Genetic Investigation of Covid 19 in Lung Disease

摘要 由SARS-CoV-2病毒引起的新冠肺炎大流行已导致全球数百万人死亡。大多数人 在新冠肺炎死亡病例中，有三分之一是由以肺泡充盈和严重低氧血症为特征的肺部疾病引起的。 对人类患者、动物模型和培养细胞的描述性研究支持了许多致病因素 新冠肺炎肺病的发病机制，包括直接上皮细胞感染、血管细胞感染和 血栓形成和急性呼吸窘迫综合征。尽管进行了密集的调查，但这些假设仍然存在。 由于缺乏因果关系的细胞功能证据而未经证实。SARS-CoV-2感染需要病毒结合 人ACE2(HACE2)细胞表面蛋白，野生型病毒不能与小鼠ACE2结合。现有hACE2- 表达的小鼠要么通过不允许条件分析的非内源性转基因来驱动疾病 或在感染SARS-CoV-2后未能确诊为严重疾病。因此，强大的小鼠遗传方法 尚未被利用来测试新冠肺炎的致病机制。为了解决我们所拥有的知识差距 产生了新的小鼠遗传模型，该模型(I)从小鼠ACE2基因座表达hACE2，水平足以 在人类患者中观察到的致命性疾病和缺氧，(Ii)允许Cre介导的hACE2丢失 表达以功能性地识别授予新冠肺炎病所需的细胞，以及(Iii)允许-Cre介导的收益 表达的hACE2，以从功能上鉴定足以应对新冠肺炎病的细胞。我们的初步研究 明确上皮细胞感染和与广泛血管内血栓形成相关的血管疾病 HACE2基因敲打动物的肺。因此我们假设新冠肺炎肺部疾病的发生是由于 肺上皮细胞和肺血管细胞的协同感染和/或功能障碍。为了测试这个中心 假设我们将(I)使用已建立的表达Cre的转基因来测试对上皮细胞和 新冠肺炎肺疾病中血管细胞类型的研究(II)人肺切片及hACE2基因敲除 (Iii)比较急性和慢性肺反应 被流感和SARS-CoV-2病毒感染，以确定导致 新冠肺炎肺病的特殊致命性。这些研究预计将产生一个功能齐全的 了解新冠肺炎肺部疾病的潜在原因，并为 开发新的治疗方法。

SUMMARY The COVID-19 pandemic due to the SARS-CoV-2 virus has resulted in millions of deaths worldwide. The majority of COVID-19 deaths are caused by lung disease characterized by alveolar filling and severe hypoxemia. Descriptive studies of human patients, animal models and cultured cells have supported numerous pathogenic mechanisms for COVID-19 lung disease, including direct epithelial cell infection, vascular cell infection and thrombosis, and acute respiratory distress syndrome. Despite intense investigation, these hypotheses remain unproven due to a lack of cellular functional evidence for causality. SARS-CoV-2 infection requires viral binding of the human ACE2 (hACE2) cell surface protein, and wild-type virus cannot bind mouse ACE2. Existing hACE2- expressing mice either drive disease through non-endogenous transgenes that do not permit conditional analysis or fail confer severe illness after infection with SARS-CoV-2. Thus, powerful mouse genetic approaches have not yet been harnessed to test COVID-19 pathogenic mechanisms. To address this gap in knowledge we have generated new mouse genetic models that (i) express hACE2 from the mouse Ace2 locus at levels sufficient to confer lethal disease and hypoxia like that observed in human patients, (ii) permit Cre-mediated loss of hACE2 expression to functionally identify cells required to confer COVID-19 disease, and (iii) permit-Cre mediated gain of hACE2 expression to functionally identify cells sufficient for COVID-19 disease. Our preliminary studies identify both epithelial cell infection and vascular disease associated with extensive intravascular thrombosis in the lungs of hACE2 knockin animals. We therefore hypothesize that COVID-19 lung disease arises due to synergistic infection and/or dysfunction of both lung epithelial and lung vascular cells. To test this central hypothesis we will (i) use established Cre-expressing transgenes to test the requirement(s) for epithelial and vascular cell types during COVID-19 lung disease, (ii) use lung slice explants from human and hACE2 knockin mouse lungs to map the cells infected by SARS-CoV-2 virus, and (iii) compare acute and chronic lung responses to infection by the influenza and SARS-CoV-2 viruses to identify the pathogenic mechanisms that underlie the exceptional lethality of COVID-19 lung disease. These studies are expected to yield a functional and integrated understanding of the events that underlie COVID-19 lung disease and provide a solid scientific foundation for the development of novel therapeutic approaches.