Isoform-specific targeting of splice variants

• US 20050048495A1
• Filed: 08/29/2003
• Published: 03/03/2005
• Est. Priority Date: 08/29/2003
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Baker, Brenda F., Vickers, Timothy A.
• Application Number:
  US10/651,772
• Publication Number:
  US 20050048495A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Compounds, compositions and methods are provided for modulating the expression of splice variants or products. The compositions comprise oligonucleotides, targeted to nucleic acid encoding splice variants or products. Methods of using these compounds for modulation of expression of splice variants or products and for diagnosis and treatment of disease associated with expression of splice variants or products are provided.

Claims

1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding galectin 9 or a galectin 9 variant, wherein said compound specifically hybridizes with said nucleic acid molecule encoding galectin 9 and inhibits the expression of galectin 9.

2. The compound of claim 1 comprising 12 to 50 nucleobases in length.

3. The compound of claim 2 comprising 15 to 30 nucleobases in length.

4. The compound of claim 1 comprising an oligonucleotide.

5. The compound of claim 4 comprising an antisense oligonucleotide.

6. The compound of claim 4 comprising a DNA oligonucleotide.

7. The compound of claim 4 comprising an RNA oligonucleotide.

8. The compound of claim 4 comprising a chimeric oligonucleotide.

9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding galectin 9 said compound specifically hybridizing to and inhibiting the expression of galectin 9.

11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding galectin 9 said compound specifically hybridizing to and inhibiting the expression of galectin 9.

12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding galectin 9 said compound specifically hybridizing to and inhibiting the expression of galectin 9.

13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding galectin 9 said compound specifically hybridizing to and inhibiting the expression of galectin 9.

14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.

15. The compound of claim 1 having at least one 2′

-O-methoxyethyl sugar moiety.

16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.

17. The compound of claim 1 having at least one 5-methylcytosine.

18. A method of inhibiting the expression of galectin 9 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of galectin 9 is inhibited.

22. A kit or assay device comprising the compound of claim 1.

23. A method of treating an animal having a disease or condition associated with galectin 9 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of galectin 9 is inhibited.

19. A method of screening for a modulator of galectin 9, the method comprising the steps of:

a. contacting a target segment of a nucleic acid molecule encoding galectin 9 with one or more candidate modulators of galectin 9, and

b. identifying one or more modulators of galectin 9expression which modulate the expression of galectin 9.

20. The method of claim 19 wherein the modulator of galectin 9 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.

21. A diagnostic method for identifying a disease state comprising identifying the presence of galectin 9 in a sample using at least one of the primers comprising SEQ ID NOs 6 or 7, or the probe comprising SEQ ID NO:

8.

24. A method of selectively reducing expression of a splice variant of a target comprising contacting said splice variant with a target specific oligonucleotide.

25. A method of modulating gene expression of a splice variant encoding a polypeptide in a cell comprising contacting the cell with an oligomeric compound comprising one or more double-stranded regions, said oligomeric compound targeted to a site of an mRNA, said mRNA encoding the polypeptide, wherein said site is unique to the splice variant.

26. A method of claim 25 wherein the mRNA site unique to the splice variant is intronic.

27. A method of claim 26 wherein the mRNA site unique to the splice variant comprises at least a portion of the donor junction of the intron.

28. A method of claim 26 wherein the mRNA site unique to the splice variant comprises at least a portion of the acceptor junction of the intron.

29. A method of claim 25 wherein said modulation is through cleavage of the mRNA of the splice variant.

30. A method of claim 25 wherein the mRNA site unique to the splice variant spans an intron/exon junction.

31. A method of claim 30 wherein at least 50% of the nucleobases of the oligomeric compound are complementary to intronic sequences of said mRNA.

32. A method of claim 25 wherein said oligomeric compound comprises:

a first segment;

a second segment; and

,

optionally a third segment comprising three or four nucleobases.

33. A method of claim 25 wherein said oligomeric compound comprises:

a first segment;

a second segment; and

,

a third segment comprising three or four nucleobases, said third portion located between said first and second segments.

34. A method of claim 33 wherein said third segment and said second segment are complementary to at least a portion of one or more introns of said mRNA.

35. A method of claim 33 wherein said first segment and said third segment are complementary to at least a portion of one or more introns of said mRNA.

36. A method of claim 25 wherein said oligomeric compound comprises a sense and antisense strand, each of which comprises from about 8 to about 80 nucleobases.

37. A method of claim 36 wherein the sense and antisense strands comprise an unequal number of nucleobases.

38. A method of claim 36 wherein the sense strand or the antisense strand comprises an overhang comprising two or more nucleobases.

