Nanotechnology Based Gene Editing to Eradicate HIV Brain Reservoir in Drug Abusers

基于纳米技术的基因编辑可根除吸毒者体内的艾滋病毒脑库

• 批准号: 9318489
• 负责人: Kamel Khalili
• 金额: $ 65.5万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318489
• 关键词: 3-Dimensional; Absence of pain sensation; Anti-Retroviral Agents; Antiviral Therapy; Area; Basal Ganglia; Binding; Biological Models; Blood - brain barrier anatomy; Brain; Cells; Central Nervous System Diseases; Charge; Chromosomes; Clinical; Collaborations; Complex; DNA; Development; Diagnostic; Drug Carriers; Drug Controls; Drug Delivery Systems; Drug Targeting; Electric Stimulation; Electrostatics; Encapsulated; Excision; Florida; Gene Expression; Genes; Genome; Guide RNA; HIV; HIV Infections; HIV-1; Image; In Vitro; Individual; Infection; International; Laboratories; Lead; Legal patent; Liposomes; Magnetic Resonance Imaging; Magnetism; Manuscripts; Medicine; Methods; Modeling; Monitor; Morphine; Motor Activity; Mus; Nanotechnology; Nerve Degeneration; Neurologic Deficit; Neurons; Opiates; Opioid; Peripheral; Pharmaceutical Preparations; Publishing; RNA; Risk Factors; Severities; Specificity; Surface; System; T-Lymphocyte; Technology; Testing; Therapeutic Agents; Time; Transgenic Mice; Transgenic Organisms; Universities; Viral Genes; Viral Genome; Virus Latency; antiretroviral therapy; base; clinical application; controlled release; design; drug abuser; drug of abuse; efficacy testing; expression vector; in vivo; magnetic field; monocyte; mouse model; nanocarrier; nanoformulation; nanoparticle; neuroAIDS; novel; novel therapeutics; opioid abuse; preclinical study; prevent; reactivation from latency; receptor; screening; vector

SUMMARY Opiate 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 needed to impede HIV-1 infection and mitigate opiate effects on the CNS. In spite of significant advances in anti-retroviral therapy (ART), the elimination of HIV-1 CNS reservoirs remains a formidable task. This is mainly attributed to the integration of the HIV-1 proviral DNA into the host genome causing viral latency in the reservoirs, including the brain. Further, the inability of ART to penetrate the BBB after systemic administration makes the brain one of the dominant HIV reservoirs. Thus, elimination of HIV-1 from the brain remains a clinically daunting and key task in the cure of HIV-1/opioid CNS disease. Most recently, we (Dr. Khalili's lab at Temple University) developed an RNA directed gene-editing strategy using Cas9/gRNA that successfully eliminates entire integrated copies of the HIV-1 genome from the host chromosome. However, delivery of this powerful Cas9/gRNA complex across the BBB is limited and an effective method for delivery and release of Cas9/gRNA is critically required to eliminate the HIV reservoir in the brain. Our laboratory (Dr. Nair's team at Florida International University) has recently patented (US patent: US20130317279 A1 and WO patent: PCT/US2013/068698) technology involving novel magneto-electro nanoparticle (MENP) based drug delivery system, which offers capability of on-demand drug release across the BBB. The collaboration of these two laboratories provided preliminary evidence that Cas9/gRNA binds to MENP, navigated across the BBB by magnetic force, and on-demand release of functionally active Cas9/gRNA by external AC stimulation. We provide evidence that morphine induced activation of HIV infection could be mitigated by methylnaltrexone (MTNX) (µ receptor antagonist). In this multi-PI application we hypothesize that efficient nanoformulations (NFs) containing Cas9/gRNA and MTNX can serve as an effective carrier to deliver Cas9/gRNA targeting HIV-1 across the BBB for the recognition and complete eradication of the HIV 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, and develop, characterize, and evaluate the delivery and on-demand release of Cas9/gRNA using an in vitro BBB-HIV infection model (Aim #1). Next, we will evaluate and pre-screen the in vivo efficacy of the developed NFs in excising integrated copies of HIV DNA in Tg26 transgenic mice harboring the entire viral genome (Aim #2). In Aim #3 we will develop and use BLT mouse model to validate and assess the in vivo efficacy of the MENP-Cas9/gRNA NFs to recognize and eradicate latently infected HIV-1 reservoirs. Finally, in Aim #4 we will examine the in vivo efficacy of the most pre-screened NFs in a BLT morphine mouse model to assess the potential excision of HIV-1 proviral DNA and morphine induced reactivation of latent HIV infection and to reverse neurological deficits by NFs containing MNTX.

概括 Opite滥用是HIV-1感染的重要危险因素，几项研究表明，结合使用 阿片类药物和HIV-1导致对大脑的损害明显更大。那是需要一种新的组合策略 阻碍HIV-1感染并减轻对中枢神经系统的影响。尽管抗逆转录病毒取得了重大进展 治疗（ART），消除HIV-1 CNS水库仍然是一项艰巨的任务。这主要归因于 HIV-1病毒DNA的整合到宿主基因组中，导致储层中的病毒潜伏期，包括 大脑。此外，系统给药后，艺术无法穿透BBB使大脑一个 主要的艾滋病毒水库。那就是消除大脑的HIV-1仍然是临床上令人生畏的关键 治愈HIV-1/阿片类中枢神经系统疾病的任务。最近，我们（坦普尔大学的哈利利博士实验室）开发了 使用CAS9/GRNA的RNA定向基因编辑策略，该策略成功地消除了整个集成的副本 宿主染色体的HIV-1基因组。但是，在跨越这种功能强大的Cas9/grna复合物 BBB是有限的，并且需要一种有效的Cas9/GRNA释放的方法以消除 大脑中的HIV水库。我们的实验室（佛罗里达国际大学的Nair博士团队）最近有了 专利（美国专利：US20130317279 A1和WO专利：PCT/US2013/068698）技术涉及小说 基于磁电纳米颗粒（Menp）的药物输送系统，该系统提供了按需药物的能力 跨BBB释放。这两个实验室的合作提供了初步证据 cas9/gRNA与Menp结合，通过磁力导航在BBB上，并按需释放功能 通过外部AC刺激的活动CAS9/GRNA。我们提供了吗啡诱导HIV激活的证据 可以通过甲基曲霉（MTNX）（µ受体拮抗剂）来减轻感染。在此多PI应用程序中 假设含有cas9/gRNA和mTNX的有效纳米制剂（NFS）可以作为有效 载体以在BBB上传递CAS9/GRNA靶向HIV-1，以识别和完全放射 脑中的HIV储存剂在大脑中，以治疗/预防吗啡感染受试者中观察到的神经系统缺陷。 为了检验我们的假设，我们建议完善我们的设计方法，并 发展，表征和评估 使用体外BBB-HIV感染模型传递和按需释放CAS9/GRNA（AIM＃1）。接下来，我们 将评估并预屏幕，以减轻HIV DNA的集成副本的开发NF的体内效率 在具有整个病毒基因组的TG26转基因小鼠中（AIM＃2）。在AIM＃3中，我们将开发和使用BLT 验证和评估Menp-Cas9/GRNA NF的体内效率的小鼠模型以识别和 根除潜在感染的HIV-1水库。最后，在AIM＃4中，我们将研究最多的体内效率 BLT吗啡小鼠模型中的预筛选NFS，以评估HIV-1病毒DNA的潜在惊喜 吗啡诱导的潜在艾滋病毒感染的重新激活，并通过含有NFS的神经系统缺陷逆转 mntx。