In vitro characterization of the activity of PF-05095808, a novel biological agent for hepatitis C virus therapy.
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 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.
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom. Helenblavender@gmail.com
Helen
H
Lavender
Kevin
K
Brady
Frances
F
Burden
Oona
O
Delpuech-Adams
Hubert
H
Denise
Amy
A
Palmer
Hannah
H
Perkins
Boris
B
Savic
Sarah
S
Scott
Caroline
C
Smith-Burchnell
Phil
P
Troke
J Fraser
JF
Wright
David
D
Suhy
Romu
R
Corbau
D001483:Base Sequence; D001681:Biological Assay; D002213:Capsid; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000628:therapy* / Q000821:virology; D006801:Humans; D008156:Luciferases; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D059367:RNA Cleavage; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012093:Replicon / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
D034741:RNA, Small Interfering; D008156:Luciferases