# siRNA Cocktail Targeting Multiple Enterovirus 71 Genes Prevents Escape Mutants and Inhibits Viral Replication

## TL;DR

Using a mix of siRNAs targeting multiple genes of Enterovirus 71 prevents resistance and stops the virus from replicating.

## Contribution

The study introduces a combination siRNA therapy that prevents the emergence of EV71 escape mutants.

## Key findings

- Combination siRNA therapy effectively suppressed EV71 escape mutants over five passages.
- Single siRNA treatment led to rapid resistance with mutations in target sites.
- Multiple siRNA targeting showed additive effects in inhibiting viral replication.

## Abstract

RNA interference (RNAi) is a powerful mechanism of post-transcriptional gene regulation in which small interfering RNA (siRNA) is utilized to target and degrade specific RNA sequences. In this study, experiments were conducted to evaluate the efficacy of combination siRNA therapy against enterovirus 71 (EV71) and the potential of this therapy to delay or prevent the emergence of resistance in vitro. siRNAs targeting multiple genes of EV71 were designed, and the effects of a cocktail of siRNAs on viral replication were assessed compared to those of single-siRNA treatment. Cotransfection of multiple siRNAs targeting different protein-coding genes of the EV71 genome effectively suppressed escape mutants resistant to RNAi. Combination therapy with siRNAs targeting multiple viral genes successfully prevented viral escape mutations over five passages. By contrast, serial passaging with a single siRNA led to the rapid emergence of resistance, with mutations identified in the siRNA target sites. The combination of siRNAs specifically targeting different regions demonstrated an additive effect and was more effective than individual siRNAs at inhibiting EV71 replication. This study supports the effectiveness of combination therapy using siRNAs targeting multiple genes of EV71 to inhibit viral replication and prevent the emergence of resistant escape mutants. Overall, the findings identify RNAi targeting multiple viral genes as a potential strategy for therapeutic development against viral diseases and for preventing the emergence of escape mutants resistant to antiviral RNAi.