Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation

• US 20030228597A1
• Filed: 03/12/2003
• Published: 12/11/2003
• Est. Priority Date: 04/13/1998
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Freier, Susan M., Baker, Brenda F., McNeil, John, Vickers, Timothy A., Wyatt, Jacqueline R., Cowsert, Lex M., Sasmor, Henri M., Ohashi, Cara, Brooks, Douglas G., Borchers, Alexander H.
• Application Number:
  US10/388,263
• Publication Number:
  US 20030228597A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

Iterative, preferably computer based iterative processes for generating synthetic compounds capable of modulation of target expression are provided. During iterations of the processes, a target nucleic acid sequence is provided or selected, and a library of candidate nucleobase sequences is generated in silico according to defined criteria. A “virtual” oligonucleotide chemistry is chosen and a library of virtual oligonucleotide compounds having the selected nucleobase sequences is generated. These virtual compounds are reviewed and compounds predicted to have particular properties are selected. The selected compounds are robotically synthesized and are preferably robotically assayed for a desired physical, chemical or biological activity. Compounds exhibiting the ability to modulate target expression are identified as target modulators. Target modulators thus generated are used in assays of parameters indicative of biological processes to effect gene function analysis and in assays of parameters indicative of diseases or disorders to effect target valid

Claims

1. A method comprising:

(a) identifying a target;

(b) generating a plurality of virtual compounds targeted to said target;

(c) robotically synthesizing a plurality of real compounds corresponding to at least some of said virtual compounds;

(d) identifying a modulator of said target from said plurality of real compounds;

(e) contacting said modulator with said target in an assay of a biochemical or biological parameter indicative of a biological process to determine one of;

an effect of modulation of said target on said parameter or a lack of an effect of modulation of said target on said parameter, thereby effecting gene function analysis.

2. The method of claim 1 wherein said plurality of real compounds corresponds to a subset of virtual compounds selected from said plurality of virtual compounds.

3. The method of claim 1 wherein said target is a gene.

4. The method of claim 3 wherein the best possible representation of the nucleotide sequence of said gene is obtained using computerized searches of available databases.

5. The method of claim 4 wherein said sequence represents a transcript isoform of said gene.

6. The method of claim 5 wherein the formation of said transcript isoform is directed by alternative splicing.

7. The method of claim 1 wherein said target is a polypeptide-encoding nucleic acid.

8. The method of claim 1 wherein said target is a non-polypeptide-encoding nucleic acid.

9. The method of claim 8 wherein said non-polypeptide-encoding nucleic acid is one of a structural RNA or an enzymatic RNA.

10. The method of claim 1 wherein said plurality of virtual compounds is targeted to functional regions of said target.

11. The method of claim 10 wherein said functional regions are selected from the group consisting of:

the transcription start site, the 5′

cap, the 5′

untranslated region, the start codon, the coding region, the stop codon, the 3′

untranslated region, 5′

splice sites, 3′

splice sites, exons, introns, exon;

intron junctions, intron;

exon junctions, exon;

exon junctions, mRNA destablization signals, mRNA destabilization signals, poly-A signals and 5′

sequences of pre-mRNA.

12. The method of claim 2 wherein said subset of virtual compounds is selected by evaluation of thermodynamic properties of said plurality of virtual compounds in silico.

13. The method of claim 1 wherein the accessibility of said target to said plurality of virtual compounds is evaluated in silico.

14. The method of claim 1 wherein said virtual compounds are 8 to 30 nucleobases in length and specifically hybridize with said target.

15. The method of claim 14 wherein said virtual compounds are antisense compounds.

16. The method of claim 15 wherein said antisense compounds are antisense oligonucleotides.

17. The method of claim 16 wherein said antisense oligonucleotides comprise at least one modified internucleoside linkage.

18. The method of claim 17 wherein said modified internucleoside linkage is a phosphorothioate linkage.

19. The method of claim 16 wherein said antisense oligonucleotides comprise at least one modified sugar moiety.

20. The method of claim 19 wherein said modified sugar moiety is a 2′

-O-methoxyethyl sugar moiety.

21. The method of claim 16 wherein said antisense oligonucleotides comprise at least one modified nucleobase.

22. The method of claim 21 wherein said modified nucleobase is a 5-methylcytosine.

23. The method of claim 14 wherein said virtual compounds are double-stranded oligomeric compounds.

24. The method of claim 23 wherein said double-stranded oligomeric compounds are double-stranded RNA oligomeric compounds.

