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球人口中牢固确立，严重 急性呼吸综合征相关冠状病毒2(SARS-CoV-2)对个人、健康构成严重威胁 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)与 具有挑战性的感染指标--在缺乏广泛的测试覆盖范围的情况下，以及缺乏已建立的 疫苗接种和治疗选择--引发了大规模和紧迫的生物医学努力，以对抗 与此相关的人类疾病就是新冠肺炎。它建立在病毒/宿主相互作用的复杂性中， 必须在翻译平台中利用对病毒或病毒材料的实验性感染，重点是 病毒和/或宿主建模，以建立预防和控制策略。在这个实验中 背景下，动物模型在抗病毒药物评估中扮演着体内宿主的核心角色， 免疫疗法和疫苗--最重要的是临床前研究，但也是平行于临床的研究。单一类型的 然而，无论是野生型还是转基因生物，都可能不足以研究所有相关的 生理机制。在这个项目中，我们提出了在小鼠身上进行反向基因设计的方法 大中型基因人源化组件，实现病毒结合和细胞感染 旨在模仿人类新冠肺炎病毒在SARS-CoV-2早期阶段的易感性 复制周期。啮齿动物物种，虽然适合作为小型动物研究对象，但通常是难治的。 在单纯感染上显示SARS和新冠肺炎的病理。解决这个物种问题的一种方法 到目前为止，边界是创造携带小规模部分人化基因的随机转基因小鼠品系 人类ACE2受体的表达单位。然而，这些模型显示了部分表型 其特征是：(A)无终末性肺结局，(B)大脑中不希望发生的复制，以及(C)缺乏多器官功能障碍。 感染时的器官衰竭(例如SARS-CoV，预计SARS-CoV-2也会出现类似的后果)。在……里面 为了能够区分肺部和其他人类表型，我们假设扩展的基因组 单独以人类ACE2受体的形式人源化(参见Spec.目标1)或结合肺- 特定的人类辅因子，即TMPRSS2/Furin(见规范目标2)-基于它们类似人类的表情 (在规范中进行验证。目标3)-将因此改善病毒感染和组织嗜性，通过及时进展来衡量 不同器官中的病毒滴度(在Spec.目标4)。荧光报告以及特定部位的重组 将在ACE2的替代Cre-重组酶融合模型中启用，而ACE2的固有功能 TMPRSS2/Furin模型将在表达时提供荧光信号。我们的非典/新冠肺炎大鼠 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 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--从而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.