PROJECT SUMMARY Combination antiretroviral therapy (cART) is highly effective, but it is weakened in the central nervous system (CNS) due to poor drug penetration. In addition, cells infected by HIV in the CNS - termed “CNS reservoirs” - are mainly constituted by myeloid lineage cells, such as microglia cells and macrophages, thus anti- HIV strategies employed are needed to be adapted to these cell types. Various approaches have been applied for controlling viral load in the CNS - for example, T cells expressing anti-HIV chimeric antigen receptor (CAR- T) or broadly neutralizing antibodies (bNAb) – and these approaches effectively target HIV or HIV-infected cells systemically but are ineffective against the CNS reservoirs since cytotoxic-T cells cannot attack myeloid-lineage cells well and antibody penetration into the CNS is extremely low. As such, there is no practical approach successful enough to eliminate these reservoirs safely. We here intend to purge the CNS reservoirs via using genetically modified macrophages expressing anti- HIV CARs (CAR-M) (Aim 1) and multidirectional bNAb (bNAb-M) (Aim 2). Both will be expressed in freshly isolated monocytes from blood sources as a carrier vehicle to the CNS and an effector cell. Importantly, monocytes will be recruited to the CNS when neuroinflammation occurs; HIV infection itself as well as some antiretroviral drugs such as Tenofovir disoproxil fumarate (TDF) or nevirapine (NVP), are known mediators of neuronal inflammation. To overcome the issue of HIV quasispecies, we will use each tool to target several different epitopes, i.e., express 2 or more anti-HIV CARs in the same cells (Aim 1) or multidirectional bNAbs in one molecule which target 3 independent epitopes on the HIV envelope protein (Aim 2). In addition, to protect the gene-modified monocytes, we will simultaneously express two anti-HIV genes with these immunotherapeutic tools; a) C46 that interferes fusion between HIV envelope and cellular membranes, thus blocking HIV infections and b) shRNA against HIV LTR (sh516) that destructs all HIV transcripts including the sequence derived from HIV LTR. Altogether, our approach will ensure CNS delivery of monocytes following gene modification to express multiple anti-HIV CARs or multidirectional bNAbs whilst protecting from HIV infections and/or productions. The monocytes will then differentiate into macrophages in the CNS and attack the infected cells via anti-HIV CAR (CAR-M) or secreting multidirectional bNAbs (bNAb-M) inducing antibody-dependent cytotoxicity or phagocytosis (ADCC/ADCP) against the infected cells. In case of the bNAb-M approach, secreted bNAbs will also work on adjacent myeloid cells which support both ADCC/ADCP. In addition, the bNAbs will neutralize new HIV infection. As such, our approach is highly unique, and, if successful, will open a new avenue to defeat HIV reservoirs in the CNS.