In vitro evaluation of antisense RNA efficacy against filovirus infection, by use of reverse genetics.

Abstract

Recent reports indicate the possibility of using small interfering RNAs (siRNAs) to treat filovirus infections; however, they also show that the effectiveness of this approach is highly dependent on target site selection. Therefore, we explored the application of minigenomes as screening tools to identify functional siRNA targets under biosafety level 2 conditions. siRNA candidates were screened using the minigenome system to identify those with potential antiviral activity, compared with controls with poor predicted function on the basis of design guidelines, or those that were noncomplementary to Zaire ebolavirus (ZEBOV). These findings were then validated in cell culture by use of a previously developed ZEBOV expressing green fluorescent protein (ZEBOV-GFP), which allowed siRNA function to be easily assessed via flow cytometry or focus formation. The most promising siRNA based on minigenome screening, targeting the nucleoprotein (NP) mRNA (ZNP1), also reduced protein expression and decreased viral titers after infection with ZEBOV-GFP to an extent similar to that reported for an siRNA recently shown to be therapeutic in guinea pigs. Minigenome screening appears to be an effective and convenient method of evaluating the therapeutic potential of siRNA targets, and findings suggest that its use would increase success rates in later stages of siRNA testing.