Robust and durable prophylactic protection conferred by RNA interference in preclinical models of SARS-CoV-2

Abstract

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral genome. We screened lipophilic small-interfering RNA (siRNA) conjugates targeting highly conserved regions of the SARS-CoV-2 genome and identified leads targeting 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. A two-siRNA combination delivered intranasally 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 to limit SARS-CoV-2 infection that may help combat emergent variants, complement existing interventions, or protect populations where vaccines are less effective. Most importantly, this strategy has implications for developing medicines that may be valuable in protecting against future coronavirus pandemics.

SciScore for 10.1101/2022.03.20.485044: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: SARS-CoV-2 infection in Syrian Golden Hamsters: Ethics Statement and Animal Exposure: Animal research was conducted under BIOQUAL Institute Institutional Animal Care and Use Committee (IACUC)-approved protocols, 20-152 in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals. Euthanasia Agents: Prior to all blood collections, animals were anesthetized using Ketamine/Xylazine.
Sex as a biological variable	siRNA dosing and SARS-CoV-2 challenge: Syrian Golden hamsters approximately 6-8 weeks of age, male (N=5-6 per group) were dosed intranasal (IN) with a luciferase siRNA (Ctl-siRNA) or COV-siRNA1+2 at Day −7, Day-14, or Day −30 prior to SARS-CoV-2 infection.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After blocking in 2% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological) at a 1:3000 dilution for 1h followerd by incubation with a secondary AlexaFluor647-labeled antibody.	SARS-CoV-2 nucleocapsid protein suggested: None
Plates were washed three times with PBS and then incubated for 1 hour at RT with 50 μL/well of goat anti-rabbit-Alexa647 (Thermo Fisher Scientific) secondary antibody at 1:1,000 in blocking buffer along with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific).	anti-rabbit-Alexa647 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Dual-luciferase assays: Cos7 cells were co-transfected with any of the reporter plasmids, and siRNAs in 384-well plates format at a density of 5×104 cells per well.	Cos7 suggested: None
SARS-CoV-2 in vitro infection assay: Vero E6 cells were reverse transfected with the corresponding concentration of siRNA in a 96-well plate format using RNAiMax.	Vero E6 suggested: None
Recombinant DNA
Sentences	Resources
The SARS-CoV-2 concatemeric sequences were synthesized and incorporated individually into the psiCHECK2 vector by Blue Heron Biotech [Bothell, WA].	psiCHECK2 suggested: RRID:Addgene_40763)
The final vectors are referred to as psiCHECK2-CoV-2A, psiCHECK2-CoV-2B, and psiCHECK-negCoV2.	psiCHECK-negCoV2 suggested: None
The siRNAs with ≥80% target reduction relative to mock-transfected controls in both psiCHECK-CoV2 plasmids were selected for further evaluations.	psiCHECK-CoV2 suggested: None
Software and Algorithms
Sentences	Resources
Illumina adapters were clipped using Trimmomatic.	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
Read alignment to SARS-CoV-2 Wuhan-Hu-1 reference (NCBI: NC_045512.2) was performed with Burrows-Wheeler Aligner (BWA).	BWA suggested: (BWA, RRID:SCR_010910)
Nucleotide variants were annotated with SnpEff to generate amino acid variants.	SnpEff suggested: (SnpEff, RRID:SCR_005191)
Extensive QCs were performed at read, alignment and variant level using FastQC, samtools, picard, mosdepth, bcftools and MultiQC.	FastQC suggested: (FastQC, RRID:SCR_014583) samtools suggested: (SAMTOOLS, RRID:SCR_002105) MultiQC suggested: (MultiQC, RRID:SCR_014982)
The siRNA sequences directly targeting the SARS-CoV-2 genomic RNA were generated from the exemplar sequence in NCBI Genbank®.	NCBI Genbank® suggested: (NCBI GenBank via FTP, RRID:SCR_010535)
The MAFFT application output is a multiple sequence alignment of the lineage sequences relative to the reference genome.	MAFFT suggested: (MAFFT, RRID:SCR_011811)
The multiple sequence alignment file was processed using a custom R script and the BioConductor Biostrings library to parse the FASTA alignment file and produce a table of insertions, deletions, and substitutions in the lineage sequences relative to the reference by simple pair-wise comparison of each aligned lineage sequence to the reference.	BioConductor suggested: (Bioconductor, RRID:SCR_006442) Biostrings suggested: (Biostrings, RRID:SCR_016949)
Statistical Analysis: Statistical analyses were performed where indicated using Prism v7.01 (GraphPad Software).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: Thank you for sharing your code.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

To address this limitation, we added a second siRNA (COV-siRNA2) as a two-siRNA combination and found this could prevent the emergence of escape mutants. This is consistent with prior results demonstrating that the use of multiple siRNAs to target other CoVs was more effective than a single siRNA (19). Using deep sequencing and a luciferase reporter assay, we confirmed that viral mutations observed with the single siRNA approach accumulated vRNA mismatches at key positions causing a reduction of siRNA efficacy. This finding highlighted the need to employ a multi-siRNA targeting distinct sites or combinations of orthogonal interventions to minimize the risk of viral resistance in any real-world therapeutic applications. Since late 2020, new SARS-CoV-2 variants have steadily replaced the spike D614G variant highly prevalent in the first wave of the pandemic. We performed an exhaustive in silico evaluation of current WHO VOI and VOC including those encoding the receptor binding motif N501Y spike mutation which emerged simultaneously in the variants Alpha, Beta, Gamma, Delta, and Omicron (20–22). While these variants encode substitutions across the viral genome, they possess similar mutation patterns associated with improved affinity for the ACE2, the known entry receptor for SARS-CoV-2. Favorably, the sequences targeted by COV-siRNA1+2 are located far away from the spike encoding region. Our in-silico analysis of public databases suggests no anticipated loss of activity (target ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
No funding statement was detected.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Robust and durable prophylactic protection conferred by RNA interference in preclinical models of SARS-CoV-2

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