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

摘要 严重急性呼吸系统综合症冠状病毒2型引发的新冠肺炎大流行 已经威胁到全球健康。疾病的严重性和SARS-CoV-2死亡人数的上升 已经引发了对有效治疗的迫切需要。除了呼吸道症状，20%-50%的患者 表现出腹泻、呕吐等胃肠道症状。此外，临床证据表明， 在直肠拭子中可以发现病毒RNA，这表明肠道可能是 SARS-CoV-2感染。在这项建议中，我们设计了一种新型的高亲和力阻断剂，用于已知的进入 SARS-CoV-2受体预防人类肠道细胞感染并追求更长远的目标 以结构为基础发现新的受体靶标。 目的1设计针对SARS-CoV-2 S蛋白与已知相互作用的阻断剂 其主要进入受体ACE2(血管紧张素转换酶2)，以及与新的共受体， CD147(单羧酸转运体辅助蛋白)，这两种蛋白都在人类中表达 小肠和结肠上皮细胞。在目标1中，我们将设计一种ACE2/CD147双特异性试剂 可同时靶向SARS-CoV-2 S蛋白受体以提高效率和 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 与野生型相比，高亲和力ACE2和CD147ECD对SARS-CoV-2 S蛋白的亲和力提高 这些类型的ECDs将被组合成包含ACE2和CD147的单个双特异性试剂 亲和力成熟的ECDs，并在人的肠道器官中进行检测。特别是，我们部署了肠道 具有“翻转的极性”的器官，其中表达ACE2的顶端方面朝向外 周围的ECM/介质，而不是朝向内腔，以更好地模拟生理性病毒 感染。在目标2中，我们将为额外的人SARS-CoV-2筛选CRISpra激活文库 秘密目标。SARS-CoV-2分泌体，即病毒编码的分泌物或表面暴露的 跨膜蛋白，也促进宿主细胞的感染，并为SARS提供新的靶点- CoV-2治疗学。该建议充分利用了Chris Garcia(家长R01的多PI)的专业知识 在蛋白质工程、免疫治疗和结构生物学方面，与Calvin Kuo(The Parent R01)在器官生成和疾病建模方面的专业知识，以设计靶向疗法 针对SARS-CoV-2。我们还利用曼努埃尔·阿米耶娃和凯瑟琳·布利什小组在 器质性心尖-基底极性反转和BSL3 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.