39. A method of claim 38 wherein the overhang is a 3′

overhang.

40. A method of claim 36 wherein the sense strand and the antisense strand both comprise an overhang comprising two or more nucleobases.

41. A method of claim 25 wherein the splice variant of the polypeptide is associated with a disease or disorder.

42. A method of claim 25 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

43. A method of claim 25 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

44. A method of claim 25 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

45. A method of claim 25 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

46. A method of claim 25 wherein the oligomeric compound has at least one 5-methylcytosine.

47. A method of claim 25 wherein the oligomeric compound comprises at least one hairpin region.

48. A method of claim 25 wherein the oligomeric compound comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one modified nucleoside.

49. A method of claim 25 wherein the oligomeric compound comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one modified nucleoside;

said modified nucleoside adapted to modulate at least one of;

binding affinity or binding specificity of said oligomeric compound comprising one or more double-stranded regions.

50. A method of claim 25 wherein the oligomeric compound modulates expression of the splice variant by at least 60%.

51. A method of claim 25 wherein the oligomeric compound has at least 2 mismatches as compared to the complement of the target RNA.

52. A method of claim 25 wherein the mismatches are internal or external base mismatches.

53. A method of claim 25 wherein the oligomeric compound is a gapmer.

54. A method of claim 25 wherein the oligomeric compound is RNA.

55. A method of modulating gene expression of a polypeptide in a cell, said gene producing at least two splice variants, said splice variants comprising at least a first and second splice variant, the method comprising contacting the cell with an oligomeric compound comprising one or more double-stranded regions, said oligomeric compound targeted to a site of the mRNA, said mRNA encoding the polypeptide, wherein the site is unique to the first splice variant.

56. A method of claim 55 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

57. A method of claim 55 wherein the oligomeric compound comprising one or more double-stranded regions has a modification at the 2′

position of at least one sugar.

58. A method of claim 55 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

59. A method of claim 55 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

60. A method of claim 55 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

61. A method of modulating the expression of at least one splice variant of a gene, wherein said gene expresses at least a first and second splice variant, the method comprising contacting the cell with an oligomeric compound comprising one or more double-stranded regions, said oligomeric compound targeted to a site of the mRNA, said mRNA encoding the polypeptide, wherein said site is unique to the first splice variant.

62. A method of claim 61 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

63. A method of claim 61 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

64. A method of claim 61 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

65. A method of claim 61 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

66. A method of claim 61 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

67. A method of selectively expressing one or more splice variants of a gene, said gene expressing at least a first and a second splice variant, said method comprising contacting a cell with one or more oligomeric compounds, said oligomeric compound comprising one or more double-stranded regions, said oligomeric compound targeted to a site of an mRNA of a first splice variant, wherein said site of the mRNA of the first splice variant is absent in an mRNA of the second splice variant.

68. A method of claim 67 further comprising aligning mRNA sequences of two or more splice variants to determine a unique target site on an mRNA of a first splice variant, wherein said target site is absent in the mRNA of said second splice variant.

69. A method of claim 67 wherein binding of the oligomeric compound to the target site of the mRNA elicits cleavage of the mRNA.

70. A method of claim 67 wherein said oligomeric compound has at least 90% sequence homology to the complement of the target site of the mRNA of the first splice variant.

71. A method of claim 67 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

72. A method of claim 67 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

73. A method of claim 67 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

75. A method of claim 67 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

76. A method of claim 67 wherein the expression of said first splice variant in the presence of the oligomeric compound is reduced by at least 50% relative to expression of the first splice variant in the absence of the oligomeric compound.

74. A method of claim D wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

77. An oligomeric compound comprising one or more double-stranded regions, said oligomeric compound specifically hybridizable with a site in an mRNA of a first splice variant wherein said site is absent in an mRNA of a second splice variant.

78. An oligomeric compound of claim 77 wherein said oligomeric compound inhibits expression of said first splice variant by at least 50% relative to expression of the first splice variant in the absence of the oligomeric compound.

79. A compound of claim 77 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

80. A compound of claim 77 wherein the oligomeric compound comprising one or more double-stranded regions has a modification at the 2′

position of at least one sugar.

81. A compound of claim 77 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

82. A compound of claim 77 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

83. A compound of claim 77 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

84. A composition comprising an oligomeric compound comprising one or more double-stranded regions, said oligomeric compound hybridized to an mRNA encoded by a gene, said gene encoding at least a first and a second splice variant, wherein said mRNA comprises a target site of the mRNA of the first splice variant, wherein said target site is absent in the mRNA of the second splice variant.

