COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 GENE
The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Eg5 gene (Eg5 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the Eg5 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by Eg5 expression and the expression of the Eg5 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Eg5 gene in a cell.
1 . A composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11 (Eg5) gene in a cell, wherein the dsRNA comprises a first sense strand comprising a first sequence and an first antisense strand comprising a second sequence complementary to a first 15 nucleotides of SEQ ID NO:1311, wherein the first sequence is complementary to the second sequence and wherein the dsRNA is between 15 and 30 base pairs in length.
2 . The composition of claim 1 , wherein the first sense strand consists of the nucleotide sequence of SEQ ID NO:135 and the first antisense strand consists of the nucleotide sequence of SEQ ID NO:136.
3 . The composition of claim 2 , wherein each strand of the first dsRNA is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”:
SEQ ID NO: 135 is ucGAGAAucuAAAcuAAcuTsT
SEQ ID NO: 136 is AGUuAGUUuAGAUUCUCGATsT
4 . A composition comprising the composition of claim 1 and a second dsRNA that inhibits expression of a human vascular endothelial growth factor (VEGF) gene in a cell, wherein the second dsRNA comprises a second sense strand comprising a third sequence and a second antisense strand comprising a fourth sequence complementary to a first 15 nucleotides of SEQ ID NO:1242, wherein the third sequence is complementary to the fourth sequence and wherein the second dsRNA is between 15 and 30 base pairs in length.
5 . The composition of claim 4 , wherein the second sense strand consists of the nucleotide sequence of SEQ ID NO:1242 and the second antisense strand consists of the nucleotide sequence SEQ ID NO:1243.
6 . The composition of claim 5 , wherein the second sense and antisense strands are modified as follows to include a 2′-0-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”:
GcAcAuAGGAGAGAuGAGCUsU
(SEQ ID NO: 1242)
AAGCUcAUCUCUCCuAuGuGCusG.
(SEQ ID NO: 1243)
7 . The composition of claim 4 , wherein the first sense strand consists of the nucleotide sequence of SEQ ID NO:135 and the first antisense strand consists of the nucleotide sequence of SEQ ID NO:136 and each strand of the first dsRNA is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”:
SEQ ID NO: 135 is ucGAGAAucuAAAcuAAcuTsT
SEQ ID NO: 136 is AGUuAGUUuAGAUUCUCGATsT;
and
wherein the second sense strand consists of the nucleotide sequence of SEQ ID NO:1242 and the second antisense strand consists of the nucleotide sequence SEQ ID NO:1243, and each strand of the second dsRNA is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”:
GcAcAuAGGAGAGAuGAGCUsU
(SEQ ID NO: 1242)
AAGCUcAUCUCUCCuAuGuGCusG.
(SEQ ID NO: 1243
8 . The composition of claim 1 , wherein the dsRNA comprises at least one modified nucleotide.
9 . The composition of claim 7 , wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
10 . The composition of claim 7 , wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
11 . The composition of claim 7 , wherein the first dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one phosphorothioate.
12 . The composition of claim 5 , wherein at least one dsRNA comprises at least one modified nucleotide.
13 . The composition of claim 12 , wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
14 . The composition of claim 12 , wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
15 . The composition of claim 12 , wherein each dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one phosphorothioate.
16 . The composition of claim 1 , wherein the composition, upon contact with a cell expressing Eg5, inhibits expression of Eg5 gene by at least 40%.
17 . The composition of claim 4 , wherein the composition, upon contact with a cell expressing Eg5 and/or VEGF, inhibits expression of the Eg5 and/or VEGF gene by at least 40%.
18 . The composition of claim 1 , wherein the first dsRNA is 19-21 base pairs in length.
19 . The composition of claim 4 , wherein the first dsRNA is 19-21 base pairs in length and the second dsRNA is 19-23 base pairs in length.
20 . An isolated cell comprising the composition of claim 1 .
21 . An isolated cell comprising the composition of claim 4 .
22 . A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the first dsRNA of the composition of claim 1 .
23 . An isolated cell comprising the vector of claim 22 .
24 . At least one vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the first dsRNA of and at least one strand of the second dsRNA of the composition of claim 4 .
25 . An isolated cell comprising the vector of claim 24 .
26 . A pharmaceutical composition for inhibiting Eg5 gene expression comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
27 . A pharmaceutical composition for inhibiting Eg5 gene expression and VEGF gene expression comprising the composition of claim 4 and a pharmaceutically acceptable carrier.
28 . A method for inhibiting Eg5 gene expression in a cell, the method comprising:
introducing into the cell the composition of claim 1 ; and
maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Eg5 gene, thereby inhibiting expression of the Eg5 gene in the cell.
29 . A method for inhibiting Eg5 gene expression and/or VEGF gene expression in a cell, the method comprising:
introducing into the cell the composition of claim 4 ; and
maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Eg5 gene and/or degradation of the mRNA transcript of the VEGF gene, thereby inhibiting expression of the Eg5 gene and/or VEGF gene in the cell.
30 . A method of treating or managing pathological processes mediated by human Eg5 expression comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the composition of claim 1 .
31 . A method of treating or managing pathological processes mediated by human Eg5 expression and/or human VEGF expression comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the composition of claim 4 .