Project Summary/Abstract Even with the advent of successful combination antiretroviral therapy (c-ART), infection by Human Immunodeficiency Virus (HIV-1) remains a global public health crisis. HIV infection of the brain results in important clinical symptoms including HIV-associated neurocognitive disorder or HAND, a constellation of symptoms affecting cognitive, behavioral, and motor functions, as well as pediatric HIV encephalopathy (PHE) in children, leading to neurodevelopmental deficits. These disorders may still occur even in patients receiving c- ART, although with less severity. The molecular and cellular mechanisms underlying the pathophysiology of HIV- HAND and PHE remain poorly understood, in large part because of limitations of the existing model systems to study them. Animal models are generally not infected by HIV, expensive and impractical or poorly recapitulate human brain infection. Cultured cell models provide some insights, but do not recapitulate many aspects of cell- cell and cell-matrix interactions found in human brain. Advances in stem cell technologies, including the use of human induced pluripotent stem cells (iPSC or iPS cells), now enable generation of patient-specific neural lineage cells and microglia (brain immune cells), as well as the assembly of these cells into 3-dimensional (3D) brain organoids. These cerebral organoids mimic important features of immune, glial and neuronal cell interactions, uniquely enabling us to examine how these interactions are affected by HIV infection and to model key aspects of HAND and PHE pathogenesis. The overarching goal of this project is to leverage our strengths in neurosciences, virology, stem cell biology and gene expression to understand the pathophysiology of HAND and PHE using a human neural-microglial system. We have established human 3D brain organoids prepared with iPS cell-derived microglial cells to study neurodevelopment and neuroimmune interactions and have successfully infected these mixed organoids with HIV. We hypothesize that microglial infection by HIV causes altered neuronal function which can be modeled in these microglial-containing brain organoids, providing a new system for mechanistic understanding of the pathogenesis of HAND and PHE. In this proposal, we will characterize the effects of HIV infection on microglia activation and cytokine production, neuronal cell populations, organization and gene expression, and the specific effects of candidate cytokine mediators. We will apply cutting- edge approaches drawn from stem cell technologies, brain organoid models, and single cell RNA sequencing to develop this model system, which will allow long-term, detailed analyses of the molecular and neurophysiologic mechanisms responsible for the pathogenesis of HIV-associated neurological disorders.