Review 2: "Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

This study aims to develop antivirals against Covid-19 that are not dependent on the immune responses to the surface antigen of SARS-CoV-2. These authors have previously reported the identification of potent siRNAs against HIV and HCV using a massively parallel sensor assay. This work tested a large number of siRNAs in an improved version of their sensor assay and identified 7 hyper-potent siRNAs against live SARS-CoV-2 infection. They further identified five siRNA pairs that exhibited a synergistic effect when used as a cocktail and showed that two such cocktails are active against multiple SARS-CoV-2 variants. Finally, they provided evidence for in vivo protection against SARS-CoV-2 infection in the Syrian hamster model by intranasal administration of one siRNA cocktail. Notably, the authors performed high
throughput mutagenesis to assess siRNA cross-reactivity. They found that the siRNA target site can tolerate a wide variety of mutations without a significant loss of potency. Together, the results from this proof-of-
principle study demonstrates the efficacy of intranasally administered siRNA cocktails as pre-exposure preventatives resilient to the mutational evolution of the RNA virus. Below are only a few minor points
for the authors to consider when revising the manuscript for publication.

1. Add a supplemental table listing the sequences of the 7 siRNAs used in Fig. 3D.
2. Are there nucleotide mutations in the region of the Omicron genome targeted by S5 and Hel14?
3. Caetano Reis e Sousa and colleagues have recently sequenced the virus-derived siRNA (vsiRNAs)
libraries from mouse brain organoids infected with SARS-CoV-2 (Science 373:213-6). It may be interesting to see whether any of these naturally produced vsiRNAs (and their relative abundance) have been tested in this study.
4. interestingly, S9-containing siRNA cocktails are also more effective to suppress SARS-CoV-2
infection than S3 alone (Fig. 3D). Table 1 indicates that the S9 target site is conserved in SARS-CoV. Is S9 target site also no variable among SARS-CoV-2 variants? N protein of both SARS-CoV and SARS-CoV-2 has been identified as dsRNA-binding viral suppressors of RNAi (J Virol 89, 9029-9043 and Sci China Life Sci 63, 1413-1416). Similar VSR activity is essential for viral infection in mammals by suppressing the natural antiviral RNAi response, including several human RNA viruses (dengue, influenza, and enterovirus A71). Thus, I am wondering whether the VSR coding sequence of viruses provides a preferred target for siRNA antivirals.