COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 GENE

Abstract

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 .