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.

摘要 阿片类药物滥用是艾滋病毒-1感染的一个重要风险因素，几项研究表明， 鸦片类药物和HIV-1对大脑的损害要大得多。因此，需要一种新的组合策略 阻止HIV-1感染，减轻阿片类药物对中枢神经系统的影响。尽管在抗逆转录病毒方面取得了重大进展 在抗逆转录病毒疗法(ART)下，消除艾滋病毒-1中枢神经系统储存库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宿主体内的病毒潜伏，包括 大脑。此外，全身给药后ART无法穿透血脑屏障，使大脑成为 主要的艾滋病毒携带者。因此，从大脑中消除艾滋病毒-1仍然是临床上令人望而生畏的关键 在治疗HIV-1/阿片类中枢神经系统疾病方面的任务。最近，我们(坦普尔大学的哈利利博士的实验室)开发出 一种使用Cas9/gRNA的RNA导向的基因编辑策略，成功地消除了 从宿主染色体中提取HIV-1基因组。然而，这种强大的Cas9/gRNA复合体的交付 BBB是有限的，迫切需要一种有效的方法来传递和释放Cas9/gRNA，以消除 大脑中的艾滋病毒蓄水池。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(美国专利：US20130317279 A1和WO专利：PCT/US2013/068698)涉及新颖的技术 基于磁电纳米颗粒(MENP)的药物输送系统，提供按需药物的能力 在BBB上释放。这两个实验室的合作提供了初步证据 Cas9/gRNA与MENP结合，通过磁力导航穿过血脑屏障，并按需释放功能 通过外部AC刺激激活Cas9/gRNA。我们提供了吗啡诱导HIV激活的证据 甲基纳曲酮(MTNX)(µ受体拮抗剂)可减轻感染。在这个多PI应用中，我们 假设含有Cas9/gRNA和MTNX的高效纳米制剂(NFS)可以作为有效的 携带者通过血脑屏障运送针对HIV-1的Cas9/gRNA，以识别和完全根除 在大脑中储存艾滋病毒，并治疗/预防在使用吗啡的艾滋病毒感染者中观察到的神经缺陷。 为了验证我们的假设，我们建议改进我们的设计方法，并 开发、表征和评估 使用体外BBB-HIV感染模型传递和按需释放Cas9/gRNA(目标1)。接下来，我们 我将评估和预先筛选开发的NFS在切除HIV DNA完整拷贝方面的体内效果 在携带整个病毒基因组的TG26转基因小鼠中(目标2)。在目标3中，我们将开发和使用BLT 小鼠模型验证和评估MENP-Cas9/gRNA NFS识别和 根除潜伏感染艾滋病毒-1的宿主。最后，在目标4中，我们将检查MOST的体内疗效 在BLT吗啡小鼠模型中预筛选神经营养因子以评估切除HIV-1前病毒DNA和 吗啡诱导潜伏的HIV感染重新激活并逆转神经功能障碍 MNTX。