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Aartsma-Rus, Annemieke, den Dunnen, Johannes Theodorus, van Ommen, Garrit-Jan Boudewijn, van Deutekom, Judith Christina Theodora
• Application Number:
  US11/233,507
• Publication Number:
  US 20060099616A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 provides a method for generating an oligonucleotide with which an exon may be skipped in a pre-mRNA and thus excluded from a produced mRNA thereof. Further provided are methods for altering the secondary structure of an mRNA to interfere with splicing processes and uses of the oligonucleotides and methods in the treatment of disease. Further provided are pharmaceutical compositions and methods and means for inducing skipping of several exons in a pre-mRNA.

Claims

1. A method for generating an oligonucleotide or an equivalent thereof comprising:

determining from a secondary structure of RNA from an exon, a region that assumes a structure that is hybridized to another part of said RNA (closed structure) and a region that is not hybridized in said structure (open structure); and

generating said oligonucleotide or said equivalent thereof, of which at least part of said oligonucleotide or said equivalent thereof is complementary to said closed structure and of which at least another part of said oligonucleotide or said equivalent thereof is complementary to said open structure.

2. The method according to claim 1, wherein said open and closed structures are adjacent to each other.

3. The method according to claim 1, wherein said oligonucleotide is complementary to a consecutive part of between 14 and 50 nucleotides of said RNA.

4. The method according to claim 1, wherein said oligonucleotide is complementary to a consecutive part of between 14 and 50 nucleotides of said RNA.

5. The method according to claim 4, wherein said RNA contains a modification, preferably a 2′

-O-methyl modified ribose (RNA) or deoxyribose (DNA) modification.

6. The method according to claim 1, wherein a pre-mRNA comprising said exon exhibits undesired splicing in a subject.

7. The method according to claim 6, wherein the absence of said exon from an mRNA produce from said pre-mRNA generates a coding region for a protein.

8. The method according to claim 6, wherein the gene from which said RNA comprising said exon is transcribed, encodes an aberrant gene selected from the group consiting of Duchenne muscular dystrophy gene (DMD), a collagen VI alpha 1 gene (COL6A1), a myotubular myopathy 1 gene (MTM1), a dysferlin gene (DYSF), a laminin-alpha 2 gene (LAMA2), an emery-dreyfuss muscular dystrophy gene (EMD) and a calpain 3 gene (CAPN3).

9. The method according to claim 8, wherein said gene is the Duchenne muscular dystrophy gene.

10. The method according to claim 9, wherein said exon comprises an exon selected from the group consisting of exons 2, 8, 9, 17, 19, 29, 40-46, 48-53, 55 and 59.

11. The method according to claim 1, wherein an equivalent of said oligonucleotide is generated comprising similar hybridization characteristics in kind, but not necessarily in amount.

12. The method according to claim 11, wherein said equivalent comprises peptide nucleic acid, locked nucleic acid and/or a morpholino phosphorodiamidate, or a combination thereof.

13. The method according to claim 12, wherein said equivalent comprises locked nucleic acid.

14. The method according to claim 1, wherein the oligonucleotide or the equivalent thereof does not overlap a splice donor and/or a splice acceptor sequence of said exon.

15. An oligonucleotide or an equivalent thereof obtainable by a method according to claim 1.

22. A method of altering recognition of an exon or exons in a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

altering recognition of said exon or exons in a pre-mRNA.

23. A method of preparing a medicament comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

preparing a medicament comprising the oligonucleotide or the equivalent thereof of claim 15.

24. A pharmaceutical preparation comprising an oligonucleotide or an equivalent thereof according to claim 15.

25. A method of preparing a medicament for the treatment of an inherited disease comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

preparing the medicament for the treatment of an inherited disease comprising the oligonucleotide or the equivalent thereof of claim 15.

26. A method of inducing exon skipping in a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

inducing exon skipping in a pre-mRNA.

27. A method of altering exon recognition in a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

altering exon recognition in a pre-mRNA.

28. A method of altering the efficiency with which a splice donor or splice acceptor sequence is used by a splicing machinery, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

altering the efficiency with which a splice donor or splice acceptor sequence is used by a splicing machinery.

29. A method of inducing exon skipping of two or more exons in a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

inducing exon skipping of two or more exons in a pre-mRNA.

30. A method of inducing exon skipping of three or more exons in a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

inducing exon skipping of three or more exons in a pre-mRNA.

