Genetic Investigation of Covid 19 in Lung Disease

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Project Summary Lethal COVID-19 disease caused by the beta-coronavirus SARS-CoV-2 is manifest as respiratory distress associated with a rapid decline in blood oxygen saturation levels, though other organ defects are often described in patients and clinical case series. Descriptive studies of human patients have proposed numerous pathogenic mechanisms for these findings, including direct epithelial cell infection, vascular cell infection and thrombosis, and released inflammatory cytokines. However, these hypotheses remain purely correlative as causality cannot be rigorously established in human patients. Mouse genetic approaches have not yet been harnessed to test COVID-19 pathogenic mechanisms. To address this gap in knowledge we generated new mouse genetic models that express hACE2 from the mouse Ace2 locus at levels sufficient to confer lethal disease, hypoxia, and pulmonary vascular thrombosis like that observed in human patients. The parent grant initially proposed to work on vascular mechanisms of these phenomenon, including to 1.2) Determine the epithelial cell lineages necessary for either acute ARDS phenotype or long term lung damage by SARS-CoV2 infection; 1.3) Define the contribution of vascular cell infection for either acute ARDS phenotype or long term lung damage by SARS-CoV2 infection; and 3) Compare and contrast the lung vascular and thrombotic effects of influenza versus SARS-CoV-2. Subsequently, we have explored the mechanism of COVID-19 on pulmonary intravascular thrombosis using the PAR1-Tango reporter allele bred with our hACE2 mice, and have observed abnormal PAR1-Tango activity in the vascular beds of multiple organs (lung, kidney, liver) following SARS-CoV-2 infection, which is unexpected since obvious thrombi are only present in the lungs. Besides these new findings, we have also learned using Cre/lox-mediated endothelial deletion of hACE2, that vascular infection by SARS-CoV-2 is not likely responsible for any major phenotypes we observed. This supplement proposes additional work in order to: a) reveal the extent and severity of vascular injury in COVID-19 disease, using PAR1-Tango specimens; b) understand the actual mechanisms of thrombin generation in SARS-CoV-2 infection by employing an extensive multi-organ immunohistological survey; and c) confirm sources of pro-thrombotic or inflammatory factors using RNA sequencing from the tissues and/or regions of interest. As the majority of specimens are already collected, but awaiting in-depth analysis, the supplement is well-suited to extend the work of the parent grant, but within its original scope. Additional equipment or reagents are not needed to pursue these studies, which instead require the time and effort of the candidate together with resources already available in the Kahn laboratory.