Identifying host factors as drug targets and drugs employing RNAi knockdown screens

discovery - We aim to identify and effectively inhibit host dependency factors being essential for SARS-CoV-2 replication and spread, following a systematic screening approach. Human epithelial cells will be seeded in "ready to transfect" multiwell plates, designed and produced in the laboratory of HE. Those plates are containing dried siRNA/transfection reagent complexes. When the cells attach to the surface, reverse transfection enables siRNA mediated knockdown of a single gene, in high-throughput. Then, the cells will be infected with clinical SARS-CoV-2 isolates collected by SC and cCytopathic effect and MTT assays performed. After inactivation by paraformaldehyde, these transportable plates will be sent to the lab of HE who will elaborately phenotype the cells using an established imaging pipeline. RK will compare the hits from the screen to time-lapse proteomics data of SARS-CoV-2 infected human epithelial host cells (data was derived from SC recently), omics data from other coronavirus infection studies, and gene associations to the Severe Acute Respiratory Syndrome. This will lead to a short list of host dependency factors for which drugs will be selected employing publicly available drug databases. Selected host factors and drugs will be validated in the lab of SC employing a broad selection of SARS-CoV-2 isolates. In addition, RK will infer a SARS-CoV-2 specific protein interaction network and identify clusters of host factors to get drug combinations, which will be experimentally validated by SC. We will yield an effective drug treatment for COVID-19 therapy and prevention.