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

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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.