31. A method of inducing skipping of at least two exons and a sequence located between said at least two exons (intervening sequence) on a pre-mRNA, said method comprising:

providing the oligonucleotide or the equivalent thereof of claim 15;

and

inducing skipping of at least two exons and a sequence located between said at least two exons (intervening sequence) on a pre-mRNA.

32. The method according to claim 31, wherein said intervening sequence comprises a further exon or exons.

33. A method for altering the efficiency with which an exon in a pre-mRNA is recognized by a splicing machinery, said pre-mRNA being encoded by a gene comprising at least two exons and at least one intron, said method comprising:

providing a transcription system comprising said splicing machinery and said gene, with a first oligonucleotide or an equivalent thereof according to claim 15;

wherein said first oligonucleotide or an equivalent thereof is capable of hybridizing to at least one of said exons; and

allowing for transcription and splicing to occur in said transcription system.

34. The method according to claim 33, wherein said gene comprises at least 3 exons.

35. The method according to claim 33, further comprising providing said transcription system with at least a second oligonucleotide, or an equivalent thereof, according to claim 15, wherein said second oligonucleotide, or equivalent thereof, is capable of hybridizing to at least another of said exons.

36. The method according to claim 35, wherein said first oligonucleotide or equivalent thereof and said second oligonucleotide or equivalent thereof are physically linked to each other.

16. A compound capable of hybridizing to at least two exons in a pre-mRNA encoded by a gene, said compound comprising at least two parts wherein a first part comprises an oligonucleotide having at least 8 consecutive nucleotides that are complementary to a first of said at least two exons, and wherein a second part comprises an oligonucleotide having at least 8 consecutive nucleotides that are complementary to a second of said at least two exons in said pre-mRNA.

17. The compound according to claim 16, wherein said compound comprises between 16 and 80 nucleotides.

18. The compound according to claim 16, wherein said first and said second exon are separated in said pre-mRNA by at least one exon to which said oligonucleotide is not complementary.

19. The compound according to claim 16, wherein stretches of nucleotides complementary to said at least two exons are separated by a linking moiety.

20. The compound according to claim 19, wherein said linking moiety comprises between 4 and 40 nucleotides.

21. The compound according to claim 16, comprising an oligonucleotide or equivalent thereof wherein the equivalent comprises similar hybridization characteristics in kind, but not necessarily in amount.

37. A method of altering recognition of an exon or exons in a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

altering recognition of said exon or exons in a pre-mRNA.

38. A method of preparing a medicament for the treatment of an inherited disease comprising administering to a subject a medicament comprising:

providing the compound of claim 21;

and

preparing a medicament comprising the compound of claim 21.

39. A pharmaceutical preparation comprising a compound according to according to claim 21.

40. A method of preparing a medicament for the treatment of an inherited disease comprising administering to a subject a medicament comprising:

providing the compound of claim 21;

and

preparing the medicament for the treatment of an inherited disease comprising the compound of claim 21.

41. A method of inducing exon skipping in a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

inducing exon skipping in a pre-mRNA.

42. A method of altering exon recognition in a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

altering exon recognition in a pre-mRNA.

43. A method of altering the efficiency with which a splice donor or splice acceptor sequence is used by a splicing machinery, said method comprising:

providing the compound of claim 21;

and

altering the efficiency with which a splice donor or splice acceptor sequence is used by a splicing machinery.

44. A method of inducing exon skipping in two or more exons in a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

inducing exon skipping in two or more exons in a pre-mRNA.

45. A method of inducing exon skipping in three or more exons in a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

inducing exon skipping in three or more exons in a pre-mRNA.

46. A method of inducing skipping of at least two exons and a sequence located between said at least two exons (intervening sequence) on a pre-mRNA, said method comprising:

providing the compound of claim 21;

and

inducing skipping of at least two exons and a sequence located between said at least two exons (intervening sequence) on a pre-mRNA.

47. The method according to claim 46, wherein said intervening sequence comprises a further exon or exons.

48. A method for altering the efficiency with which an exon in a pre-mRNA is recognized by a splicing machinery, said pre-mRNA being encoded by a gene comprising at least two exons and at least one intron, said method comprising:

providing a transcription system comprising said splicing machinery and said gene, with a first compound according to claim 21;

wherein said first compound is capable of hybridizing to at least one of said exons; and

allowing for transcription and splicing to occur in said transcription system.

49. The method according to claim 48, wherein said gene comprises at least 3 exons.

50. The method according to claim 48, further comprising providing said transcription system with at least a second compound according to claim 21, wherein said second compound is capable of hybridizing to at least another of said exons.

51. The method according to claim 50, wherein said first compound and said second compound are physically linked to each other.