SUMMARYOpiate abuse is a significant risk factor for HIV-1 infection and several studies have shown that, in combination,opiates and HIV-1 lead to significantly greater damage to the brain. Thus, a new combinatory strategy is neededto impede HIV-1 infection and mitigate opiate effects on the CNS. In spite of significant advances in anti-retroviraltherapy (ART), the elimination of HIV-1 CNS reservoirs remains a formidable task. This is mainly attributed tothe integration of the HIV-1 proviral DNA into the host genome causing viral latency in the reservoirs, includingthe brain. Further, the inability of ART to penetrate the BBB after systemic administration makes the brain oneof the dominant HIV reservoirs. Thus, elimination of HIV-1 from the brain remains a clinically daunting and keytask in the cure of HIV-1/opioid CNS disease. Most recently, we (Dr. Khalili's lab at Temple University) developedan RNA directed gene-editing strategy using Cas9/gRNA that successfully eliminates entire integrated copies ofthe HIV-1 genome from the host chromosome. However, delivery of this powerful Cas9/gRNA complex acrossthe BBB is limited and an effective method for delivery and release of Cas9/gRNA is critically required to eliminatethe HIV reservoir in the brain. Our laboratory (Dr. Nair's team at Florida International University) has recentlypatented (US patent: US20130317279 A1 and WO patent: PCT/US2013/068698) technology involving novelmagneto-electro nanoparticle (MENP) based drug delivery system, which offers capability of on-demand drugrelease across the BBB. The collaboration of these two laboratories provided preliminary evidence thatCas9/gRNA binds to MENP, navigated across the BBB by magnetic force, and on-demand release of functionallyactive Cas9/gRNA by external AC stimulation. We provide evidence that morphine induced activation of HIVinfection could be mitigated by methylnaltrexone (MTNX) (µ receptor antagonist). In this multi-PI application wehypothesize that efficient nanoformulations (NFs) containing Cas9/gRNA and MTNX can serve as an effectivecarrier to deliver Cas9/gRNA targeting HIV-1 across the BBB for the recognition and complete eradication of theHIV reservoir in brain and to treat/prevent neurological deficits observed in morphine-using HIV infected subjects.To test our hypothesis, we propose to refine our design method, anddevelop, characterize, and evaluate thedelivery and on-demand release of Cas9/gRNA using an in vitro BBB-HIV infection model (Aim #1). Next, wewill evaluate and pre-screen the in vivo efficacy of the developed NFs in excising integrated copies of HIV DNAin Tg26 transgenic mice harboring the entire viral genome (Aim #2). In Aim #3 we will develop and use BLTmouse model to validate and assess the in vivo efficacy of the MENP-Cas9/gRNA NFs to recognize anderadicate latently infected HIV-1 reservoirs. Finally, in Aim #4 we will examine the in vivo efficacy of the mostpre-screened NFs in a BLT morphine mouse model to assess the potential excision of HIV-1 proviral DNA andmorphine induced reactivation of latent HIV infection and to reverse neurological deficits by NFs containingMNTX.