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

摘要 由严重急性呼吸道综合征冠状病毒2型（SARS-CoV-2）引起的COVID-19大流行 已经威胁到全球健康。疾病的严重性和SARS-CoV-2死亡人数的增加 迫切需要有效的治疗方法。除了呼吸道症状，20-50%的患者 出现腹泻和呕吐等胃肠道症状。此外，临床证据显示， 在直肠拭子中可以发现病毒RNA，这表明肠道可能是感染的关键靶点。 SARS-CoV-2感染。在这项提案中，我们设计了新的高亲和力阻断剂，用于已知的进入 SARS-CoV-2的受体，以防止感染人类肠道细胞，并追求长期目标 基于结构发现新的受体靶点。 目的1设计靶向SARS-CoV-2S蛋白与SARS-CoV-2S蛋白相互作用的阻断剂， 其主要进入受体ACE 2（血管紧张素转换酶2），以及一种新的辅助受体， CD 147（单羧酸转运蛋白的辅助蛋白），两者均在人类中表达 小肠和结肠上皮细胞。在目标1中，我们将设计ACE 2/CD 147双特异性试剂 可以同时靶向SARS-CoV-2S蛋白受体，提高效率， 病毒阻断的特异性。我们利用酵母细胞表面展示的体外蛋白质进化来产生 高亲和力ACE 2和CD 147 ECDs与SARS-CoV-2 S蛋白相比， 这些将组合成含有ACE 2和CD 147的单一双特异性试剂 亲和力成熟的ECD并在人肠类器官中测定。特别是，我们将肠道 具有“翻转极性”的类器官，其中顶端ACE 2表达方面向外朝向 周围的ECM/介质，而不是朝向内腔，以更好地模拟生理病毒 感染在Aim 2中，我们将筛选CRISPRa激活文库，以获得额外的人SARS-CoV-2 分泌靶点。SARS-CoV-2分泌组，即病毒编码的分泌型或表面暴露型 跨膜蛋白，也有助于感染宿主细胞，并提供新的目标，SARS- CoV-2治疗。本提案利用Chris Garcia（父代R 01的多PI）的专业知识 与卡尔文郭（Multi-PI of the 亲本R 01）在类器官生成和疾病建模方面的专业知识，以设计靶向治疗方法 SARS-CoV-2。我们还利用Manuel Amieva和Catherine Blish团队的合作， 类器官顶部-基底极性反转和BSL 3 SARS-CoV-2感染。

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.