Respiratory Delivery of Highly Conserved Antiviral siRNAs Suppress SARS-CoV-2 Infection

COVID-19 has resulted in over 777 million confirmed cases and more than 7 million deaths globally. While vaccination offers protection for individuals with a functional immune system, immunocompromised populations will not generate sufficient responses, highlighting the critical need for new antiviral treatments. Here we evaluated four highly conserved anti-COVID siRNAs targeting the ORF1a-Nsp1, Membrane, and Nucleocapsid regions by identifying their antiviral efficacy in vitro and investigated the direct delivery of naked siRNAs to the respiratory tract of mice via intranasal instillation to provide proof-of-concept evidence of their in vivo antiviral activity. Dose-response analysis of siRNAs revealed a range of IC50 0.02 nM to 0.9 nM. Intranasal administration of naked anti-COVID siRNA-18 in a K18-hACE2 transgenic SARS-CoV-2 mouse model was capable of reducing viral mRNA levels and disease severity. While anti-COVID siRNA-30 induced modest interferon-stimulated gene expression in vitro and immune cell infiltration in vivo, these effects were markedly reduced by 2'-O-methyl-AS456 chemical modification, which preserved antiviral efficacy against SARS-CoV-2 while minimizing off-target immune activation. These results demonstrate the feasibility of direct respiratory siRNA administration for in vivo viral suppression and highlight the benefit of using conserved target sequences and chemical modification to enhance therapeutic safety and efficacy.