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.
We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63-66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action.


OBJECTIVE
This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle, if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.
SomaGenics, Inc., Santa Cruz, California, USA.
Anne
A
Dallas
Heini
H
Ilves
Han
H
Ma
Daniel J
DJ
Chin
Ian
I
Maclachlan
Klaus
K
Klumpp
Brian H
BH
Johnston
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism*; D004195:Disease Models, Animal; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology; D008297:Male; D051379:Mice; D009154:Mutation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D017421:Sequence Analysis
D000998:Antiviral Agents; D034741:RNA, Small Interfering