Human RNase III and compositions and uses thereof

• US 20030044941A1
• Filed: 02/20/2002
• Published: 03/06/2003
• Est. Priority Date: 06/06/1996
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Crooke, Stanley T.
• Application Number:
  US10/079,185
• Publication Number:
  US 20030044941A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/316 Phosphonothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• 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 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/26003 : Ribonuclease III (3.1.26.3)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides polynucleotides encoding human RNase III and polypeptides encoded thereby. Methods of using said polynucleotides and polypeptides are also provided.

Claims

1. A method for eliciting modification of a selected RNA target in a cell comprising:

(a) providing an RNA-like polynucleotide hybridizable with said RNA target;

(b) hybridizing the RNA-like polynucleotide and the RNA to form a polynucleotide-target duplex; and

(c) contacting the duplex with a polypeptide comprising an RNase III domain, under conditions selected to effect modification of the duplex by the polypeptide, and modification of the RNA target thereby.

2. The method of claim 1 wherein said modification of the RNA target occurs in the cell'"'"'s nucleus.

3. The method of claim 1 wherein the polypeptide comprising an RNase III domain is an RNase III polypeptide.

4. The method of claim 1 wherein the RNase III polypeptide is a human RNase III polypeptide.

5. The method of claim 1 wherein modification of the selected RNA target is cleavage of the RNA target.

6. The method of claim 1 wherein the polypeptide comprising an RNase III domain is present in enriched amounts.

7. The method of claim 6 wherein the polypeptide comprising an RNase III domain present in enriched amounts is overexpressed or exogenously added.

8. The method of claim 1 wherein the polypeptide comprising an RNase III domain is a purified RNase III polypeptide.

9. The method of claim 1 wherein the RNA-like polynucleotide has a modification at the 2′

position of at least one sugar.

10. The method of claim 1 wherein step (c) is performed within a cell.

11. The method of claim 1 wherein step (b) is performed within a cell.

12. The method of claim 1 wherein step (b) is performed outside a cell.

13. The method of claim 1 wherein at least one furanosyl moiety of the RNA-like polynucleotide is a ribofuranosyl moiety.

14. The method of claim 13 wherein a majority of the furanosyl moieties of the RNA-like polynucleotide are ribofuranosyl moieties.

15. A method for promoting gene silencing in a cell comprising providing to the cell, in an amount effective to promote said gene silencing, a polypeptide comprising an RNase III domain.

16. The method of claim 15 wherein said promotion of gene silencing occurs in the cell'"'"'s nucleus.

17. The method of claim 15 wherein the polypeptide comprising an RNase III domain is an RNase III polypeptide.

18. The method of claim 15 wherein the RNase III polypeptide is a human RNase III polypeptide.

19. The method of claim 15 wherein the RNase III polypeptide is exogenously added.

20. The method of claim 15 wherein the RNase III polypeptide is provided through upregulation of endogenous production of the polypeptide.

21. The method of claim 15 wherein said RNase III polypeptide is a purified RNase III polypeptide.

22. The method of claim 15 wherein said RNase III polypeptide is expressed by an exogenously added vector encoding said RNase III polypeptide.

23. The method of claim 15 wherein said cell is a mammalian cell.

24. The method of claim 15 wherein said cell is a human cell.

25. A method for promoting gene silencing in a cell comprising enriching the amount or activity of RNase III polypeptide in said cell to a level effective to promote said gene silencing.

26. The method of claim 25 wherein said promotion of gene silencing occurs in the cell'"'"'s nucleus.

27. The method of claim 25 wherein said enriching is by exogenous addition of RNase III polypeptide.

28. The method of claim 27 wherein said exogenously added RNase III polypeptide is a purified RNase III polypeptide.

29. The method of claim 25 wherein the RNase III polypeptide is provided through upregulation of endogenous production of the polypeptide.

