Defining therapeutic drug targets for SARS-CoV-2-specific and pan-coronavirus inhibition

项目摘要 由严重呼吸窘迫综合征冠状病毒2型（SARS-CoV-2）引起的COVID-19疾病是 正在肆虐世界人类感染引起的症状范围从 无症状感染者至严重呼吸窘迫和死亡。尽管疫苗和 目前正在研究重新利用的药物，但尚不确定它们是否有效，因此努力 开发额外的治疗和预防方案至关重要。这项研究将利用结果 从已经在实验室进行的多个批量CRISPR敲除筛选中， SARS-CoV-2和其他人类冠状病毒复制所需的。这些筛选利用了来自 两种组织来源，肺和肝，四种人类冠状病毒，包括SARS-CoV-2，两种温度 模拟上下呼吸道的CRISPR文库，以及五种不同的CRISPR文库， 基因组文库，一个专注于人类因素的文库，最近发现与人类基因组中的蛋白质相互作用。 SARS-CoV-2和全基因组文库。在第一个目标中，在每个屏幕中被识别为命中的因素 将交叉比较并优先分为三类：1）SARS-CoV-2特异性，2） SARS-CoV-2和至少一种其他冠状病毒，或3）对任何病毒（Aim 1a）。来自这些类别的命中将使用半阵列CRISPR方法进行细化 (Aim 1b）以及药物和小分子抑制试验（Aim 1c）， 冠状病毒在第二个目标中，将在初级阶段进行优先目标的基因破坏。 人肺细胞（Aim 2a）以及一组冠状病毒对复制和细胞杀伤的影响， 包括SARS-CoV-2在内的所有疾病的发病率（目标2b）。目标1c中发现的抗病毒药物和化合物 将在原代人类肺细胞中测试其对冠状病毒的抗病毒活性。 经验证的因素，可能是SARS-CoV-2特异性的，或可能是 冠状病毒，将是未来深入机制研究和药物开发的目标。

Project Summary COVID-19 disease, caused by severe respiratory distress syndrome coronavirus 2 (SARS-CoV-2), is currently ravaging the world. Infection of humans causes symptoms spanning the spectrum from asymptomatic infection to severe respiratory distress and death. Despite the fact that vaccines and repurposed drugs are currently under study, it is uncertain whether they will be efficacious and so efforts to develop additional treatment and preventative options are crucial. This study will leverage results from multiple bulk CRISPR knockout screens already performed in the lab that identify host factors that SARS-CoV-2 and other human coronaviruses require for replication. These screens utilized cells from two tissue sources, lung and liver, four human coronaviruses including SARS-CoV-2, two temperatures that mimic the upper and lower airway, and five different CRISPR libraries including three druggable genome libraries, a library focused on human factors recently discovered to interact with proteins of SARS-CoV-2 and a full genome library. In the first aim, factors identified as hits in each of the screens will be cross compared and prioritized into three categories: 1) specific to SARS-CoV-2, 2) common to SARS-CoV-2 and at least one other coronavirus, or 3) specific to the lung for any of the viruses (Aim 1a). Hits from these categories will be subjected to refinement using a semi-arrayed CRISPR approach (Aim 1b) as well as drug and small molecule inhibition assays (Aim 1c) in the context of a panel of coronaviruses. In the second aim, gene disruption of the prioritized targets will be performed in primary human lung cells (Aim 2a) and the effect on the replication and cell killing by a panel of coronaviruses, including SARS-CoV-2 will be determined (Aim 2b). Drugs and compounds found as antiviral in Aim 1c will be tested in the primary human lung cells for their antiviral activity against the coronavirus panel. Validated factors, which could be SARS-CoV-2-specific, or possibly factors required generally for coronaviruses, would be targets for future in depth mechanistic studies and drug development.