Mechanistic Characterization of the IBD Risk Gene, PTPN2, as a Novel Susceptibility Marker for Increased SARS-CoV-2 Infection

总结/摘要 2019年全球冠状病毒病（COVID-19）大流行影响了220个国家的7000多万人 国家（www.example.com）。COVID-19的一个主要临床混杂因素是缺乏对宿主因素的了解， 增加对SARS-CoV-2感染的易感性，并使部分患者出现更严重的胃肠道症状。 此外，也缺乏减轻这些风险的干预措施。我们的初步研究表明， 对几种肠道疾病易感性的遗传标记，rs1893217功能丧失PTPN 2变体， 炎症性肠病肠活检中SARS-CoV-2受体ACE2水平升高 (IBD)患者我们假设携带PTPN 2功能缺失变异的个体可能比携带PTPN 2功能缺失变异的个体更多。 容易感染SARS-CoV-2。我们已经在功能上验证了我们在IBD中的初步发现 PTPN 2基因活性丧失导致ACE2表达增加和SARS-Cov-2 刺突蛋白细胞进入，使用体外和/或体内模型。我们的总体目标是 机械地确定PTPN 2活性丧失如何通过上调促进SARS-CoV-2细胞进入 ACE2表达的敏感性，以及这种敏感性是否可以通过临床批准的JAK抑制剂托法替尼减轻。 目的1将阐明PTPN 2如何限制ACE 2和其他宿主病毒进入辅因子的表达， 限制病毒进入PTPN 2降低或表达IBD风险的人肠上皮细胞（IEC）系 PTPN2变体rs1893217，以及来自Ptpn 2缺陷小鼠的类肠。目标2将确定PTPN 2如何 IEC的缺乏改变了小鼠适应的SARS-CoV-2感染的严重程度，以及肠道 与COVID-19患者的肺部症状相关的结果。目标3将机械地确定JAK是否 抑制剂托法替尼可以在体外、体内使ACE2水平正常化，以减少IEC、人和小鼠中的病毒进入 类肠杆菌、Ptpn 2缺陷型小鼠和来自PTPN 2基因型IBD患者的细胞。我们还将确定托法替尼是否 可以改变IBD患者的ACE2水平。我们已经建立了新的小鼠品系和体外模型系统， 本研究我们将在一系列创新和成熟的方法中使用这些模型系统，使我们能够 机制定义PTPN 2调节SARS-CoV-2和上皮细胞之间的基本相互作用， 这些细胞是病毒进入并随后导致COVID-19的细胞。这些实验代表了 令人兴奋的新方向，协同调查小组的专业知识。这些研究的结果是 准备产生重大进展，以确定1）如何遗传风险变异的高度相关性，以几个 NIDDK疾病可以增加对SARS-CoV-2感染的易感性; 2）SARS-CoV-2如何破坏上皮细胞 完整性和体内平衡，以促进腹泻; 3）如何将批准的IBD治疗药物重新用于 降低这些风险。因此，这项研究的临床意义包括确定一种新的机制， 可以被现有的JAK抑制剂靶向用于预防性或治疗性给药， 进行SARS-CoV-2疫苗试验。

SUMMARY/ABSTRACT The global coronavirus disease 2019 (COVID-19) pandemic has affected over 70 million individuals in 220 countries (www.who.int). A major clinical confounder of COVID-19 is the lack of knowledge of host factors that promote susceptibility to SARS-CoV-2 infection and more severe gastrointestinal symptoms in some patients. Moreover, there is also a lack of interventions to mitigate these risks. Our preliminary studies identified that a genetic marker of susceptibility to several intestinal diseases, the rs1893217 loss-of-function PTPN2 variant, increased levels of the SARS-CoV-2 receptor, ACE2, in intestinal biopsies from inflammatory bowel disease (IBD) patients. We hypothesize that individuals harboring PTPN2 loss-of-function variants may be more susceptible to SARS-CoV-2 infection. We have already functionally validated our initial findings in IBD patients that loss of activity of the PTPN2 gene results in increased ACE2 expression and SARS-Cov-2 spike protein cellular entry, using in vitro and/or in vivo models. Our overall objective will be to mechanistically determine how PTPN2 loss-of-activity promotes SARS-CoV-2 cellular entry through upregulation of ACE2 expression, and if this susceptibility can be mitigated by the clinically approved JAK inhibitor, tofacitinib. Aim 1 will address how PTPN2 restricts expression of ACE2, and other host virus entry co-factors, to functionally restrict virus entry in human intestinal epithelial cell (IEC) lines with reduced PTPN2 or expressing the IBD risk PTPN2 variant rs1893217, as well as enteroids from Ptpn2-deficient mice. Aim 2 will o determine how PTPN2 deficiency in IECs alters the severity of infection with a mouse-adapted SARS-CoV-2, as well as intestinal outcomes relevant to diarrheal symptoms in COVID-19 patients. Aim 3 will mechanistically determine if the JAK inhibitor, tofacitinib can normalize ACE2 levels in vitro, in vivo, to reduce virus entry in IEC, human and mouse enteroids, Ptpn2-deficient mice, and cells from PTPN2-genotyped IBD patients. We will also identify if tofacitinib can modify ACE2 levels in IBD patients. We have established novel mouse lines and in vitro model systems for this study. We will use these model systems in a series of innovative and established approaches, to allow us to mechanistically define PTPN2 regulation of the fundamental interactions between SARS-CoV-2 and epithelial cells that are required for virus entry and subsequently cause COVID-19. These experiments represent an exciting new direction that synergizes the expertise of the investigative team. The results from these studies are poised to generate significant advances in identifying 1) how a genetic risk variant of high relevance to several NIDDK diseases can increase susceptibility to SARS-CoV-2 infection; 2) how SARS-CoV-2 can disrupt epithelial integrity and homeostasis to promote diarrhea; 3) how an approved IBD therapeutic can be repurposed to mitigate these risks. Therefore, the clinical implications of this study include identifying a novel mechanism that can be targeted by existing JAK inhibitors for prophylactic or therapeutic administration, and genetic screening of individuals for SARS-CoV-2 vaccine trials.