30. The method of claim 25 wherein said enriching is by addition of a vector encoding the RNase III polypeptide.

31. The method of claim 25 wherein said cell is a mammalian cell.

32. The method of claim 25 wherein said cell is a human cell.

33. A method for promoting gene silencing of a gene in a cell comprising:

(a) providing to said cell a polynucleotide hybridizable with a target RNA encoded by a selected gene whose expression is to be silenced;

(b) hybridizing said polynucleotide and said target RNA to form a polynucleotide-target duplex; and

(c) contacting said duplex with a polypeptide comprising an RNase III domain, under conditions selected to effect cleavage or modification of the target RNA strand of the polynucleotide-target RNA duplex by the polypeptide comprising an RNase III domain, and silencing of the gene thereby.

34. The method of claim 33 wherein said promotion of gene silencing occurs in the cell'"'"'s nucleus.

35. The method of claim 33 wherein the polypeptide comprising an RNase III domain is an RNase III polypeptide.

36. The method of claim 33 wherein the RNase III polypeptide is a human RNase III polypeptide.

37. The method of claim 36 wherein the human RNase III polypeptide comprises an amino acid sequence with at least 90% homology to SEQ ID NO:

2.

38. The method of claim 33 wherein the polynucleotide is provided as a single stranded polynucleotide.

39. The method of claim 33 wherein the polynucleotide is provided as part of a double stranded nucleic acid structure.

40. The method of claim 33 wherein the polynucleotide is an antisense oligonucleotide.

41. The method of claim 33 wherein the polynucleotide is an RNA-like polynucleotide.

42. The method of claim 33 wherein at least one sugar moiety of the polynucleotide is a ribofuranosyl sugar moiety.

43. The method of claim 42 wherein at least 50% of the sugar moieties of the polynucleotide are ribofuranosyl sugar moieties.

44. The method of claim 33 wherein the polynucleotide has at least one modification of the base, sugar or internucleoside linkage.

45. The method of claim 44 wherein the polynucleotide has a modification at the 2′

position of at least one sugar.

46. The method of claim 33 wherein the RNase III polypeptide is present in enriched amounts.

47. The method of claim 46 wherein the RNase III polypeptide present in enriched amounts is overexpressed or exogenously added.

48. The method of claim 46 wherein the RNase III polypeptide is a purified RNase III polypeptide.

49. The method of claim 46 wherein said enriching is by addition of a vector encoding said RNase III polypeptide.

50. The method of claim 46 wherein the RNase III polypeptide is provided through upregulation of endogenous production of the polypeptide.

51. The method of claim 33 wherein said cell is a mammalian cell.

52. The method of claim 33 wherein said cell is a human cell.

53. The method of claim 33 wherein said polynucleotide-target RNA duplex forms inside the cell.

54. The method of claim 33 wherein said polynucleotide-target RNA duplex forms outside the cell.

55. A method for inhibiting the expression of a gene in a cell comprising providing to said cell an agent effective to elicit RNase III modification of double-stranded RNA in a cell.

56. The method of claim 55 wherein said inhibition of gene expression occurs in the cell'"'"'s nucleus.

57. The method of claim 55 wherein said agent is a nucleic acid which is hybridizable with an RNA encoded by the gene whose expression is to be inhibited.

58. The method of claim 55 wherein said RNase III modification is RNase III cleavage.

59. The method of claim 55 wherein the polynucleotide is provided as a single stranded polynucleotide.

60. The method of claim 55 wherein the polynucleotide is provided as part of a double stranded nucleic acid structure.

61. The method of claim 55 wherein the polynucleotide is an antisense oligonucleotide.

62. The method of claim 55 wherein the polynucleotide is an RNA-like polynucleotide.

63. The method of claim 55 wherein at least one sugar moiety of the polynucleotide is a ribofuranosyl sugar moiety.

64. The method of claim 63 wherein at least 50% of the sugar moieties of the polynucleotide are ribofuranosyl sugar moieties.

65. The method of claim 55 wherein the polynucleotide has at least one modification of the base, sugar or internucleoside linkage.

