In Vitro Characterization of the Activity of PF-05095808, a Novel Biological Agent for Hepatitis C Virus Therapy

Abstract

PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adenoassociated 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 understand 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 shRNA molecules required for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

“…To address the issue HCV-infected cells were transduced with a lentiviral vector and we observed effective viral inhibition for both single and dual shRNA (Fig.7a-d, 8a, b). Few contemporary studies have also shown similar synergistic effects on HCV inhibition upon transduction with multiple shRNA expressing adenoviral or adeno-associated viral vectors [14,15,27,28].…”

Inhibition of Hepatitis C Virus in Chimeric Mice by Short Synthetic Hairpin RNAs: Sequence Analysis of Surviving Virus Shows Added Selective Pressure of Combination Therapy

“…In the present context, additional sshRNAs could be combined with SG220 and SG273 to target the most common escape mutants revealed in our study or to target independent, conserved sites. Such combinations of siRNAs targeting different conserved regions have been shown to mitigate the risk of resistance in vitro (60,67). In the case of morbilliviruses, a combination of three siRNAs targeting conserved regions was found to prevent viral escape, but two were insufficient (67).…”

“…The ability of viruses with error-prone polymerases to escape inhibition of siRNA and shRNA agents in vitro is well known (57)(58)(59)(60)(61)(62)(63)(64)(65). Strategies for mitigating resistance selection include targeting highly conserved target sites, increasing the inhibitory quotient, and using combination therapy.…”

Prospects for nucleic acid-based therapeutics against hepatitis C virus

“…Long shRNA can be processed by the host cell machinery into two or more siRNAs. A vector that directs expression of three shRNAs targeting the 5'-UTR and two NS5B regions of the HCV genome showed sequence-specific antiviral activity in the HCV replicon and in infectious virus systems [125,126] . When using a mutant virus with a genome containing an escape mutation against one siRNA, the remaining two siRNAs that could target the mutant virus displayed fully active and effective anti-HCV effects.…”

“…As vector-derived shRNAs are difficult to modify chemically, many researchers have manipulated the shRNA structure [127] and expression strategies by using tissue specific or inducible promoter to improve their usefulness as antivirals [129] . To test siRNA as an anti-HCV therapeutic in animal models, viral delivery systems have been employed [125][126][127]129] . Sakamoto et al [130] used adenovirus to deliver an shRNA expression vector into the livers of transgenic mice that could be induced to express HCV structural proteins by the Cre/loxP switching system.…”