From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis

Abstract

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal administration of this siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the gold-standard Syrian hamster model. Our results pave the way for the development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

Biochemistry-informed design selects potent siRNAs against SARS-CoV-2

“…To make the comparison as fair and uniform as possible, we compiled the results of ex vivo assessment of siRNAs against SARS-CoV-2 and flagged ‘high activity’ those siRNAs with asignificant repressive effect on viral RNA accumulation or viral plaque forming units exceeding 50% in the tested conditions (selecting conditions with siRNA concentrations close to 10 nM and post-transfection incubation close to 48 h whenever possible). The proportion of designed siRNAs with high activity ranges from 0 [ 57 , 59 ] to 10/11 [ 61 ] and 1/1 [ 55 ] (see Figure 4 ; note that siRNAs designed by [ 58 ] could not be analysed because they were tested ex vivo against SARS-CoV-2 only at a100 nM concentration). Our approach, yielding 6/8 siRNAs with high activity, proved efficient in designing potent siRNAs with apure bioinformatics approach, without requiring high-throughput experimental screening.…”

“…The main obstacle to siRNA usage invivo lies in the difficulty of delivery to the target organs (in the case of COVID-19: respiratory epithelia). But the administration of anebulized siRNA solution by inhalation is under development [ 30 ] and it gave promising results with chemically-modified or unmodified siRNAs directed against SARS-CoV-2 [ 58 , 61 ]. Optimal timing for siRNA administration would also need to be optimized ( e.g ., administering the siRNAs at various time points before or after viral exposure, then scoring clinical outcomes), because the amplitude of viral replication and its anatonomical localization vary greatly in the course of COVID-19 infection [ 67 ].…”