Induction of exon skipping in eukaryotic cells

• US 20090228998A1
• Filed: 03/30/2009
• Published: 09/10/2009
• Est. Priority Date: 09/21/2000
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: van Ommen, Garrit-Jan Boudewijn, van Deutekom, Judith Christina, T., den Dunnen, Johannes Theodorus
• Application Number:
  US12/383,897
• Publication Number:
  US 20090228998A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 15/86 Viral vectors
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• 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 2320/33 : Alteration of splicing
• CPC Class Codes: C12N 2750/14142 : virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Abstract

The present invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, by inducing so-called exon skipping in the cell. Exon-skipping results in mature mRNA that does not contain the skipped exon, which leads to an altered product of the exon codes for amino acids. Exon skipping is performed by providing a cell with an agent capable of specifically inhibiting an exon inclusion signal, for instance, an exon recognition sequence, of the exon. The exon inclusion signal can be interfered with by a nucleic acid comprising complementarity to a part of the exon. The nucleic acid, which is also herewith provided, can be used for the preparation of a medicament, for instance, for the treatment of an inherited disease.

Claims

1-24. -24. (canceled)

25. A method for directing splicing of a dystrophin pre-mRNA in a cell having dystrophin pre-mRNA, the method comprising:

contacting the dystrophin pre-mRNA in the cell with an antisense-oligonucleotide having between 14-40 nucleotides, capable of specifically inhibiting an exon inclusion signal of exon number 2, 8, 29, 43, 44, 45;

46, 50, 51, 52, or 53 in the pre-mRNA, and

allowing splicing of the pre-mRNA.

26. The method according to claim 25, wherein the cell is a cell from an individual suffering from Duchenne Muscular Dystrophy (DMD).

27. The method according to claim 26, wherein the dystrophin pre-mRNA comprises a DMD deletion of one or more exons selected from the groups consisting of exons 3-7, 4-7, 5-7, 6-7, 18-44, 35-43, 44, 44-47, 45, 45-54, 45-52, 50, 50-52, 45-50, 46-47, 46-48, 46 49, 46-51, 46-53, 48-52, 48-50, 49-50, 49-52, 52, 52-63, 51, 51-55, 53, and 53-55.

28. The method according to claim 25, wherein the antisense-oligonucleotide exhibiting the specific inhibition of an exon inclusion signal is obtainable by a method comprising:

providing a second cell with the antisense-oligonucleotide, the cell having pre-mRNA containing the exon,

culturing the second cell to form mRNA in the second cell from the pre-mRNA in the second cell, and

determining whether the exon is absent from the thus formed mRNA in the second cell.

29. The method according to claim 25, further comprising allowing translation of an mRNA produced from splicing of the dystrophin pre-mRNA.

30. The method according to claim 29, wherein the mRNA encodes a functional dystrophin protein.

31. The method according to claim 30, wherein the functional dystrophin protein comprises at least two domains, wherein at least one of the domains is encoded by the mRNA as a result of skipping of at least part of an exon in the dystrophin pre-mRNA.

32. The method according to claim 25, wherein contacting results in activation of a cryptic splice site in a contacted exon.

33. The method according to claim 25, wherein the exon inclusion signal is present in an exon comprising a strong splice donor/acceptor pair.

34. The method according to claim 29, wherein the translation results in a mutant dystrophin protein or a normal dystrophin protein.

35. The method according to claim 34, wherein the mutant dystrophin protein is equivalent to a dystrophin protein of a Becker patient.

36. The method according to claim 25, wherein the antisense-oligonucleotide is capable of specifically inhibiting an exon inclusion signal of exon number 51.

37. The method according to claim 25, wherein the antisense-oligonucleotide comprises a nucleic acid.

38. The method according to claim 37, wherein the nucleic acid comprises a 2′

-O-methyl-oligoribonucleotide or a 2′

-O-methyl-phosphorothioate.

39. The method according to claim 25, wherein the antisense-oligonucleotide contains between 15-25 nucleotides.

40. The method according to claim 25, further comprising:

providing the cell with another antisense-oligonucleotide capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.

41. A method for at least in part decreasing the production of an aberrant dystrophin protein in a cell, the cell comprising dystrophin pre-mRNA comprising exons coding for the aberrant dystrophin protein, the method comprising:

providing the cell with an antisense-oligonucleotide having between 14-40 nucleotides, capable of specifically inhibiting an exon inclusion signal of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53, and

allowing translation of mRNA produced from splicing of the dystrophin pre-mRNA.

42. The method according to claim 41, wherein the antisense-oligonucleotide exhibiting the specific inhibition of an exon inclusion signal is obtainable by a method comprising:

providing a second cell with the antisense-oligonucleotide, the cell having pre-mRNA containing the exon,

culturing the second cell to form mRNA in the second cell from the pre-mRNA in the second cell, and

determining whether the exon is absent from the thus formed mRNA in the second cell.

