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

基本信息

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
项目类别：
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

项目摘要

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.

概括阿片类药物滥用是 HIV-1 感染的一个重要危险因素，多项研究表明，综合而言，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。尽管在抗逆转录病毒方面取得了重大进展，但仍可阻止 HIV-1 感染并减轻阿片类药物对中枢神经系统的影响。治疗（ART）中，消除HIV-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，导致病毒在储存库中潜伏，包括此外，全身给药后 ART 无法穿透血脑屏障，这使得大脑成为其中之一。因此，从大脑中消除 HIV-1 仍然是临床上令人畏惧的关键。最近，我们（天普大学 Khalili 博士的实验室）开发了治疗 HIV-1/阿片类中枢神经系统疾病的任务。使用 Cas9/gRNA 的 RNA 定向基因编辑策略，成功消除了完整的整合拷贝然而，这种强大的 Cas9/gRNA 复合物的传递却来自宿主染色体。 BBB 有限，迫切需要一种有效的 Cas9/gRNA 递送和释放方法来消除我们的实验室（佛罗里达国际大学奈尔博士的团队）最近研究了大脑中的艾滋病毒储存库。专利（美国专利：US20130317279 A1 和 WO 专利：PCT/US2013/068698）技术涉及新颖基于磁电纳米颗粒（MENP）的药物输送系统，提供按需给药的能力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）可以减轻感染。发现含有 Cas9/gRNA 和 MTNX 的高效纳米制剂 (NF) 可以作为有效的载体将靶向 HIV-1 的 Cas9/gRNA 跨 BBB 传递，以识别并彻底根除 HIV-1 大脑中的艾滋病毒储存库并治疗/预防在使用吗啡的艾滋病毒感染者中观察到的神经功能缺损。为了检验我们的假设，我们建议改进我们的设计方法，并且开发、表征和评估使用体外 BBB-HIV 感染模型交付和按需释放 Cas9/gRNA（目标#1）。将评估和预筛选已开发的 NF 在切除 HIV DNA 整合拷贝方面的体内功效在携带整个病毒基因组的 Tg26 转基因小鼠中（目标#2），我们将开发并使用 BLT。小鼠模型来验证和评估 MENP-Cas9/gRNA NF 识别和评估的体内功效最后，在目标 4 中，我们将检查大多数病毒的体内功效。在 BLT 吗啡小鼠模型中预先筛选 NF，以评估 HIV-1 前病毒 DNA 的潜在切除和吗啡诱导潜伏 HIV 感染的重新激活，并通过含有 NF 的神经功能逆转神经功能缺损 MNTX。