Project Summary Extracellular vesicle mediated epigenetic silencing of HIV in the brain Human Immunodeficiency Virus type 1 (HIV) is a lentivirus that causes a persistent viral infection and results in the demise of immune regulatory cells. Clearance of HIV infection by the immune system is inefficient, and integration of provirus into the genome of host cells provides a means for long-term persistence and latency which require lifelong anti-retroviral therapy. Moreover, it is becoming apparent that HIV-infected monocyte/macrophages represent a sanctuary for HIV-1 in central nervous system (CNS), where they appear to contribute to HIV-associated neurological disorders (HAND). A methodology that can specifically target and epigenetically silence HIV provirus within virus infected microglial cells in the brain could be one means by which to develop a functional cure and possibly a treatment for HAND. We recently developed a zinc finger epigenetic repressor that can epigenetically silence HIV in the brain when delivered by extracellular vesicles (EVs) intravenously. We propose here to contrast this recombinant zinc finger approach EV approach with a small hairpin RNA (shRNA) EV approach, which is also targeted to the LTR to epigenetically silence HIV transcription. The premise of this proposal is that cellular-derived EVs can be used to deliver novel anti-HIV zinc finger or LTR targeted transcriptional modulating shRNAs to the brain and epigenetically silence HIV. We propose 3 aims here to test the hypothesis that cellular derived receptor targeted EVs containing anti-HIV zinc finger and the LTR directed shRNA, both regulators of HIV transcription that utilize endogenous cellular epigenetic silencing mechanisms (3-5, 14), can spread systemically in vivo and stably silence HIV transcription. We will test this hypothesis here in vivo using a modular extracellular vesicle (EV) delivery approach, whereby by neural stem cells (NSC) will be engineered such that they constitutively generate anti-HIV EVs capable of cell directed stable epigenetic silencing of HIV. If successful the approach outlined here may not only result in the epigenetic silencing of HIV in the brain but also help usher in a new generation of EV-RNA therapies that can operate seamlessly with endogenous cellular mechanisms to target epigenetic regulation of gene transcription.