of: Proposed COVID-19-related supplement to In vivo PET imaging of novel engineered AAVs informs capsid design There is an urgent need to develop tools to assess viral pathogenesis and the efficacy of potential therapeutics for novel viruses such as SARS-CoV-2. While organoid and cell-based assays have been broadly used to assess the candidate receptor of such viruses, these assays cannot answer key questions as to: 1) whether multiple receptors for the virus exist, 2) the in vivo receptor affinity of the virus and accumulation within the upper respiratory tract, 3) transport of the virus into the vascular system and ultimately to the heart and kidneys, and 4) the resulting transfection of these various sites. Our laboratory has previous developed methods to label adeno-associated viruses and track their transport following systemic injection. We found that these engineered viruses carry cargo attached to the capsid across the blood brain barrier and the cargo accumulates deep within the brain. Combined optical and PET studies have suggested that binding of the virus to its receptor results in transcytosis of the intact capsid. We hypothesize that coronaviruses may possess similar capabilities to be transported across the lung epithelium. We plan to address key issues by assessing receptor binding and transduction using PET and optical imaging. Our resulting specific aims are the following: 1) development and validation of tagging strategies to image pseudotype viral particles at BSL2, 2) development and validation of reporter gene strategies to image transduction of engineered viruses, and 3) application and dissemination of these dual strategies to assess viral transport, transduction and susceptibility to available therapies including a) antibodies, b) protease inhibitors, and c) fusion inhibitors. We propose to develop and image engineered viruses expressing the spike protein and a reporter gene and track these viruses within a model of lung fibrosis and a mouse model with a humanized ACE2 receptor. We will leverage the capabilities to label and track viral capsids and transduction developed within this R01 and key capabilities of Stanford University. Pseudotype viruses based on vesicular stomatitis virus (VSV) and lentivirus have been developed with spike proteins corresponding to SARS-CoV or SARS-CoV2. Further, replicons with intact viral proteases have been engineered. We propose to collaborate with those developing and testing engineered viruses and therapeutics at Stanford, including Jan Carette and Catherine Blish in addition to the key personnel on our parent project. At the conclusion of each phase of this project, we will disseminate strategies for the incorporation of a PET tag, a reporter gene, and dual PET imaging protocols. We hypothesize that these tools can be disseminated and rapidly modified to assess both SARS-CoV-2 and future viruses. We will make our technology available through commercial and scientific partners.
This proposal seeks to understand the mechanistic basis for SARS-CoV-2 related lung, cardiac and renal pathologies by applying PET imaging to elucidate the pharmacokinetics and transduction of pseudotype viral particles mimicking the SARS-CoV and SARS-CoV-2 capsids. We develop the novel imaging methods required to understand these mechanisms.
