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: 10155985
• 负责人: WEI WENG
• 金额: $ 29.72万
• 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/10155985
• 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 disease; 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.

随着第一波人畜共患传播在全世界人群中牢固确立，严重的 急性综合征呼吸道相关冠状病毒 2 (SARS-CoV-2) 对个人、健康构成严重威胁 护理系统和社会的不同程度的疾病严重程度（从无症状到致命）。 具有挑战性的感染指标——在缺乏广泛的检测覆盖范围以及缺乏既定的 疫苗接种和治疗方案——已大规模引发紧急生物医学努力来应对 与人类疾病相关的 COVID-19 是在病毒/宿主相互作用的复杂性中发现的。 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，以便在本实验中建立预防和控制策略。 在环境中，动物模型作为体内宿主在抗病毒药物的评估中发挥着核心作用， 免疫疗法和疫苗——最重要的是临床前研究，但也包括与临床平行的研究。 然而，生物体，无论是野生型还是转基因生物，可能不足以研究所有相关的 在这个项目中，我们提出了通过引入小鼠的反向遗传设计。 大中型基因人源化成分，实现病毒结合和细胞感染 目的是模拟 SARS-CoV-2 早期阶段人类 COVID-19 疾病的易感性 啮齿动物物种虽然作为小动物研究对象很受欢迎，但通常难以控制。 在简单感染后显示 SARS 和 COVID-19 病理学是解决该物种问题的一种方法。 迄今为止的边界是创建携带小规模部分人源化基因的随机转基因小鼠品系 然而，这些模型显示了部分表型。 其特征是：(a) 没有终末肺结果，(b) 大脑中出现不需要的复制，以及 (c) 缺乏多 感染后出现器官衰竭（以 SARS-CoV 为例，预计 SARS-CoV-2 会出现类似的结果）。 为了实现独特的肺和其他人类表型，我们追求扩展的基因组 单独以人 ACE2 受体形式人源化（参见规格目标 1）或与肺结合 特定的人类辅助因子，即 TMPRSS2/Furin（参见规范目标 2）——基于其类人表达 （在规范目标 3 中验证）——从而改善病毒感染和通过及时进展测量的组织向性 不同器官中的病毒滴度（在规范目标 4 中）。 将在 ACE2 的替代 Cre 重组酶融合模型中启用，而 ACE2 的内在特征 TMPRSS2/Furin 模型将在表达后提供荧光信号。 模型平台实用程序（由第一阶段的三个单独模型组成）将极大地支持跨领域 对疾病发展的物种转化研究和干预措施的测试 由生物医学领域的专家进行研究——从而满足他们的短期和长期研究需求。