MMRRC COVID-19 variant testing in humanized mouse models

摘要和工作范围 美国加州大学戴维斯分校突变小鼠资源与研究中心（MMRRC-UC Davis） 很高兴提交这份行政补充，以响应ORIP的 参与PA-20-272，“对现有NIH赠款和合作协议的行政补充” 特别是与严重急性呼吸道综合征冠状病毒2型（SARS-CoV-2）疾病（COVID-19）有关。 该申请解决了呼吁支持COVID-19相关研究的一些既定目标， 包括开发和表征COVID-19急性后后遗症（PASC）的动物模型， 研究现有COVID-19动物模型对新出现的病毒基因组变异的易感性。具体地说， 该应用将产生多基因人源化小鼠模型，用于病毒攻击、验证和后处理。 使用成熟的测试，在年轻和老龄小鼠中观察COVID-19（PASC）的急性后遗症， 在ABSL 3环境中筛选平台。这个项目将建立在我们成功的努力， 针对几种基因（ACE 2、TMPRSS 2和TMPRSS 3）的单基因人源化敲入/鼠敲除小鼠系。 FURIN）参与SARS-CoV-2的结合、进入和激活。有了这些经验，我们现在 建议在新的多基因人源化小鼠中确定感染性和传播，以评估 它们可以用作人类PASC的合适模型。具体来说，在这个项目中，我们将1）使用 体外受精（IVF）扩增和我们现存的单基因模型的交叉，以快速产生 hACE 2/hTMPRSS 2和hACE 2/hTMPRSS 2/hFURIN的多基因人源化小鼠模型，2）验证 病理生理学效应，并评估当前占优势的循环（B.1.1.7;菌株： USA/CA_CDC_5574/2020）在多基因人源化小鼠的年轻和老龄雄性和雌性队列中进行。 ABSL 3条件，以及3）建立人源化小鼠的繁殖集落和冻存种质 用于存档和分发给生物医学研究界的模型。验证研究将涉及 病毒载量和清除率、体重动力学和肺部炎症的系统表征， SARS-CoV-2对雄性和雌性小鼠群的攻击;阳性结果将传达给 ACTIV-Preclinical Working Group和NIH的其他人。此外，还需要进行观察和病理学筛查， 存活的老龄小鼠将进行筛选PASC的证据;有希望的发现将是 与PASC倡议和研究者联盟（OTA-21- 015 A和B）的成员进行了沟通， 其他NIH工作人员确保快速翻译人类研究和动物功能研究的结果 模型此外，我们将确保我们的小鼠模型和测试平台随时可供使用， 其他研究人员使用它来快速评估不仅是新出现的SARS-CoV-2的体内后果， 这些变异不仅逃脱了目前的治疗和疫苗策略，而且也逃脱了未来病毒的类似高- 影响大流行潜力。这项研究对于克服小鼠和小鼠之间的遗传差异至关重要。 并填补现有COVID-19小动物模型中阻碍研究转化的关键空白 改善人类健康的发现，包括了解发展，治疗， PASC的预防、抗病毒治疗的有效性和疾病预防疫苗的可靠性 战略布局

ABSTRACT & SCOPE OF WORK The Mutant Mouse Resource and Research Center at the University of California, Davis (MMRRC-UC Davis) is pleased to submit this administrative supplement for up to 1 year of support in response to ORIP’s participation in PA-20-272, “Administrative Supplements to Existing NIH Grants and Cooperative Agreements” specifically related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19). This application addresses a number of the stated objectives of the call to support COVID-19 related research, including to develop and characterize animal models for post-acute sequelae of COVID-19 (PASC) and to study the susceptibility of existing COVID-19 animal models to emerging viral genomic variants. Specifically, this application will generate polygenic humanized mouse models for virus challenge, validation, and post- acute sequellae of COVID-19 (PASC) in both young and aging mice using well-established testing and screening platforms in an ABSL3 environment. This project will build upon our successful efforts to generate monogenic humanized knockin/murine knockout mouse lines for several genes (ACE2, TMPRSS2, and FURIN) involved in SARS-CoV-2 binding, entry, and activation. With this prior experience in hand, we now propose to determine infectivity and transmission in new polygenic humanized mice to assess the extent to which they can be used as suitable models of PASC in humans. Specifically in this project we will 1) use in vitro fertilization (IVF) expansion and intercrossing of our extant monogenic models to rapidly generate polygenic humanized mouse models of hACE2/hTMPRSS2 and hACE2/hTMPRSS2/hFURIN, 2) validate the pathophysiological effects and assess PASC after challenge with currently dominant circulating (B.1.1.7; strain: USA/CA_CDC_5574/2020) in young and aging male and female cohorts of polygenic humanized mice under ABSL3 conditions, and 3) establish breeding colonies and cryopreserved germplasm of humanized mouse models for archiving and distribution to the biomedical research community. Validation studies will involve systematic characterization of viral load and clearance, body weight kinetics, and lung inflammation after SARS-CoV-2 challenge of male and female cohorts of mice; positive results will be communicated to the ACTIV-Preclinical Working Group and others at NIH. In addition, observational and pathological screening of surviving aging mice will be conducted to screen for evidence of PASC; promising findings will be communicated with members of the PASC Initiative and Investigator Consortium (OTA-21-015A and B) and other NIH staff to ensure rapid translation of findings for human studies and functional studies in animal models. Further, we will ensure that our mouse models and testing platform will be made readily available for use by other researchers to swiftly assess the in vivo consequences of not only newly appearing SARS-CoV-2 variants that escape current therapeutic and vaccine strategies but also of future viruses with similarly high- impact pandemic potential. This study is essential to overcome genetic discrepancies between mouse and human and to fill crucial gaps in existing small animal models of COVID-19 that hinder translation of research findings to improvements in human health, including understanding the development, treatment, and prevention of PASC, the effectiveness of antiviral therapies, and the reliability of disease-prevention vaccine strategies.