Genetic Investigation of Covid 19 in Lung Disease

总结 由SARS-CoV-2病毒引起的COVID-19大流行已导致全球数百万人死亡。大多数 的COVID-19死亡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性 COVID-19肺部疾病的机制，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。尽管深入调查，这些假设仍然存在 由于缺乏因果关系的细胞功能证据而无法证实。SARS-CoV-2感染需要病毒结合 的人ACE 2（hACE 2）细胞表面蛋白，和野生型病毒不能结合小鼠ACE 2。现有hACE 2- 表达小鼠或者通过不允许条件分析的非内源性转基因驱动疾病 或感染SARS-CoV-2后未能导致严重疾病。因此，强大的小鼠遗传方法 尚未被用于测试COVID-19的致病机制。为了填补这一知识空白， 产生了新的小鼠遗传模型，（i）从小鼠Ace 2基因座表达hACE 2，其水平足以 导致致死性疾病和缺氧，如在人类患者中观察到的，（ii）允许Cre介导的hACE 2损失 表达以功能性鉴定赋予COVID-19疾病所需的细胞，以及（iii）EST-Cre介导的增益 hACE 2表达，以在功能上鉴定足以治疗COVID-19疾病的细胞。我们的初步研究 鉴别与广泛血管内血栓形成相关的上皮细胞感染和血管疾病， hACE 2基因敲入动物的肺。因此，我们假设COVID-19肺病是由于 协同感染和/或肺上皮细胞和肺血管细胞功能障碍。为了测试这个中央 假设我们将（i）使用已建立的表达Cre的转基因来测试上皮细胞的需要， 在COVID-19肺病期间的血管细胞类型，（ii）使用来自人类的肺切片外植体和hACE 2敲入 小鼠肺，以绘制被SARS-CoV-2病毒感染的细胞，以及（iii）比较急性和慢性肺反应 流感病毒和SARS-CoV-2病毒感染，以确定 COVID-19肺部疾病的异常致命性。这些研究预计将产生一个功能和综合的 了解COVID-19肺部疾病背后的事件，并为以下方面提供坚实的科学基础： 新的治疗方法的发展。

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.