Single molecule functional validation of neurogenomic alterations in opioid-exposed HIV brain

About This Grant

Neurogenomic studies by the SCORCH (Single Cell Opioid Response in the Context of HIV) consortium strongly suggest that brains of individuals living with HIV in the context of opioid or (polys)substance use disorder (OUD/SUD) comorbidity harbor a molecular environment permissive for HIV viral replication and risk for cytotoxic damage. This conclusion also applied to donors who showed systemic, antiretroviral drug- mediated suppression of the virus. There was a stepwise progression of transcriptomic dysregulation in OUD+HIV+ brain, culminating in widespread neuronal pathology and pronounced inflammatory signatures in microglia from individuals with poor viral suppression. The goals of the current project are two-fold. First, we aim for single molecule validation of SCORCH single cell results, by analyzing~12-20kb single molecule fiber- seq libraries from cingulate cortex of SCORCH brains carefully annotated for OUD/SUD and systemic (HIV) suppression status. We will embark on single fiber-level multiomic profiling with differential analyses to uncover effects of HIV infection on nucleosome phasing, positioning and offset at transcription start sites, together with endogenous m5CpG methylation and transcription factor footprints. Integrating single cell (RNA+ATAC-seq) data already generated from the same set of SCORCH brain cohort, with our new single molecule multiomic fiber-seq mappings is expected to provide unprecedented neurogenomic insights into the HIV and substance-exposed brain. Second, we aim for additional functional validation of SCORCH data, by employing HIV-induced lineage tracing (HILT) in human induced pluripotent stem cell (hiPSC)-derived Neuron-Astrocyte-Microglia (hiPSC N-A-Mg) tricultures, in conjunction with CRISPRi for multiplexed microglial promoter repression focused on genes that are both (i) dysregulated in SCORCH SUD+ postmortem brain and (ii) implicated in HIV expression or replication. We will assess viral integration frequency, numbers and proportions of infected microglia, and compare transcriptomes and chromatin of infected/integrated HIV+ microglia with those from exposed bystander cells. This project is a first step to validate SCORCH genomic discoveries on the single molecule level, followed by the design of novel therapeutic tools targeting opioid/substance-dysregulated genes that could foster HIV infection and spread in the brain.