Antisense oligonucleotides targeting the SARS-CoV-2 nucleocapsid gene decrease viral titers in hamsters

SARS-CoV-2 is a positive, single-stranded RNA coronavirus responsible for the COVID-19 pandemic. The emergence of new variants and the limited efficacy of current antivirals demonstrate the need for novel therapeutic strategies. Here, we present the design, screening, and evaluation of antisense oligonucleotides (ASOs) targeting the SARS-CoV-2 genomic and sub-genomic RNA. By employing a combination of luminescence-based reporter assays and quantitative reverse-transcription PCR (RT-qPCR) in human cells, we selected ASO-N1, directed against the nucleocapsid RNA, as the best candidate to be validated <i>in vivo</i>. Hamsters infected with SARS-CoV-2 were treated with an initial intranasal ASO dose, followed by daily systemic administration. ASO-treated animals showed significantly improved clinical signs, indicated by increased food consumption and reduced weight loss. ASO-N1 induced a sustained reduction in viral RNA and inflammatory cytokine expression in the nasal mucosa and efficiently decreased infectious viral titers in nasal swabs at day 3 post infection. Further optimization of the ASO-N1 sequence with a combination of RNase-H1-dependent and steric blockage chemistries synergistically increased the antiviral efficacy <i>in vitro</i>. Finally, the target site of ASO-N1 remained completely conserved across all major SARS-CoV-2 variants over the past 4 years, demonstrating the potential of nucleocapsid RNA-targeting ASO as a robust antiviral strategy against SARS-CoV-2.