A novel mechanism that regulates CNS viral load and reservoir upon substance use

About This Grant

Project Summary: HIV infection and cocaine use are comorbid conditions that can exacerbate each other's harmful effects, as ART is not sufficient to fully suppress HIV transcription or prevent latency reactivation. HIV gene products (RNA and proteins) greatly contribute to perpetual immune activation and inflammation throughout the system, primarily in the central nervous system (CNS) of people with HIV (PWH). Additionally, viral latency, the main cause of HIV persistence, is also largely regulated at the transcriptional level. Therefore, defining all the mechanisms that control HIV transcription is essential before utilizing them therapeutically to limit HIV persistence and associated comorbidities, such as neuroinflammation, which is prevalent even in well-controlled PWH. DNA breaks are a common occurrence during the transcription of genes, which are required to be repaired via transcription-coupled repair (TCR). Without TCR, during transcription of a gene, breaks in the template DNA cause frequent RNAPII pausing or degradation, which halts transcription. Interestingly, upon cocaine exposure to microglia, we noted both the increased DNA-dependent protein kinase (DNA-PK) activity and corresponding enhanced HIV transcription and replication. Additionally, we found that DNA-PK knockdown results in the dissociation of TCR machinery from LTR, showing a direct role of DNA-PK. However, in microglia, the major HIV reservoir in the CNS, the impact of DNA-PK-mediated TCR during HIV transcription in the context of ART and cocaine, and the potential therapeutic reversal using clinically tested DNA-PK inhibitors (DNA-PKi), remains largely unexplored. This study aims to address this critical knowledge gap, using brain organoids harboring microglia. Together, these facts offer a strong premise for the hypothesis that DNA-PK plays a crucial role during TCR and neutralization of negative factors in relieving RNAPII pausing during HIV transcription, and cocaine further supports these effects by stimulating both the level and activity of DNA-PK; however, clinically tested DNA-PKi can reverse this process, limiting viral load and rebound after ART interruption. Aim 1 will investigate the role of cocaine-induced DNA-PK in relieving RNAPII pausing via TCR in brain organoids and iPSC-derived microglia +/- ART. Aim 2 will define the impact of DNA-PK mediated regulation of negative transcription factors (NFs) during RNAPII pause-release. Aim 3 will test the efficacy of clinically tested DNA-PKi in blocking HIV transcription and viral rebound upon ART interruption in the context of cocaine +/- ART in brain organoids. The proposed high-risk/high-payoff exploratory study with great translational potential is closely aligned with the main objective of this FOA: 1) we propose to develop new tools and techniques in our lab - DNA curtain methodology/assay and human brain organoids harboring microglia; 2) define a novel mechanism, DNA-PK- mediated TCR, in regulating HIV transcription, latency, and rebound in the context of cocaine+/- ART.