Viral RNA-Targeting Locked Nucleic Acid Gapmers: Design and In Vitro Evaluation.

Recurrent viral outbreaks, such as coronavirus disease 2019 (COVID-19), seasonal influenza, and flavivirus infections, highlight the urgent need for rapid-response therapies against viral diseases. These outbreaks are mainly caused by viruses with RNA genomes. Accordingly, antisense oligonucleotides (ASOs), which bind target RNA in a sequence-specific manner to modulate its functions, represent a promising modality for developing antiviral agents. Recent studies, particularly those on RNase H-recruiting ASOs incorporating the nucleic acid analogue locked nucleic acid (LNA), known as LNA gapmers, have demonstrated effective antiviral activity via targeted viral RNA degradation. Since the approval of fomivirsen, the first ASO drug targeting cytomegalovirus RNA, in 1998, the development of LNA gapmers has positioned viral RNA degradation as a promising approach for clinical application. Here, using Japanese encephalitis virus (JEV), a flavivirus of major public health concern, as a model system, we describe a step-by-step protocol for the design and in vitro evaluation of the antiviral efficacy and cytotoxicity of JEV RNA-targeting LNA gapmers. The approaches presented here provide a basis for the design and screening of antiviral LNA gapmers and can be applied to other viruses.