title: Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo.
abstract: 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 approximately 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.
authors.affiliation: Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ely
authors.forename: Tanusha
authors.initials: T
authors.lastname: Naidoo
authors.forename: Steven
authors.initials: S
authors.lastname: Mufamadi
authors.forename: Carol
authors.initials: C
authors.lastname: Crowther
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D035683:MicroRNAs; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D014162:Transfection; D014779:Virus Replication
chemical_list: D035683:MicroRNAs