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.

总结 阿片类药物滥用是HIV-1感染的一个重要风险因素，几项研究表明， 阿片类药物和HIV-1会对大脑造成更大的损害。因此，需要一种新的组合策略 以阻止HIV-1感染并减轻阿片类药物对CNS的影响。尽管抗逆转录病毒治疗取得了重大进展， 尽管HIV-1的中枢神经系统储库在抗逆转录病毒疗法（ART）中的应用仍然是一项艰巨的任务。这主要归因于 HIV-1前病毒DNA整合到宿主基因组中，导致病毒在宿主中潜伏，包括 大脑此外，ART在全身给药后不能穿透BBB，这使得脑成为一个 艾滋病病毒的主要宿主。因此，从大脑中消除HIV-1仍然是临床上令人生畏的关键， HIV-1/阿片类中枢神经系统疾病的治疗任务。最近，我们（坦普尔大学哈利利博士的实验室）开发了 使用Cas9/gRNA的RNA指导的基因编辑策略，成功消除了 HIV-1基因组与宿主染色体分离然而，将这种强大的Cas9/gRNA复合物递送到不同的细胞中是不可能的。 BBB是有限的，并且迫切需要用于递送和释放Cas9/gRNA的有效方法来消除 大脑中的艾滋病病毒库。我们的实验室（奈尔博士在佛罗里达国际大学的团队）最近 专利技术（美国专利：US 20130317279 A1和WO专利：PCT/US 2013/068698），涉及新颖的 基于磁电纳米颗粒（MENP）的药物递送系统，其提供按需给药的能力 通过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，通过磁力导航穿过BBB，并按需释放功能性的Cas9/gRNA。 通过外部AC刺激激活Cas9/gRNA。我们提供的证据表明，吗啡诱导激活艾滋病毒 甲基纳洛酮（MTNX）（μ受体拮抗剂）可减轻感染。在这个多PI应用程序中，我们 假设含有Cas9/gRNA和MTNX的有效纳米制剂（NF）可以作为有效的治疗剂， 载体递送靶向HIV-1的Cas9/gRNA穿过BBB，以识别和完全根除HIV-1。 脑中的HIV储库，并治疗/预防在使用吗啡的HIV感染受试者中观察到的神经功能缺损。 为了验证我们的假设，我们建议改进我们的设计方法， 开发、表征和评估 使用体外BBB-HIV感染模型的Cas9/gRNA的递送和按需释放（目标#1）。接下来我们 将评估和预筛选开发的NF在切除HIV DNA整合拷贝中的体内功效 在携带整个病毒基因组的Tg 26转基因小鼠中（目标#2）。在目标3中，我们将开发和使用BLT 小鼠模型，以验证和评估MENP-Cas9/gRNA NF识别和 根除潜伏感染的HIV-1宿主。最后，在目标#4中，我们将检查大多数药物的体内功效。 在BLT吗啡小鼠模型中预筛选NF以评估HIV-1前病毒DNA的潜在切除， 吗啡诱导潜伏的HIV感染的再激活，并通过含有 MNTX。