High resistance barrier and prophylactic protection in preclinical models of SARS-CoV-2 with two siRNA combination

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 the 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 medicines that may protect against future coronavirus pandemics. To determine the barrier to drug resistance for the lead siRNAs, we used in vitro viral serial passaging method. SARS-CoV-2 virus was passaged five times in the presence of either single-siRNA (COV-siRNA1 or COV-siRNA2) or the two-siRNA combination (COV-siRNA1+2) at 5x, 10x, and 20x of EC50 determined from earlier experiments.