Transcriptional silencing of HIV by LTR targeting RNAi - One of the primary impediments to curing HIV is the existence of viral reservoirs in distinct compartments and cell types that are unaffected by current antiretroviral therapies (ART). In the central nervous system (CNS), reservoirs are comprised primarily of myeloid cells, such as microglia and perivascular macrophages. Because HIV enters the CNS rapidly after initial infection, these reservoirs are established prior to initiation of ART. While ART can enter the CNS and block the infection of new myeloid cells, it does not stop viral replication from an integrated provirus. Thus, most of our current antiretrovirals have no effect on viral replication from chronically infected myeloid cells allowing long-lived, infected CNS microglia to drive ongoing neuroinflammation and provide a source of HIV for viral recrudescence. To prevent this, it is critical to precisely target viral replication in these cells. Existing strategies to eliminate this reservoir have shown some promise in pre-clinical studies but have not yet proven successful. This proposal pursues a novel, alternative strategy: employing RNA interference (RNAi) to durably silence HIV transcription in myeloid cells. This will stop viral replication from these cells and functionally eliminate the CNS reservoir through the “block and lock” strategy. To do this, we will evaluate an approach that uses small RNA to induce transcriptional gene silencing (TGS) of the integrated LTR. Our preliminary studies demonstrate the efficacy of inducing TGS through RNAi and also show changes in viral output in chronically infected myeloid populations treated with ART. The state of viral output in myeloid cells is distinct from T-cell latency and is increased by exposure to benzodiazepines. We hypothesize that treatment with RNAi will induce a stable heterochromatic state, blocking proviral transcription and eliminating production of new virions in chronically infected microglia. Thus, we have proposed studies that will develop our RNAi based strategy to eliminated virion production in infected myeloid populations, providing an effective strategy by which to nullify the CNS myeloid reservoir. Successful completion of this screen will provide necessary data for further applications exploring specific applications of this technology and examination of transcriptional dynamics in microglia using the described systems. To carry out these studies, we propose the following Aim I: To identify and optimize LTR targeting RNAi for transcriptional silencing of HIV in myeloid cells and Aim II: To determine the efficacy of RNAi candidates in iPSC-derived human microglia and durability after LRA and benzodiazepine treatment.