25. The method of claim 24 wherein said double-stranded RNA oligomeric compounds are siRNAs.

26. The method of claim 23 wherein said double-stranded oligomeric compounds comprise at least one two-nucleobase overhang of deoxythymidine.

27. The method of claim 23 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified internucleoside linkage.

28. The method of claim 27 wherein said modified internucleoside linkage is a phosphorothioate linkage.

29. The method of claim 23 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified sugar moiety.

30. The method of claim 29 wherein said modified sugar moiety is a 2′

-O-methoxyethyl sugar moiety.

31. The method of claim 23 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified nucleobase.

32. The method of claim 31 wherein said modified nucleobase is a 5-methylcytosine.

33. The method of claim 1 wherein said target is expressed in a sample capable of exhibiting said parameter wherein said sample is selected from the group consisting of:

a cell culture, a cell-free extract, a tissue and an animal.

34. The method of claim 1 wherein said modulator is identified by a computer-controlled real-time polymerase chain reaction or a computer-controlled enzyme-linked immunosorbent assay.

35. The method of claim 1 wherein said parameter is the expression of at least one gene related to said biological process.

36. The method of claim 1 wherein said parameter is determined by an assay selected from the group consisting of:

a caspase activity assay, a cell cycle assay, a matrix metalloproteinase activity assay and a tube formation assay.

37. The method of claim 1 wherein the value of said parameter is increased as a result of modulation of said target.

38. The method of claim 1 wherein the value of said parameter is decreased as a result of modulation of said target.

39. The method of claim 1 wherein said biological process is selected from the group consisting of apoptosis, inflammation and angiogenesis.

40. A method comprising:

(a) identifying a target;

(b) generating a plurality of virtual compounds targeted to said target;

(c) robotically synthesizing a plurality of real compounds corresponding to at least some of said virtual compounds;

(d) identifying a modulator of said target from said real compounds;

(e) contacting said modulator with said target in an assay of a biochemical or biological parameter indicative of a disease or disorder to determine one of;

an effect of modulation of said target on said parameter or a lack of an effect of modulation of said target on said parameter, thereby effecting target validation.

41. The method of claim 40 wherein said plurality of real compounds corresponds to a subset of virtual compounds selected from said plurality of virtual compounds.

42. The method of claim 40 wherein said target is a gene.

43. The method of claim 42 wherein the best possible representation of the nucleotide sequence of said gene is obtained using computerized searches of available databases.

44. The method of claim 43 wherein said sequence represents a transcript isoform of said gene.

45. The method of claim 44 wherein the formation of said transcript isoform is directed by alternative splicing.

46. The method of claim 40 wherein said target is a polypeptide-encoding nucleic acid.

47. The method of claim 40 wherein said target is a non-polypeptide-encoding nucleic acid.

48. The method of claim 47 wherein said non-polypeptide-encoding nucleic acid is one of a structural RNA or an enzymatic RNA.

49. The method of claim 40 wherein said plurality of virtual compounds is targeted to functional regions of said target.

50. The method of claim 49 wherein said functional regions are selected from the group consisting of:

the transcription start site, the 5′

cap, the 5′

untranslated region, the start codon, the coding region, the stop codon, the 3′

untranslated region, 5′

splice sites, 3′

splice sites, specific exons, specific introns, exon;

intron junctions, intron;

exon junctions, exon;

exon junctions, mRNA destablization signals, mRNA destabilization signals, poly-A signals and 5′

sequences of known pre-mRNA.

51. The method of claim 41 wherein said subset is selected by evaluation of thermodynamic properties of said plurality of virtual compounds in silico.

52. The method of claim 40 wherein the accessibility of said target to said plurality of virtual compounds is evaluated in silico.

53. The method of claim 40 wherein said virtual compounds are 8 to 30 nucleobases in length targeted to a nucleic acid molecule encoding said target and specifically hybridize with said target.

54. The method of claim 53 wherein said virtual compounds are antisense compounds.

55. The method of claim 54 wherein said antisense compounds are antisense oligonucleotides.

56. The method of claim 55 wherein said antisense oligonucleotides comprise at least one modified internucleoside linkage.

57. The method of claim 56 wherein said modified internucleoside linkage is a phosphorothioate linkage.

58. The method of claim 55 wherein said antisense oligonucleotides comprise at least one modified sugar moiety.

59. The method of claim 58 wherein said modified sugar moiety is a 2′

-O-methoxyethyl sugar moiety.

