HIV infection and cocaine use are comorbid conditions that can amplify each other's detrimental effects on people with HIV (PWH) despite efficient antiretroviral therapy (ART), as ART is not effective in restricting transcription and latency-reactivation of HIV. Additionally, viral latency is the primary cause of HIV persistence and is largely regulated at the transcriptional level. Therefore, defining and targeting the mechanisms that regulate HIV transcription is crucial for developing more effective therapeutic interventions aimed at permanently silencing HIV transcription. These methods could lead to a functional cure through a "block and lock" strategy, the main target area of the FOA. Hence, approaches are needed that not only restrict HIV infection of new cells but also target HIV transcription. This gap in knowledge is addressed here, as Upon conclusion, we will reveal the crucial role of DNA-PK in regulating HIV transcription and latency; and establish the translational potential of in-human tested DNA-PKi in reversing this process. We have shown the vital role of DNA-PK in supporting HIV transcription and latency-reactivation in both lymphoid and myeloid cells. However, mechanistically very little is known about HIV latency/persistence in microglial cells, the major contributor to viral reservoirs in the central nervous system (CNS). We found that partial DNA-PK inhibition, using less than one-fifth amount of clinically tested DNA-PK inhibitor (DNA-PKi), as cancer therapeutics, was sufficient to restrict HIV transcription without any cell toxicity in patients’ PBMCs, suggesting the therapeutic potential of DNA-PKi in permanently silencing HIV transcription. The objective of this grant is to define the role of cocaine-induced DNA-PK in supporting HIV transcription and latency reactivation using both iPSCs-derived microglia and brain-organoids containing microglial cells; and test the therapeutic potential of in-human tested DNA-PKi in limiting viral load and CNS reservoir. Based on our several publications on this subject, we hypothesize that cocaine, by stimulating DNA-PK activity, augments HIV transcription, leading to enhanced HIV reservoirs due to higher HIV replication; however, clinically evaluated DNA-PK inhibitors can reverse this process. To test our hypothesis, using iPSCs-derived microglia and brain-organoids; In Aim 1, we will evaluate the cooperative impact of cocaine on DNA-PK-mediated upregulation of HIV transcription, replication, and latency-reactivation. In Aim 2, we will test the therapeutic potential of in-human tested DNA-PKi in restricting HIV load, reservoir, and viral rebound upon ART interruption. The proposed research is innovative because data will be obtained using different high throughput approaches in iPSC-derived microglia and confirmation in a brain organoid model. This contribution is significant since we will test the clinical relevance of DNA-PK inhibitors in controlling HIV and define the mechanisms regulating HIV transcription and latency in microglia. This will enable the designing of optimized therapies targeting HIV reservoirs in the CNS and contributing to cure approaches.