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

用于 COVID-19 下游疾病和治疗研究的新小鼠模型中 ACE2 和相关引发蛋白酶的人源化

• 批准号: 10322764
• 负责人: WEI WENG
• 金额: $ 29.27万
• 依托单位: INGENIOUS TARGETING LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322764
• 关键词: 2019-nCoV; ACE2; Address; Animal Experimentation; Animal Model; Animals; Antiviral Agents; Bacterial Artificial Chromosomes; Binding; Body Weight decreased; Brain; COVID-19; Clinical Research; Clone Cells; Communities; Complementary DNA; Containment; Coronavirus; Detection; Development; Disease; Disease Outbreaks; Disease Progression; Disease susceptibility; Embryo; Enabling Factors; Engineering; Enterobacteria phage P1 Cre recombinase; Etiology; Evaluation; Exons; Future; Gene Expression; Genes; Genetic; Genetic Recombination; Genetically Engineered Mouse; Genome; Genomics; Genotype; Health; Healthcare Systems; Human; Immunotherapy; Individual; Infection; Infectious Agent; Integration Host Factors; Intervention; Introns; Investigation; Investigational Therapies; K-18 conjugate; Kinetics; Knowledge; Laboratories; Licensing; Life Cycle Stages; Logic; Lung; Measures; Mediating; Medical; Modeling; Modification; Monitor; Multiple Organ Failure; Mus; Organ; Organism; Outcome; Pathogenesis; Pathology; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Play; Population; Predisposition; Prevention; Preventive; Preventive measure; Production; Protein Isoforms; Protein S; Proteins; Public Health; RNA Splicing; Refractory; Regulation; Regulatory Element; Reporter; Reporting; Reproducibility; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Severity of illness; Shapes; Signal Transduction; Site; Small Business Innovation Research Grant; Societies; Specialist; Structure; System; TMPRSS2 gene; Testing; The Jackson Laboratory; Time; Transcript; Transgenic Mice; Transgenic Organisms; Tropism; Vaccination; Vaccines; Validation; Viral; Viral Load result; Virion; Virus; Virus Diseases; Zoonoses; base; cofactor; design; drug discovery; embryonic stem cell; human disease; improved; in vivo; innovation; interest; mouse genome; mouse model; novel coronavirus; offspring; pandemic coronavirus; particle; pathogen; pre-clinical; promoter; receptor; response; reverse genetics; socioeconomics; stressor; tissue tropism; tool; transmission process; vaccine development; vaccine discovery; vector; virus host interaction

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.

随着人口世界中最初的人畜共患传播的最初浪潮，严重 急性呼吸道综合症相关的冠状病毒2（SARS-COV-2）对个人有巨大威胁，健康 护理系统和社会。各种程度的疾病严重程度（从无症状到致死）结合 具有挑战性的感染指标 - 在没有宽度测试覆盖范围以及缺乏既定的情况下 疫苗接种和治疗方案 - 引发了巨大和紧急的生物医学努力，以对抗 相关的人类疾病，即19岁。建立在病毒/宿主相互作用的复杂性中，是 必须在翻译平台中利用病毒或病毒材料的实验感染，重点 病毒和/或宿主建模，以建立预防和控制策略。在这个实验中 设置，动物模型是用于评估抗病毒药物的体内宿主的核心作用， 免疫疗法和疫苗 - 临床前最重要的，但也是平行于临床的研究。一种类型的 然而 生理机制。在这个项目中，我们通过引入鼠标中提出反向遗传设计 大中和中等尺度上的遗传人文化成分，使病毒结合和细胞感染能够 旨在模仿SARS-COV-2的早期阶段中的人类共vid-19疾病敏感性 复制周期。啮齿动物物种虽然作为小动物研究对象有利，但通常是难治性的 在简单感染后显示SARS和COVID-19病理。解决该物种的一种方法 到目前为止的边界是创建带有小规模部分人源化基因的随机转基因小鼠线 人ACE2受体的表达单位。但是，这些模型显示部分表型 特征是：（a）没有末端肺结果，（b）大脑中不希望的复制，（c）缺乏多种多样的 感染后的器官衰竭（以SARS-COV为例，SARS-COV-2的预期相似）。 为了实现独特的肺和其他人类表型，我们假设扩展基因组 单独以人ACE2受体的形式进行人性化（请参阅规格AIM 1）或与肺部结合 特定的人类辅助因子，即tmprss2/furin（请参见Spec。Aim2） - 基于它们的人类表达 （在规格AIM 3中进行了验证） - 因此，将改善通过及时进展来衡量的病毒感染和组织往流 不同器官中的病毒滴度（在规格AIM 4中）。荧光报告以及特定地点重组 将在ACE2的替代Cre-cobinase融合模型中启用，而固有特征 TMPRSS2/FURIN模型将在表达时提供荧光信号。我们广泛的SARS/COVID-19鼠标 模型平台实用程序（由I阶段阶段的三个单独模型组成）将大大支持交叉 物种翻译研究疾病发展和干预测量的测试 生物医学领域的专家 - 因此，解决了他们的短期和长期研究需求。