鉴于全球范围内爆发/重新出现呼吸道病毒的风险，迫切需要建立新的抗病毒策略。在这项研究中，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 gapmer 反义寡核苷酸 (ASO)，可有效抑制病毒复制。我们合成了大约 300 个gapmer ASO，旨在针对各种 SARS-CoV-2 RNA 区域，并评估它们在基于细胞的测定中的活性。通过细胞培养系统中的多步筛选，我们发现 ASO#41 靶向病毒主要蛋白酶的编码区，可降低受感染细胞中的 SARS-CoV-2 RNA 水平，并抑制病毒诱导的细胞病变效应。在 iPS 细胞衍生的人肺类器官中也观察到了 ASO#41 的抗病毒作用。 ASO#41 在基因组复制过程中以内源 RNaseH 依赖性方式耗尽细胞内病毒 RNA。 ASO#41 对相关 SARS-CoV-2 变体（包括 Alpha、Delta 和 Omicron）表现出广泛的抗病毒活性。对小鼠进行鼻内给药后，ASO#41 在肺部出现细胞内积聚，并显着降低了病毒感染滴度，并且由于 SARS-CoV-2 感染而导致的体重减轻较轻。含磷酰胍主链的进一步化学修饰提高了抗 SARS-CoV-2 活性，50% 抗病毒抑制浓度为 23.4 nM，是迄今为止报道的最强的抗 SARS-CoV-2 ASO 之一。我们的研究提出了一种识别针对 SARS-CoV-2 的活性 ASO 的方法，该方法可能有助于通过针对呼吸道病毒的基因组 RNA 来建立抗病毒策略。版权所有 © 2024。由 Elsevier B.V. 出版。

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.Copyright © 2024. Published by Elsevier B.V.