Screening and Evaluation Scheme for the Identification of Antisense Oligonucleotides with Antiviral Activity Against SARS-CoV-2.

Antisense oligonucleotides (ASOs) are advantageous for the development of antiviral drugs against respiratory viruses. However, the methodology for efficiently identifying antiviral ASOs has not been well established, and to date no ASOs have successfully been developed for treating viral respiratory diseases. In this chapter, we describe a scheme that consists of three stepwise evaluation assays to identify ASOs having antiviral effects against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19). The primary evaluation employs a luciferase-based assay without virus infection, which can be applied to high-throughput screening without the need for a high biosafety level facility. The secondary evaluation exploits a cell-based infection assay that assesses antiviral activity by detecting SARS-CoV-2-induced cytopathic effects, viral RNA, and proteins. Candidate ASOs are then further evaluated in a mouse SARS-CoV-2 infection model by detecting viral propagation and pathogenesis in vivo. This scheme permits systematic identification of antiviral agents starting from a library with a large number of ASOs.