Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo

The use of RNA interference ( RNAi) to inhibit gene expression is potentially applicable in the treatment of viral infections such as hepatitis B virus ( HBV) persistence. Although efficient HBV gene silencing by short hairpin RNA ( shRNA) expressed from RNA polymerase ( Pol) III promoters has been reported, constitutive high-level transcription may cause harmful side effects. Here, we report an approach that allows the use of a Pol II promoter to improve transcription regulation of expressed RNAi effecters. Pol II [ cytomegalovirus ( CMV)] or Pol III ( U6) promoter cassettes that transcribe anti-HBV primary microRNA ( pri-miR)-122 and pri-miR-31 shuttles were generated. In cultured cells both types of pri-miR-like sequences effected knockdown of markers of viral replication (> 80%) and were processed to form intended 21-nucleotide guides. The concentration of CMV-expressed miRs was similar to 85-fold lower than the U6 shRNA-derived guide RNA. When cells were co-transfected with pri-miR expression cassettes, attenuation of independent RNAi mediated gene silencing was not observed, which is in contrast to the action of U6 shRNA expression cassettes. The efficacy of the anti-HBV pri-miR shuttles in vivo was verified using the murine hydrodynamic injection model. Employing Pol II-expressed pri-miR mimics may be useful in the treatment of HBV infection, and potentially also for generic application in RNAi-based therapy.