Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; Department of Applied Biological Sciences, Tokyo University of Science, Noda, 278-8510, Japan.
Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan.
Antiviral Res. 2024 Oct;230:105992. doi: 10.1016/j.antiviral.2024.105992. Epub 2024 Aug 23.
Given the worldwide risk for the outbreak of emerging/re-emerging respiratory viruses, establishment of new antiviral strategies is greatly demanded. In this study, we present a scheme to identify gapmer antisense oligonucleotides (ASOs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA that efficiently inhibit viral replication. We synthesized approximately 300 gapmer ASOs designed to target various SARS-CoV-2 RNA regions and evaluated their activity in cell-based assays. Through a multistep screening in cell culture systems, we identified that ASO#41, targeting the coding region for viral main protease, reduced SARS-CoV-2 RNA levels in infected cells and inhibited virus-induced cytopathic effects. Antiviral effect of ASO#41 was also observed in iPS cell-derived human lung organoids. ASO#41 depleted intracellular viral RNAs during genome replication in an endogenous RNaseH-dependent manner. ASO#41 showed a wide range of antiviral activity against SARS-CoV-2 variants of concern including Alpha, Delta, and Omicron. Intranasal administration to mice exhibited intracellular accumulation of ASO#41 in the lung and significantly reduced the viral infectious titer, with milder body weight loss due to SARS-CoV-2 infection. Further chemical modification with phosphoryl guanidine-containing backbone linkages provided an elevation of anti-SARS-CoV-2 activity, with 23.4 nM of 50% antiviral inhibitory concentration, one of the strongest anti-SARS-CoV-2 ASOs reported so far. Our study presents an approach to identify active ASOs against SARS-CoV-2, which is potentially useful for establishing an antiviral strategy by targeting genome RNA of respiratory viruses.
鉴于新发/再发呼吸道病毒在全球范围内爆发的风险，急需建立新的抗病毒策略。在本研究中，我们提出了一种方案，以鉴定针对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）RNA 的有效抑制病毒复制的 Gapmer 反义寡核苷酸（ASO）。我们合成了大约 300 种针对各种 SARS-CoV-2 RNA 区域的 Gapmer ASO，并在细胞测定中评估了它们的活性。通过在细胞培养系统中的多步筛选，我们鉴定出靶向病毒主要蛋白酶编码区的 ASO#41 可降低感染细胞中的 SARS-CoV-2 RNA 水平并抑制病毒诱导的细胞病变效应。ASO#41 在 iPS 细胞衍生的人类肺类器官中也表现出抗病毒作用。ASO#41 通过内源性 RNaseH 依赖性方式在基因组复制过程中消耗细胞内病毒 RNA。ASO#41 对包括 Alpha、Delta 和 Omicron 在内的 SARS-CoV-2 关切变异株表现出广泛的抗病毒活性。鼻内给药可使 ASO#41 在肺部细胞内蓄积，并显著降低病毒感染滴度，因 SARS-CoV-2 感染导致的体重减轻减轻。用含有磷酰胍骨架连接的化学修饰进一步提高了抗 SARS-CoV-2 活性，其 50%抗病毒抑制浓度的 50%抑制浓度为 23.4 nM，是迄今为止报道的最强抗 SARS-CoV-2 ASO 之一。我们的研究提出了一种鉴定针对 SARS-CoV-2 的有效 ASO 的方法，这对于通过靶向呼吸道病毒的基因组 RNA 建立抗病毒策略可能是有用的。
