PROJECT SUMMARY This funding request is a one-year administrative supplement to NIH Grant U42 OD012210 for the UC Davis Mutant Mouse Resource and Research Center (MMRRC). It will be used to create and validate a unique and “highly” impactful polygenic humanized mouse model for human immunodeficiency virus type one (HIV-1) therapeutics, prevention, and cure. Prior attempts to generate an HIV-1 mouse model by random transgenesis have failed. What is now proposed through CRISPR/Cas9 will overcome past limitations. This strategy enables genomic insertion of human coding sequence into orthologous mouse loci, rendering gene expression under promoter and enhancer elements in a physiologically spatial and temporal manner. These modifications will, for the first time, recapitulate what is required for human cell susceptibility to productive and restrictive HIV-1 infection. Such unimpeded viral growth in mouse cells will facilitate novel preventative, therapeutic and cure strategies. To that end, we will generate knockins of human coding sequences for CD4 and CCR5 and an aspartic acid to glycine (D106G) modification of C1qbp in the orthologous genomic mouse loci. These genetic modifications will result in a humanized polygenic (hCD4/hCCR5/C1qbp[D106G]) expressing mouse with deactivation of the corresponding mouse genes. This mouse will permit, for the first time, productive viral infection in CD4+ T cell and monocyte-macrophages, immune suppression, end organ disease, and response(s) to antiretroviral and immune-based preventative therapies as seen after human infection. Model creation and deposition into the MMRRC (K Lloyd, UC Davis) and validation for therapeutics, prevention and cure (H Gendelman, UNMC) will be completed by January 31, 2023. Both creation and validation of the new HIV-1 mouse models as proposed herein are within the scope of the parent award to the MMRRC when such models are not available in public repositories, there is strong scientific justification, and research and validation expertise is not broadly available. There are four unique aspects to this project: (1) Creation of a unique polygenic humanized mouse model unavailable elsewhere in the world that express together all three proteins (CD4, CCR5, and C1qbp[D106G]) necessary for HIV binding, entry, and activation in human cells; (2) Verification of expression of human coding sequences into the orthologous mouse loci and the absence of expression of the corresponding mouse genes following the highest quality control standards and principles of scientific rigor and reproducibility; (3) Validation of the model by testing in studies of viral prevention, therapeutics and cure studies, and (4) Sharing of this new and fully validated HIV mouse model, data, and testing platforms with the biomedical research community without limitations or restrictions. The outcomes of this project…unique model, experimental data, validation protocols and conditions will contribute significantly to our a...