85. A composition of claim 84 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

86. A composition of claim 84 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

87. A composition of claim 84 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

88. A composition of claim 84 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

89. A composition of claim 84 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

90. A composition of claim 84 further comprising a pharmaceutically acceptable carrier or excipient.

91. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the 5′

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

untranslated region of the mRNA.

92. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the 5′

untranslated region.

93. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the start codon region.

94. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the coding region.

95. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the stop codon region.

96. A composition of claim 84 wherein the oligomeric compound specifically hybridizes to the 3′

untranslated region.

97. A composition of claim 84 wherein the oligomeric compound comprises SEQ ID NO:

16, 17, 18, 19, 20, 21, 22, or 23.

98. A method of selectively expressing a second splice variant of a gene while inhibiting expression of a first splice variant, comprising contacting a cell with one or more oligomeric compounds comprising one or more double-stranded regions, said oligomeric compound targeted to a site on a mRNA of said first splice variant, wherein said site on the mRNA of the first splice variant is absent in the mRNA of the second splice variant, thereby expressing the second splice variant while inhibiting expression of the first splice variant.

99. A method of claim 98 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

100. A method of claim 98 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

101. A method of claim 98 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

102. A method of claim 98 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

103. A method of claim 98 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

104. A method of modulating a disease or disorder associated with the expression of a first splice variant of a gene, said gene encoding at least a first and a second splice variant, said method comprising contacting a cell with one or more oligomeric compounds comprising one or more double-stranded regions, said oligomeric compound targeted to a site of an mRNA of the first splice variant, wherein said target site is absent in an mRNA of the second splice variant.

105. A method of claim 104 wherein the disease or disorder associated with the expression of a first splice variant is selected from the group consisting of hyperproliferative diseases and cancers, inflammatory conditions, diseases related to cell differentiation and homeostasis, autoimmune disorders;

movement disorders, CNS disorders, infections, metabolic and cardiovascular diseases and disorders and hormonal disorders.

106. A method of claim 104 wherein the gene is FGFR1, FGFR2, FGFR3, FGFR4, CD44, BDNF, HER2, Trx-1, PTPN6, Bcl-x, Bax, FHIT, p73, mdm2, BRCA1, BRCA2, CRK or XPG.

107. A method of claim 104 wherein said disease or disorder is not associated with the expression of said second splice variant.

108. A method of claim 104 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

109. A method of claim 104 wherein the oligomeric compound comprising one or more double-stranded regions has a modification at the 2′

position of at least one sugar.

110. A method of claim 104 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

111. A method of claim 104 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

112. A method of claim 104 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

113. A method of modulating a disease or disorder associated with the expression of a first splice variant of a gene, said gene encoding at least a first and a second splice variant, said method comprising contacting a cell with one or more oligomeric compounds comprising one or more double-stranded regions, said oligomeric compound targeted to a site of an mRNA of said first splice variant, wherein said target site is absent in the mRNA of the second splice variant.

114. A method of claim 113 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

115. A method of claim 113 wherein the oligomeric compound comprising one or more double-stranded regions has a modification at the 2′

position of at least one sugar.

116. A method of claim 113 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

117. A method of claim 113 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

118. A method of claim 113 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.

127. A method of any one of claims 25, 55, 61, 67, 98, 113 or 119 wherein the gene is galectin 9, FGFR1, FGFR2, FGFR3, FGFR4, CD44, BDNF, HER2, Trx-1, PTPN6, Bcl-x, Bax, FHIT, p73, mdm2, BRCA1, BRCA2, CRK or XPG.

119. A method of inhibiting the expression of a first splice variant of a gene in a cell without inhibiting the expression of a second splice variant, said method comprising contacting the cell with one or more oligomeric compounds comprising one or more double-stranded regions, said oligomeric compound targeted to a site of an mRNA of the first splice variant, said target site absent in the mRNA of a second splice variant.

120. A method of claim 119 wherein expression of the first splice variant is reduced at least 50% while expression of the second splice variant is reduced by no more than 20%.

121. A method of claim 119 wherein expression of the first splice variant is reduced at least 70% while expression of the second splice variant is reduced by no more than 10%.

122. A method of claim 119 wherein the oligomeric compound has at least one modified internucleoside linkage, sugar moiety, or nucleobase.

123. A method of claim 119 wherein the oligomeric compound has a modification at the 2′

position of at least one sugar.

124. A method of claim 119 wherein the oligomeric compound has at least one 2′

-O-methoxyethyl sugar moiety.

125. A method of claim 119 wherein the oligomeric compound has at least one phosphorothioate internucleoside linkage.

126. A method of claim 119 wherein the oligomeric compound modulates expression of the first splice variant by at least 60%.