66. The method of claim 65 wherein the polynucleotide has a modification at the 2′

position of at least one sugar.

67. A method for promoting inhibition of expression of a gene in a cell comprising:

(a) providing to said cell a polynucleotide hybridizable with a target RNA encoded by the gene whose expression is to be inhibited;

(b) hybridizing the polynucleotide and the target RNA to to form a polynucleotide-target duplex; and

(c) contacting the duplex with a polypeptide comprising an RNase III domain, under conditions effective to effect cleavage or modification of the target RNA strand of the polynucleotide-target RNA duplex by the RNase III polypeptide, and inhibition of expression of the gene thereby.

68. The method of claim 67 wherein said promotion of inhibition of gene expression occurs in the cell'"'"'s nucleus.

69. The method of claim 67 wherein the polypeptide comprising an RNase III domain is an RNase III polypeptide.

70. The method of claim 69 wherein the RNase III polypeptide is a human RNase III polypeptide.

71. The method of claim 70 wherein the human RNase III polypeptide comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:

2.

72. The method of claim 67 wherein the polynucleotide is provided as a single stranded polynucleotide.

73. The method of claim 67 wherein the polynucleotide is provided as part of a double stranded nucleic acid structure.

74. The method of claim 67 wherein the polynucleotide is an antisense oligonucleotide.

75. The method of claim 67 wherein the polynucleotide is an RNA-like polynucleotide.

76. The method of claim 67 wherein at least one sugar moiety of the polynucleotide is a ribofuranosyl sugar moiety.

77. The method of claim 76 wherein at least 50% of the sugar moieties of the polynucleotide are ribofuranosyl sugar moieties.

78. The method of claim 67 wherein the polynucleotide has at least one modification of the base, sugar or internucleoside linkage.

79. The method of claim 78 wherein the polynucleotide has a modification at the 2′

position of at least one sugar.

80. The method of claim 67 wherein the polypeptide comprising an RNase III domain is present in enriched amounts.

81. The method of claim 80 wherein the polypeptide comprising an RNase III domain and present in enriched amounts is overexpressed or exogenously added.

82. The method of claim 81 wherein the polypeptide comprising an RNase III domain and present in enriched amounts is a purified RNase III polypeptide.

83. The method of claim 81 wherein said enriching is by addition of a vector encoding said polypeptide comprising an RNase III domain.

84. The method of claim 67 wherein said cell is a human cell.

85. The method of claim 67 wherein step (c) is performed within a cell.

86. The method of claim 67 wherein step (b) is performed within a cell.

87. The method of claim 67 wherein step (b) is performed outside a cell.

88. A cell having enhanced RNase III activity over an activity exhibited by a second cell, said second cell not enriched with respect to the amount or activity of RNase III polypeptide.

89. The cell of claim 88 wherein said enhanced RNase III activity is detectable in the cell'"'"'s nucleus.

90. The cell of claim 88 wherein said enhanced RNase III activity is due to overexpression of RNase III.

91. The cell of claim 88 wherein the RNase III polypeptide is provided through upregulation of endogenous production of the RNase III polypeptide.

92. The cell of claim 88 wherein said enhanced RNase III activity is due to exogenously added RNase III.

93. A method for eliciting modification of an RNA target in a cell comprising:

(a) providing an RNA-like polynucleotide hybridizable with said RNA target;

(b) hybridizing the RNA-like polynucleotide and the RNA to form a polynucleotide-target duplex; and

(c) contacting the duplex with a polypeptide comprising an RNase III domain, under conditions selected to effect modification of the duplex by the polypeptide, and modification of the RNA target thereby.

94. A hybrid RNase III comprising at least one domain from a human RNase III and at least one domain from an RNase III of an organism other than human.

95. The hybrid RNase III of claim 94 wherein the non-human RNase III domain is derived from an organism selected from the group consisting of E. coli, S. pombe, C. elegans and S. cerevisiae.