The emergence of highly pathogenic coronaviruses (CoVs) like SARS-CoV, MERS-CoV, and SARS-CoV-2 has underscored the critical need for effective antiviral therapies. Traditional antiviral strategies employing small-molecule inhibitors have faced challenges due to the limited availability of specific targets and low efficacy against diverse viral strains. In contrast, RNA interference (RNAi) offers a promising alternative by directly targeting and destroying viral RNA as well as exploiting the conserved nature of viral RNA sequences essential for replication. In this study, we present the development of a broad-spectrum small interfering RNA (siRNA) therapeutic targeting multiple human coronaviruses.

Through comprehensive bioinformatic analysis, we identified highly conserved genomic regions across human coronaviruses. Employing an innovative high-throughput virus infection model based on a recombinant SARS-CoV-2 strain expressing GFP, we screened 347 siRNAs and identified two potent siRNAs with picomolar IC50 values. These siRNAs demonstrated remarkable antiviral efficacy against SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-NL63, and HCoV-OC43 targets in a Dual-Luciferase reporter system. In an in vitro infection model, the two selected siRNAs exhibited significant antiviral activity, reducing viral genomic RNA by more than 1000-fold for HCoV-OC43, SARS-CoV-2 and SARS-CoV-1. In a next step, the efficacy of the two siRNAs will be further assessed in an air-liquid interface (ALI) HCoV-OC43 infection model, utilizing biodegradable polymer nanoparticles for siRNA delivery.

This study advances the development of broad-spectrum antiviral therapies against coronaviruses. By harnessing siRNA-based RNA interference, our findings contribute to global efforts in pandemic preparedness and response to emerging viral threats