Abstract

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral RNA. We identified potent lipophilic small interfering RNA (siRNA) conjugates targeting highly conserved regions of SARS-CoV-2 outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single siRNA approach. Viral resistance to single siRNA treatment occurred due to emergence of point mutations at critical positions required for siRNA-mediated target binding and cleavage, which led to a loss of siRNA efficacy. With a two-siRNA combination, emergence of mutations within the siRNA binding site was abolished. When delivered intranasally, two-siRNA combination protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach with high resistance barrier to counteract SARS-CoV-2 emergent variants and complement vaccination. Most importantly, given that the siRNAs can be rapidly developed from a new pathogen sequence, this strategy has implications as a new type of preventive medicine that may protect against future coronavirus pandemics.