43. The method according to claim 41, wherein the antisense-oligonucleotide is capable of specifically inhibiting an exon inclusion signal of exon number 51.

44. The method according to claim 41, wherein the dystrophin pre-mRNA comprises a Duchenne Muscular Dystrophy (DMD) deletion of one or more exons selected from the groups consisting of exons 3-7, 4-7, 5-7, 6-7, 18-44, 35-43, 44, 44-47, 45, 45-54, 45-52, 50, 50-52, 45-50, 46-47, 46-48, 46-49, 46-51, 46-53, 48-52, 48-50, 49-50, 49-52, 52, 52-63, 51, 51 55, 53, and 53-55.

45. The method according to claim 41, wherein the antisense-oligonucleotide comprises a nucleic acid.

47. The method according to claim 45, wherein the antisense-oligonucleotide contains between 15-25 nucleotides.

48. The method according to claim 41, further comprising:

providing the cell with another antisense-oligonucleotide capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.

49. A method for determining whether a compound is able specifically inhibit an exon inclusion signal of an exon of a dystrophin pre-mRNA, the exon being selected from the group consisting of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53 and combinations thereof of the dystrophin pre-mRNA, wherein the compound has complementarity to a part of the selected exon, the method comprising:

providing a cell having a dystrophin pre-mRNA containing the exon with the compound,

culturing the cell to allow the formation of an mRNA from the dystrophin pre-mRNA, and

determining whether the exon is absent from the mRNA.

50. The method according to claim 49 further comprising determining in vitro the relative binding affinity of the compound to an RNA molecule comprising the exon.

51. A compound identified by the method according to claim 49.

52. A nucleic acid delivery vehicle comprising the compound of claim 51, or the complement thereof.

53. A nucleic acid delivery vehicle comprising the compound of claim 51.

54. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising:

administering to the subject the compound of claim 51.

55. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising administering to the subject the nucleic acid delivery vehicle of claim 52.

59. A non-human animal provided with the compound of claim 51.

60. The non-human animal of claim 59, further comprising a nucleic acid encoding a human protein.

61. The non-human animal of claim 60, further comprising a silencing mutation in a gene encoding an animal homologue of the human protein.

72. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising administering to the subject the nucleic acid delivery vehicle of claim 53.

56. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising:

administering to the subject an antisense-oligonucleotide, having between 14-40 nucleotides, and having complementarity to an element located within an exon in the pre-mRNA, the exon being selected from the group consisting of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53 and combinations thereof of a dystrophin gene encoding an aberrant protein,

wherein the antisense-oligonucleotide specifically inhibits and interferes with an exon inclusion signal of the exon in the pre-mRNA, thus forming a final mRNA excluding the exon, but encoding a mutant dystrophin protein having Becker mutant'"'"'s functionality.

57. The method according to claim 56, wherein the antisense-oligonucleotide that specifically inhibits and interferes with an exon inclusion signal of the exon in the pre-mRNA is obtainable by a method comprising:

providing a cell with the antisense-oligonucleotide, the cell having a pre-mRNA containing the exon, culturing the cell to form mRNA in the cell from the pre-mRNA in the cell, and determining whether the exon is absent from the thus formed mRNA in the cell.

58. The method according to claim 56, wherein the antisense-oligonucleotide contains between 15-25 nucleotides.

62. A composition comprising:

a nucleic acid or a functional equivalent thereof capable of inhibiting an exon inclusion signal in exon 2, 8, 29, 43, 44, 45, 46, 50, 51 or 53 of a dystrophin pre-mRNA, and having between 14-40 nucleotides.

63. The composition of claim 62, further comprising:

a further nucleic acid or a functional equivalent thereof capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.

64. The composition of claim 62, wherein the nucleic acid is an antisense-oligonucleotide containing between 15-25 nucleotides.

65. A nucleic acid delivery vehicle comprising:

an antisense-oligonucleotide capable of inhibiting an exon-inclusion signal in at least one of exons 2, 8, 29, 43, 44, 45, 50, 51, 52 or 53 of a dystrophin pre-mRNA, wherein the antisense-oligonucleotide contains between 14-40 nucleotides, or

the complement of said antisense-oligonucleotide.

66. The nucleic acid delivery vehicle of claim 65, wherein the antisense-oligonucleotide contains between 15-25 nucleotides.

67. An antisense-oligonucleotide comprising one a nucleic acid sequence selected from the group consisting of

68. The antisense-oligonucleotide of claim 67, containing between 14-40 nucleotides.

69. A nucleic acid delivery vehicle capable of expressing the antisense-oligonucleotide of claim 67.

70. The nucleic acid delivery vehicle of claim 69, wherein the nucleic acid delivery vehicle is a single stranded virus.

71. The nucleic acid delivery vehicle of claim 70, wherein said single stranded virus comprises an adeno-associated virus.