60. The method of claim 55 wherein said antisense oligonucleotides comprise at least one modified nucleobase.

61. The method of claim 60 wherein said modified nucleobase is a 5-methylcytosine.

62. The method of claim 53 wherein said virtual compounds are double-stranded oligomeric compounds.

63. The method of claim 62 wherein said double-stranded oligomeric compounds are double-stranded RNA oligomeric compounds.

64. The method of claim 63 wherein said double-stranded RNA oligomeric compounds are siRNAs.

65. The method of claim 62 wherein said double-stranded oligomeric compounds comprise at least one two-nucleobase overhang of deoxythymidine.

66. The method of claim 62 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified internucleoside linkage.

67. The method of claim 66 wherein said modified internucleoside linkage is a phosphorothioate linkage.

68. The method of claim 62 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified sugar moiety.

69. The method of claim 68 wherein said modified sugar moiety is a 2′

-O-methoxyethyl sugar moiety.

70. The method of claim 62 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified nucleobase.

71. The method of claim 70 wherein said modified nucleobase is a 5-methylcytosine.

72. The method of claim 40 wherein said target is expressed in a sample capable of exhibiting said parameter wherein said sample is selected from the group consisting of:

a cell culture, a cell-free extract, a tissue and an animal.

73. The method of claim 40 wherein said modulator is identified by a computer-controlled real-time polymerase chain reaction or a computer-controlled enzyme-linked immunosorbent assay.

74. The method of claim 40 wherein said parameter is the expression of at least one gene related to said disease or disorder.

75. The method of claim 40 wherein said parameter is the level of a biochemical component selected from the group consisting of cholesterol, triglyceride, lipoprotein, glucose, insulin and PEPCK.

76. The method of claim 40 wherein said parameter is measured in a rodent.

77. The method of claim 76 wherein said parameter measured in a rodent is selected from the group consisting of survival rate, spleen weight, liver weight and fat pad weight.

78. The method of claim 40 wherein the valuse of said parameter is decreased as a result of modulation of said target.

79. The method of claim 40 wherein the value of said parameter is increased as a result of modulation of said target.

80. A method comprising:

(a) identifying a target;

(b) generating a plurality of virtual compounds designed to modulate said target;

(c) robotically synthesizing a plurality of real compounds corresponding to at least some of said virtual compounds;

(d) identifying at least one modulator of said target by contacting said target with said real compounds and measuring the extent of modulation of said target using an automated means;

(e) performing an automated assay of at least one biological or biochemical parameter indicative of one of;

(i) a biological process, thereby effecting gene function analysis or (ii) a disease or disorder, thereby effecting target validation.

81. A method comprising:

(a) identifying a target;

(b) generating a plurality of virtual double-stranded oligomeric compounds designed to modulate said target;

(c) robotically synthesizing a plurality of real double-stranded oligomeric compounds corresponding to at least some of said virtual double-stranded oligomeric compounds;

(d) identifying at least one modulator of said target by contacting said target with said real double-stranded oligomeric compounds and measuring the extent of modulation of said target using an automated means;

(e) performing an automated assay of at least one biological or biochemical parameter indicative of one of;

(i) a biological process, thereby effecting gene function analysis or (ii) a disease or disorder, thereby effecting target validation.

82. The method of claim 81 wherein said double-stranded oligomeric compounds are double-stranded RNA oligomeric compounds.

83. The method of claim 82 wherein said double-stranded RNA oligomeric compounds are siRNAs.

84. The method of claim 81 wherein said double-stranded oligomeric compounds are 15 to 30 nucleobases in length.

85. The method of claim 81 wherein said double-stranded oligomeric compounds are 20 to 25 nucleobases in length.

86. The method of claim 81 wherein said double-stranded oligomeric compounds comprise at least one two-nucleobase overhang of deoxythymidine.

87. The method of claim 81 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified internucleoside linkage.

88. The method of claim 87 wherein said modified internucleoside linkage is a phosphorothioate linkage.

89. The method of claim 81 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified sugar moiety.

90. The method of claim 89 wherein said modified sugar moiety is a 2′

-O-methoxyethyl sugar moiety.

91. The method of claim 81 wherein both strands of said double-stranded oligomeric compounds comprise at least one modified nucleobase.

92. The method of claim 91 wherein said modified nucleobase is a 5-methylcytosine.