Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly 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 with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to 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

“…Several studies have already described the design and experimental evaluation of siRNAs against SARS-CoV-2, with some siRNAs being more active than others Khaitov et al, 2021; Idris et al, 2021; Tolksdorf et al, 2021; Niktab et al, 2021; Sohrab et al, 2022b; Yogev et al, 2022; Sohrab et al, 2022a; Friedrich et al, 2022; Chang et al, 2022; Becker et al, 2022; Ambike et al, 2022; Nabiabad et al, 2022. Comparing success rates among diverse studies is not straightforward because of methodological differences between studies.…”

“…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 a significant 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 Sohrab et al, 2022b; Sohrab et al, 2022a to 10/11 Chang et al, 2022 and 1/1 Tolksdorf et al, 2021 (see Figure 4; note that siRNAs designed by Yogev et al, 2022 could not be analyzed because their sequence was not released). Our approach, yielding 6/8 siRNAs with high activity, proved efficient in designing potent siRNAs with a pure bioinformatics approach, without requiring high-throughput experimental screening.…”

“…Significance was assessed using Dunnett's test, with the "∅" siRNA as a reference; p-values are indicated above each graph, with p-values lower than 0.05 being shown in red. designed siRNAs with high activity ranges from 0 Sohrab et al, 2022b ;Sohrab et al, 2022a 4; note that siRNAs designed by Yogev et al, 2022 could not be analyzed because their sequence was not released). Our approach, yielding 6/8 siRNAs with high activity, proved efficient in designing potent siRNAs with a pure bioinformatics approach, without requiring high-throughput experimental screening.…”

“…Several studies have already described the design and experimental evaluation of siRNAs against SARS-CoV-2, with some siRNAs being more active than others Khaitov et al, 2021 ;Idris et al, 2021 ;Tolksdorf et al, 2021 ;Niktab et al, 2021 ;Sohrab et al, 2022b ;Yogev et al, 2022 ;Sohrab et al, 2022a ;Friedrich et al, 2022 ;Chang et al, 2022 ;Becker et al, 2022 ;Ambike et al, 2022 ;Nabiabad et al, 2022. Comparing success rates among diverse studies is not straightforward because of methodological differences between studies. 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 a significant 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).…”

RNA Interference Approach Is a Good Strategy against SARS-CoV-2

“…Because the shRNAs used in this study were designed in May 2020 and the Omicron strain appeared in Nov 2021, the retrospective comparison in Figure 1 showed that the mutation of the SP conserved sequence can also evade shRNA targeting. Therefore, we suggest using the shRNA cocktail to provide better protection or therapeutic effects for COVID-19 [ 31 ]. Four siRNAs have been demonstrated to bind complementary sequences in the SARS-CoV-2 genome and presented in every clinically relevant published SARS-CoV-2 genome, including variants α, β, γ, and Omicron, effectively reducing viral load [ 32 ].…”