In Silico and In Vitro Development of novel small interfering RNAs (siRNAs) to inhibit SARS-CoV-2

Abstract One of the deadliest pandemics of the 21st century is being driven by SARS-CoV-2, a significant betacoronavirus, causing severe to moderate respiratory tract infections and represents a major public health threat than other human coronaviruses like severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome (MERS), which has been ravaging the world’s health, social life, and the economy. In response to the sixth wave of SARS-CoV-2, we aim to develop novel innovative viral replication inhibitor therapeutics. We achieved highly specific siRNAs by optimizing RNAi efficacy and reducing potential side effects and considering various factors such as target RNA variations, thermodynamics, accessibility of the siRNA, and off-target effects. Out of 258 siRNAs targeting conserved regions, four siRNAs (siRNA1, siRNA2, siRNA3, siRNA4) were chosen based on their predicted potency and high specificity that target critical highly conserved areas (NSP8, NSP12, and NSP14) in the viral genomes of SARS, MERS, and SARS-CoV2 with no predicted human genome off-targets. We assess the effectiveness of the four siRNAs on SARS-CoV2 strain hCoV-19/Egypt/NRC-03/2020. In VeroE6 cells, the selected siRNAs at a concentration 100nM had no cellular toxicity. siRNA2 significantly reduced viral replication with a knockdown percentage of 98% after 24 hr post-infection. In addition, siRNA4 had a statistical significance and knockdown percentage, in S gene and ORF1b gene, of 94% in viral replication. SiRNA2 and siRNA4 could be considered as potential siRNA therapy for SARS-CoV-2 infection.