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PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assess in vitro activity are well defined; we developed new assays to assess this RNA interference agent and so to under-stand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNAmolecules re-quired for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNAmolecules needed to drive antiviral activity proved to be independent of the vector delivery system used fo