Structure-based Bioengineering of Wnt Surrogates for Intestinal Stem Cell Biology and Therapy

Abstract: The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)has threatened global health. The severity of disease and rising number of deaths from SARS-CoV-2have raised an urgent need for effective therapies. Besides respiratory symptoms, 20-50% of patientsexhibit gastrointestinal symptoms such as diarrhea and emesis. In addition, clinical evidence showsthat viral RNA can be found in rectal swabs, indicating that the intestine may be a critical target ofSARS-CoV-2 infection. In this proposal, we engineer novel high-affinity blocking agents for known entryreceptors of SARS-CoV-2 to prevent infection of human intestinal cells and pursue a longer-term goalof structure-based discovery of novel receptor targets. Aim 1 designs blocking agents that target the known interaction of SARS-CoV-2 S protein withits primary entry receptor ACE2 (angiotensin-converting enzyme 2), as well as with a novel co-receptor,CD147 (accessory protein for monocarboxylate transporters), both of which are expressed in humansmall intestinal and colon epithelial cells. In Aim 1 we will engineer an ACE2/CD147 bi-specific agentthat can simultaneously target both SARS-CoV-2 S protein receptors to improve the efficiency andspecificity of viral blockade. We utilize in vitro protein evolution by yeast cell surface display to generatehigh-affinity ACE2 and CD147 ECDs with improved affinity for SARS-CoV-2 S protein versus the wild-type ECDs These will be combined into a single bispecific agent containing both ACE2 and CD147affinity-matured ECDs and assayed in human intestinal organoids. In particular, we deploy intestinalorganoids with a "flipped polarity" where the apical ACE2-expressing aspect faces outwards towardsthe surrounding ECM/media instead of towards the interior lumen to better model physiologic viralinfection. In Aim 2, we will screen a CRISPRa activating library for additional human SARS-CoV-2secretome targets. The SARS-CoV-2 secretome, i.e. virus-encoded secreted or surface-exposedtransmembrane proteins, also facilitates infection of host cells and provides novel targets for SARS-CoV-2 therapeutics. This proposal leverages expertise of Chris Garcia (Multi-PI of the parental R01)in protein engineering, immunotherapeutics, and structural biology with Calvin Kuo (Multi-PI of theparental R01) expertise in organoid generation and disease modelling to design targeted therapeuticsfor SARS-CoV-2. We also utilize collaboration from the Manuel Amieva and Catherine Blish groups inorganoid apical-basal polarity inversion and BSL3 SARS-CoV-2 infection, respectively.