Laboratory of Preventive Veterinary Medicine and Animal Health, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.
Nihon University Veterinary Research Center, Fujisawa, Kanagawa 252-0880, Japan.
Int J Mol Sci. 2023 Oct 2;24(19):14846. doi: 10.3390/ijms241914846.
RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.
依赖 RNase H 的 gapmer 反义寡核苷酸（ASO）是一种有前途的治疗方法，通过与靶 RNA 特异性结合并降解靶 RNA。然而，抗病毒 gapmer ASO 的疗效和机制仍不清楚。在这里，我们研究了含有锁核酸（LNA gapmer）的 gapmer ASO 对高致死性增殖的蚊传黄病毒、日本脑炎病毒（JEV）的抑制作用。我们设计了几种针对 JEV 基因组 RNA 的 3'非翻译区的 LNA gapmer。使用 Vero 细胞通过蚀斑测定进行的体外筛选表明，针对茎环结构的 LNA gapmer 可有效抑制 JEV 的增殖。使用错配的 LNA gapmer 的基于细胞和 RNA 切割的测定表明，抑制病毒产生的机制是 LNA gapmer 通过序列和修饰依赖性方式降解 JEV RNA。令人鼓舞的是，LNA gapmer 在没有明显细胞毒性的情况下，强烈抑制了人神经母细胞瘤细胞中五种主要基因型 I 和 III 的 JEV 株的增殖。数据库搜索显示，我们的 LNA gapmer 与人 RNA 发生非靶标结合的可能性较低。这里观察到的靶病毒 RNA 序列保守性突出了它们对不同 JEV 基因型/株的广谱抗病毒潜力。这项工作将促进用于 JEV 和其他黄病毒感染的抗病毒 LNA gapmer 治疗的开发。
