A recent study posted to the bioRxiv* preprint server demonstrated that RNA interference (RNAi) could prophylactically protect against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
Background
Coronavirus disease 2019 (COVID-19) vaccines have been developed at an unprecedented pace; nonetheless, challenges in global supply, adherence, and the high viral transmission have contributed to the appearance of novel SARS-CoV-2 variants resulting in continual COVID-19 waves. Hence, there is a pressing need to develop novel antiviral tools/therapies to curb the spread of SARS-CoV-2 and enhance vaccination efforts.
The study and findings
In the present study, researchers showed that small-interfering RNAs (siRNAs) conjugated with 2’-O-palmityl (C16) confer prophylactic protection against SARS-CoV-2. The authors screened conserved genomic sequences of SARS-CoV-2, SARS-CoV, and the Middle East respiratory syndrome CoV (MERS-CoV) viruses. More than 1500 conserved sites were identified between SARS-CoV and SARS-CoV-2 genomes and nine sequences between MERS-CoV and SARS-CoV-2. About 349 RNAi target sequences were selected, and siRNAs corresponding to these sites were developed as conjugates of C16.
In vitro activity was assessed by transfection of dual-luciferase reporters. The target sites on the plus-strand of SARS-CoV-2 were introduced into the 3′ untranslated region of Renilla luciferase as concatemers, with firefly luciferase serving as the control. Ninety-one siRNAs were identified with this assay, and the most effective siRNAs were evaluated in vitro in a live virus infection assay.
The researchers transfected Vero E6 cells with siRNAs before SARS-CoV-2 infection, and viral quantification was done using quantitative reverse transcription polymerase chain reaction. (RT-qPCR). The live virus assay detected 23 siRNAs with more than 2-log reduction of viral RNA at 10nm concentration and four siRNAs with > 3-log reduction at 0.1nm (lowest concentration). No siRNA targeting the minus strand of SARS-CoV-2 RNA demonstrated activity.
Next, the two leading siRNA candidates (COV-siRNA1 and COV-siRNA2) targeting the ORF1ab were further assessed using SARS-CoV-2 N protein immunofluorescence assay. These siRNAs demonstrated potent antiviral activity in a dose-dependent manner. The intracellular N protein was undetectable when the candidate siRNAs were used alone or in combination at 10 nm concentration.
Half-maximal effective concentration (EC50) for COV-siRNA1 was 42pm and 86pm for COV-siRNA2. Drug resistance barriers for these siRNAs were determined by passaging the virus five times with COV-siRNA1 alone or both siRNAs at 5x, 10x, and 20x EC50. Escape mutants of the virus appeared with single siRNA treatment. Still, combination treatment presented a barrier to the emergence of escape mutants, indicating that combining two siRNAs targeting the conserved sites is essential to create a barrier to SARS-CoV-2 escape.
Further, the siRNA binding sites in the SARS-CoV-2 genome were deep-sequenced to understand the decreased susceptibility of single siRNA treatment. Point mutations were observed in the binding site of COV-siRNA1 due to serial passaging and not in that of the COV-siRNA2. Mostly, these mutations emerged as triple thymine mutation (TTM) at 6 – 8 antisense nucleotides (nt. 6 – 8), and in higher passages, two other mutations appeared at nt. 10 and nt. 11. The mutations were non-synonymous and synonymous base substitutions; contrastingly, no mutations were observed with the combination treatment at any siRNA binding sites.
The COV-siRNA1 and CoV-siRNA2 target sequences across publicly available 4386 SARS-CoV-2 genomes were evaluated in silico to determine if any mutations appeared at the target sites. The authors observed no mutations at the target sites of COV-siRNA 1 and 2, indicating that these loci have remained conserved without any significant variation, including in the SARS-CoV-2 variants.
Lastly, the efficacy of siRNA combination treatment was investigated in vivo in Syrian hamsters. The two siRNAs were administered seven days before infection, and luciferase-targeting siRNA was the control. SARS-CoV-2 was inoculated prophylactically at day 0. siRNA treatment resulted in dose-dependent protection of hamsters from weight loss.
Additionally, hamsters were separately treated with two siRNAs one day before infection. These animals lost weight at the same rate as the control group due to the delayed onset of the mechanism. Hamsters treated with siRNAs 30 days before infection showed similar results consistent with animals treated seven days before infection. Moreover, double siRNA treatment reduced viral genomic and subgenomic RNA in the lung tissues.
Conclusions
The present study identified several siRNAs with binding sites spanning the entire SARS-CoV-2 genome and observed that the combination of CoV-siRNA1 and CoV-siRNA2 targets the conserved sequences within ORF1ab. This indicated that the conservation of those sites was critical for viral fitness. The preclinical models demonstrated promising results and warrant further clinical research. Overall, the present study’s findings suggest that the combination siRNA treatment could be crucial in mitigating COVID-19 and might serve as potential prophylaxis against severe disease.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
