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.

PROJECT NARRATIVE This proposal from the Mutant Mouse Resource and Research Center at UC Davis (MMRRC-UC Davis) seeks to use an established screening and testing platform in an ABSL3 environment for rapid and reliable determination of viral susceptibility, pathogenesis, and pathophysiological consequences of infection after challenge with SARS-CoV-2 variants in polygenic humanized mouse models of COVID-19 in response to NIH PA-20-272, Administrative Supplements to Existing NIH Grants and Cooperative Agreements. This project will build upon our previous successful efforts to produce, validate, and distribute monogenic humanized COVID- 19 relevant mouse models. We will conduct studies to validate the models as human surrogates for the study of SARS-CoV-2 infection (e.g., viral load and clearance, body weight kinetics, lung inflammation) and to assess their suitability as models for the evaluation of post-acute sequelae of COVID-19 (PASC) in humans. We will also establish breeding colonies of mouse models for distribution by the MMRRC and ensure that the testing platform will be available to researchers to assess the in vivo consequences of SARS-CoV-2 variants and of future viruses with similar high-impact (e.g., pandemic) potential. Promising results will be communicated with the ACTIV-Preclinical Working Group, the PASC Initiative and Investigator Consortium, and other NIH program scientists and staff.