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

点击翻译按钮获取中文摘要

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-2 have raised an urgent need for effective therapies. Besides respiratory symptoms, 20-50% of patients exhibit gastrointestinal symptoms such as diarrhea and emesis. In addition, clinical evidence shows that viral RNA can be found in rectal swabs, indicating that the intestine may be a critical target of SARS-CoV-2 infection. In this proposal, we engineer novel high-affinity blocking agents for known entry receptors of SARS-CoV-2 to prevent infection of human intestinal cells and pursue a longer-term goal of structure-based discovery of novel receptor targets. Aim 1 designs blocking agents that target the known interaction of SARS-CoV-2 S protein with its 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 human small intestinal and colon epithelial cells. In Aim 1 we will engineer an ACE2/CD147 bi-specific agent that can simultaneously target both SARS-CoV-2 S protein receptors to improve the efficiency and specificity of viral blockade. We utilize in vitro protein evolution by yeast cell surface display to generate high-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 CD147 affinity-matured ECDs and assayed in human intestinal organoids. In particular, we deploy intestinal organoids with a “flipped polarity” where the apical ACE2-expressing aspect faces outwards towards the surrounding ECM/media instead of towards the interior lumen to better model physiologic viral infection. In Aim 2, we will screen a CRISPRa activating library for additional human SARS-CoV-2 secretome targets. The SARS-CoV-2 secretome, i.e. virus-encoded secreted or surface-exposed transmembrane 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 the parental R01) expertise in organoid generation and disease modelling to design targeted therapeutics for SARS-CoV-2. We also utilize collaboration from the Manuel Amieva and Catherine Blish groups in organoid apical-basal polarity inversion and BSL3 SARS-CoV-2 infection, respectively.