Targeting Inflammasome with stable endocannabinoid ligand AMG315. CRISPR/Cas9 and nanotechnology study in the context of HIV and cannabinoid

以稳定的内源性大麻素配体AMG315为靶向的炎症体：CRISPR/Cas9和 在艾滋病毒和大麻素的背景下进行纳米技术研究。 项目摘要(摘要)： 尽管将大麻用于医疗目的在治疗某些疾病方面显示出很大的前景 在医疗条件下，滥用大麻类药物会对神经认知和行为造成严重损害 在感染艾滋病毒的患者中，这些功能和影响会加剧。研究表明，即使在 HIV-1抑制联合抗逆转录病毒疗法(CART)，HIV-1 TAT是从大脑中产生的 前病毒DNA与潜伏感染和炎症性小体发展的病原体有关 HIV感染患者的介导性神经炎症。CRISPR/Cas9基因编辑技术已经 我们和其他人表明，它可以有效地切除整合到宿主基因组中的艾滋病毒基因组。我们的 初步研究使用了 新近发现的代谢稳定的内源性大麻类似物 AMG315证明这种合成的大麻素通过抑制 NLRP3炎症体与HIV感染。因此，我们假设艾滋病毒-1Tat基因的消除 TAT特异性CRISPR/Cas9对中枢神经系统细胞的作用及CB1特异性稳定剂对炎症小体的抑制作用 内源性大麻素类似物AMG315可消除活动性HIV感染/诱导永久潜伏期，防止 神经退行性变。 然而，AMG-315和CRISPR对大脑的透过性足够 预防艾滋病毒感染、炎症体激活和随后的神经退化所需的数量。 为了克服这一点，我们将使用我们的专利磁电纳米颗粒(MENP)技术和 提供CRISPR/Cas9和AMG315的脂质体，以及按需控制释放。对于持续的 为了释放CRISPR并防止溶酶体降解，与MENP结合的CRISPR和AMG315将被 被包裹在脂质体中。因此，在具体目标#1中，我们将开发、描述和评估 用MENP药物通过体外血脑屏障传递TAT特异性CRISPR Cas9/gRNA和AMG315 切除HIV-1 Tat基因，减轻大麻素和TAT诱导的炎症的递送方法， 分别进行了分析。在特定目标#2中，我们将研究MENP纳米制剂的体内治疗效果 包含CRISPR和AMG315，使用多西环素诱导的HIV-1 TAT转基因小鼠(ITAT)作为 HIV/神经艾滋病和大麻素给药动物模型。在具体的目标3中，我们将验证效果 这些纳米制剂在体内对神经元可塑性和神经认知功能的影响。成功完成 这笔赠款将在预防艾滋病毒-1TAT和大麻素中介方面具有翻译意义 HIV感染的大麻素滥用患者的神经变性。这一多学科的新突破 以稳定的内源性大麻素配体AMG315和TAT特异性靶向脑内炎症小体的概念 CRISPR/CAS9使用基于MENP的技术是对RFA-DA-20-026的响应，将对 抑制HIV复制、NLRP3炎症体活性及治疗大麻素诱导的神经元 艾滋病毒感染的大麻素滥用者的损害。

Targeting Inflammasome with stable endocannabinoid ligand AMG315: CRISPR/Cas9 and nanotechnology study in the context of HIV and cannabinoid. PROJECT SUMMARY (ABSTRACT): Although the use of cannabis for medical purposes has shown great promise for the treatment of certain medical conditions, cannabinoid abuse exerts significant impairments in neurocognitive and behavioral functions and these effects are exacerbated in patients with HIV infection. Studies suggest that even after HIV-1 suppressing combined antiretroviral therapy (cART), HIV-1 Tat is being produced in the brain from proviral DNA and implicated as a causative agent for latent infection and development of inflammasome mediated neuroinflammation in HIV infected patients. CRISPR/Cas9 gene-editing technology has been shown by us and others to be effective for excising the HIV genome integrated into the host genome. Our preliminary studies using a recently discovered and potent metabolically stable endocannabinoid analog AMG315 demonstrate that this synthetic cannabinoid exerts anti-inflammatory properties by suppressing NLRP3 inflammasome and HIV infection. Accordingly, we hypothesize that elimination of the HIV-1 Tat gene in CNS cells using Tat specific CRISPR/Cas9 and suppression of inflammasome with CB1-specific stable endocannabinoid analog AMG315 can eliminate active HIV infection/induce permanent latency and prevent neurodegeneration, respectively. However, AMG-315 and CRISPR are impenetrable to the brain in sufficient quantities necessary to prevent HIV-infection, inflammasome activation, and subsequent neurodegeneration. To overcome this, we will use our patented magneto-electric nanoparticles (MENP) technology and liposomes to deliver CRISPR/Cas9 and AMG315, and for on-demand controlled-release. For the sustained release and to protect CRISPR from lysosomal degradation, MENP-bound CRISPR and AMG315 will be encapsulated in liposomes. Accordingly, in Specific Aim # 1, we will develop, characterize, and evaluate the delivery of Tat-specific CRISPR Cas9/gRNA and AMG315 across the in vitro BBB using MENP-based drug delivery approach to excise HIV-1 Tat gene, and attenuate cannabinoid and Tat-induced inflammasome, respectively. In Specific Aim # 2, we will study the in vivo therapeutic efficacy of MENP nanoformulation containing CRISPR, and AMG315 using doxycycline-inducible HIV-1 Tat transgenic mice (iTat) as an HIV/neuroAIDS and cannabinoid administration animal model. In Specific Aim-3, we will validate the effects of these nanoformulations on neuronal plasticity and neurocognitive functions in vivo. Successful completion of this grant will have a translational significance in preventing HIV-1 Tat and cannabinoid-mediated neurodegeneration in HIV infected cannabinoid-abusing patients. This multidisciplinary new break-through concept targeting inflammasome in the brain with stable endocannabinoid ligand AMG315 and Tat specific CRISPR/Cas9 using MENP-based technology is in response to RFA-DA-20-026 and will be useful for the suppression of HIV replication, NLRP3 inflammasome activity and to treat cannabinoid-induced neuronal impairments in HIV infected cannabinoid abusers.