Humanization of ACE2 and Associated Priming Proteases in New Mouse Models for Downstream Disease and Therapy Investigations of COVID-19

随着第一波人畜共患病传播在全世界人群中牢固确立， 急性呼吸道综合征相关冠状病毒2型（SARS-CoV-2）对个人、健康 护理系统和社会。不同程度的疾病严重程度（从无症状到致命）， 在缺乏广泛的测试覆盖率以及缺乏既定的 疫苗接种和治疗方案已经引发了大规模和紧急的生物医学努力，以应对 与人类疾病相关的新冠肺炎它建立在病毒/宿主相互作用的复杂性之上， 必须在翻译平台中利用病毒或病毒材料的实验感染，重点是 病毒和/或宿主建模，以建立预防和控制策略。本实验 背景，动物模型作为用于评价抗病毒药物目的的体内宿主起着中心作用， 免疫疗法和疫苗不仅在临床前研究中是最重要的，而且在平行于临床的研究中也是如此。单一类型的 然而，野生型或遗传修饰的生物体可能不足以用于所有相关的研究。 生理机制。在这个项目中，我们提出了反向遗传设计的小鼠， 大规模和中等规模的基因人源化成分，使病毒结合和细胞感染成为可能 目的是在SARS-CoV-2的早期阶段模拟人类COVID-19疾病的易感性 复制周期啮齿类动物，虽然作为小动物研究对象是有利的，但通常是难治性的 显示SARS和COVID-19病理在简单的感染。解决这个物种的一种方法 到目前为止，我们的目标是建立随机的转基因小鼠品系， 人ACE 2受体的表达单位。然而，这些模型显示部分表型 其特征在于：（a）没有终末肺结果，（B）在脑中不期望的复制和（c）缺乏多- 感染后器官衰竭（以SARS-CoV为例，预期SARS-CoV-2的结局相似）。在 为了使不同的肺和其他人类表型，我们假设， 人源化以单独的人ACE 2受体的形式（参见Spec. Aim 1）或与肺- 特定的人辅因子，即，TMPRSS 2/Furin（参见Spec. Aim 2）基于它们的类人表达而被克隆 （在质量标准目的3中验证）因此，通过及时进展测量，顺铂将改善病毒感染和组织嗜性 不同器官中的病毒滴度（见质量标准目的4）。荧光报告以及位点特异性重组 将在ACE 2的替代Cre重组酶融合模型中实现，而重组酶的内在特征将在ACE 2的替代Cre重组酶融合模型中实现。 TMPRSS 2/弗林蛋白酶模型将在表达时提供荧光信号。我们的宽SARS/COVID-19小鼠 模型平台实用程序（在第一阶段由三个单独的模型组成）将显著支持跨 对疾病发展的物种转化调查和干预措施的测试 由生物医学领域的专家组成，以满足他们的短期和长期研究需求。

With the initial wave of zoonotic transmission firmly established in the human population worldwide, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) poses an eminent threat to individuals, health care systems and societies. Various degrees of disease severity (from asymptomatic to lethal) combined with challenging infection metrics ‒ in the absence of widespread testing coverage, as well as lack of established vaccination and treatment options ‒ have triggered massive and urgent biomedical efforts to counter the associated human disease that is COVID-19. Founded in the complexities of virus/host interactions, it is imperative to utilize experimental infections with virus or viral material in translational platforms with a focus on viral and/or host modeling in order to establish preventive as well as control strategies. In this experimental setting, animal models play a central role as in vivo hosts for evaluation purposes of antiviral drugs, immunotherapy and vaccines ‒ foremost in preclinical, but also in parallel-to-clinical, studies. A single type of organism, either wildtype or genetically-modified, will however likely not be sufficient for studies of all relevant physiological mechanisms. In this project, we propose reverse genetic designs in the mouse by introducing genetically humanized components on large and medium scales, enabling viral binding and cellular infection with the aim to mimic human COVID-19 disease susceptibility during early stages of the SARS-CoV-2 replication cycle. Rodent species, although favorable as small animal research objects, are generally refractory to displaying SARS and the COVID-19 pathology upon simple infection. One way to address this species boundary so far was to create random transgenic mouse lines carrying small-scale partially humanized gene expression units for the human ACE2 receptor. These models, however, display partial phenotypes characterized by: (a) no terminal-lung outcomes, (b) undesired replication in the brain and (c) lack of multi- organ failure upon infection (exemplified by SARS-CoV, with similar outcomes expected for SARS-CoV-2). In order to enable a distinct lung and other human phenotypes, we hypothesize that extended genomic humanization in the form of the human ACE2 receptor alone (see Spec. Aim 1) or in combination with lung- specific human cofactors, i.e., TMPRSS2/Furin (see Spec. Aim 2) ‒ based on their human-like expression (verified in Spec. Aim 3) ‒ will thus improve viral infection and tissue tropism measured by timely progression of viral titers in different organs (in Spec. Aim 4). Fluorescent reporting as well as site-specific recombination will be enabled in an alternative Cre-recombinase fusion model of ACE2, while intrinsic features of the TMPRSS2/Furin model will provide a fluorescent signal upon expression. Our broad SARS/COVID-19 mouse model platform utility (consisting of three individual models at the Phase I stage) will significantly support cross- species translational investigations into the development of disease and the testing of intervention measures by specialists in the biomedical field ‒ thus, addressing their short-term and long-term research needs.