Multiple rna polymerase iii promoter expression constructs

Abstract

Expression constructs comprising at least two different RNA polymerase III promoters, wherein each promoter is operably linked to a nucleic acid sequence encoding an RNA effector molecule, are disclosed herein. Further provided are expression constructs comprising multiple polymerase III promoters operably linked to sequences encoding short hairpin RNA molecules, which may comprise single and/or multiple fingers. The provided constructs are useful for in vivo delivery of RNA molecules effective in gene silencing, including of viral genes including HBV and HCV.

1 . An expression construct comprising an RNA polymerase III promoter of SEQ ID NO:5. 
     
     
         2 . The expression construct of  claim 1 , wherein the promoter of SEQ ID NO: 5 is operatively linked to a nucleic acid sequence encoding an RNA effector molecule wherein the RNA effector molecule is capable of decreasing the expression of a target gene. 
     
     
         3 . The expression construct of  claim 2 , wherein the RNA effector molecule is a short hairpin RNA sequence. 
     
     
         4 . The expression construct of  claim 1 , wherein the RNA polymerase III promoter comprises SEQ ID NO: 5 except that nucleotides 241 to 244 comprise gggg. 
     
     
         5 . The expression construct of  claim 3 , wherein the short hairpin RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; wherein U is substituted for T. 
     
     
         6 . The expression construct of  claim 2 , wherein the target gene is a viral gene. 
     
     
         7 . The expression construct of  claim 6 , wherein said viral gene is an HBV, HCV, HIV, HPV, or West Nile Virus gene. 
     
     
         8 . The expression construct of  claim 1 , wherein a termination signal is operably linked to the RNA polymerase LEI promoter of SEQ ID NO:5. 
     
     
         9 . The expression construct of  claim 1  further comprise a bacterial origin of replication and a bacterial antibiotic resistance gene. 
     
     
         10 . A pharmaceutical composition comprising the expression construct of  claim 1 . 
     
     
         11 . A composition comprising one or more expression constructs, wherein one expression construct is that of  claim 1  with an RNA polymerase III promoter comprising SEQ ID NO:5. 
     
     
         12 . The composition of  claim 11 , wherein the promoter of SEQ ID NO: 5 is operatively linked to a nucleic acid sequence encoding an RNA effector molecule wherein the RNA effector molecule is capable of decreasing the expression of a target gene. 
     
     
         13 . The composition of  claim 11 , wherein the RNA effector molecule is a short hairpin RNA sequence. 
     
     
         14 . The composition of  claim 11 , wherein the RNA polymerase III promoter comprises SEQ ID NO: 5 except that nucleotides 241 to 244 comprise gggg. 
     
     
         15 . The composition of  claim 13 , wherein the short hairpin RNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; wherein U is substituted for T. 
     
     
         16 . The composition of  claim 12 , wherein the target gene is a viral gene. 
     
     
         17 . The composition of  claim 16 , wherein said viral gene is an HBV, HCV, HIV, HPV, or West Nile Virus gene. 
     
     
         18 . The composition of  claim 11 , wherein no more than one copy of the same RNA polymerase III promoter is present in the composition. 
     
     
         19 . A pharmaceutical composition comprising the composition of  claim 11 .