PROJECT SUMMARY This application is submitted in response to RFA-D-21-019: Single Cell Opioid Responses in the Context of HIV (SCORCH) Program Expansion: CNS Data Generation for Chronic Opioid, Methamphetamine, and/or Cocaine Exposures. We have assembled an outstanding team of investigators with world-class expertise in HIV virology, addiction neurobiology, and single cell transcriptomics. We will leverage state-of-the-art single-nuclei RNA sequencing (snRNA-seq) coupled to single-nuclei epigenetics (snATAC-seq) to define the transcriptional and epigenetic landscape of neuronal and nonneuronal cells in addiction-relevant brain cells of HIV-infected mice that compulsively self-administer cocaine. In this manner we will identify cell types and brain sites in which HIV and cocaine interact to exacerbate the negative impact of HIV infection on the brain and contribute to the persistence of cocaine use disorder in infected individuals. To facilitate this goal, we have developed an extended access intravenous cocaine self-administration procedure for mice that precipitates compulsive-like response for cocaine that is resistant to negative outcome (contingently delivered noxious foot shocks). We will utilize EcoHIV, which is a modified HIV strain that can infect conventional immunocompetent mice. The Chromium Single Cell Multiome ATAC and Gene Expression assay and Multiplexed error-robust fluorescence in situ hybridization (MERFISH) will be used to identify cocaine and/or HIV-responsive cells in the brain with unprecedented cellular and spatial resolution. We will confirm our major findings using postmortem brain tissues from cocaine- experienced HIV-infected individuals. Our goals will be accomplished through the following four specific aims. In Aim 1, we will characterize compulsive-like cocaine intake in control and HIV-infected mice and determine the impact of antiretroviral treatment (ART) on cocaine response in these mice. In Aim 2, we will use snRNA-seq and Mulitome assays to identify those cells that show the most robust transcriptional and epigenetic plasticity to cocaine consumption and HIV infection. In Aim 3, we will use MERFISH to confirm and validate our major snRNA- seq and Multiome assay findings, and generate more refined spatially resolved maps of those cells most impacted by cocaine and/or HIV. In Aim 4, we will confirm our major findings using post-mortem human brain tissues. This highly innovative, collaborative and multidisciplinary program of research promises to yield fundamental new insights into disease-related interactions between HIV infection and cocaine use.

PROJECT NARRATIVE Cocaine use disorder increases the risk of HIV infection and HIV infection increases cocaine use, suggesting that these conditions share important reciprocal interactions such that each can exacerbate the other; however, the molecular and cellular bases for cocaine-HIV interactions are poorly understood. Here, we will use cutting- edge single-cell sequencing, single-cell epigenetics, and spatially resolved transcriptomics technologies to characterize transcriptional responses to HIV infection and/or volitional cocaine consumption in a cell type- specific manner in addiction-relevant brain regions of mice, to determine the functional interactions between cocaine use in HIV infection and these cell type-specific transcriptomic profiles. This program of research promises to deliver maps of brain gene expression relevant to cocaine addiction and HIV infection with unprecedented cellular resolution.