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

用于肠干细胞生物学和治疗的 Wnt 替代物的基于结构的生物工程

• 批准号: 10176894
• 负责人: Kenan Christopher GARCIA
• 金额: $ 55.4万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10176894
• 关键词: 2019-nCoV; Affinity; Apical; Biological Assay; Biomedical Engineering; COVID-19; COVID-19 pandemic; Cell Therapy; Cell surface; Cells; Cessation of life; Clinical; Collaborations; Colon; Diarrhea; Disease; Disease model; Engineering; Epithelial Cells; Evolution; Exhibits; Extracellular Matrix; Face; Generations; Goals; Human; Immunotherapeutic agent; In Vitro; Infection; Infection prevention; Integral Membrane Protein; Intestines; Libraries; Organoids; Patients; Peptidyl-Dipeptidase A; Protein Engineering; Proteins; Respiratory Signs and Symptoms; SARS coronavirus; Severity of illness; Small Intestines; Specificity; Structure; Surface; Swab; Therapeutic; Viral; Virus; Virus Diseases; Vomiting; Yeasts; base; cellular engineering; design; effective therapy; gastrointestinal symptom; global health; improved; novel; pandemic disease; physiologic model; prevent; receptor; rectal; respiratory; stem cell biology; structural biology; targeted agent; targeted treatment; viral RNA

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.

抽象的 由严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 引起的 COVID-19 大流行 已威胁到全球健康。 SARS-CoV-2 疾病的严重性和死亡人数的上升 迫切需要有效的治疗方法。除了呼吸道症状外，20-50% 的患者 出现腹泻、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，这表明肠道可能是病毒的关键目标 SARS-CoV-2 感染。在本提案中，我们为已知的进入设计了新型高亲和力阻断剂 SARS-CoV-2 受体预防人类肠道细胞感染并追求长期目标 基于结构的新型受体靶标的发现。 目标 1 设计阻断剂，针对已知的 SARS-CoV-2 S 蛋白与 它的主要进入受体 ACE2（血管紧张素转换酶 2），以及一个新的辅助受体， CD147（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，我们将设计一种 ACE2/CD147 双特异性试剂 可以同时靶向 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生成 高亲和力 ACE2 和 CD147 ECD 与野生型相比，对 SARS-CoV-2 S 蛋白的亲和力有所提高 类型 ECD 这些将被组合成包含 ACE2 和 CD147 的单一双特异性试剂 亲和力成熟的 ECD 并在人肠道类器官中进行了测定。特别是，我们部署肠道 具有“翻转极性”的类器官，其中表达 ACE2 的顶端面朝外 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。在目标 2 中，我们将筛选 CRISPRa 激活文库以寻找其他人类 SARS-CoV-2 分泌蛋白组目标。 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，还促进宿主细胞的感染，并为 SARS 提供新的靶标 CoV-2 疗法。该提案利用了 Chris Garcia（父母 R01 的多 PI）的专业知识 与卡尔文·郭 (Calvin Kuo) 一起研究蛋白质工程、免疫治疗和结构生物学（Multi-PI of the 父母 R01）在类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 Manuel Amieva 和 Catherine Blish 团队的合作 分别是类器官顶端-基底极性反转和 BSL3 SARS-CoV-2 感染。