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

用稳定的内源性大麻素配体AMG 315靶向炎症体：CRISPR/Cas9和 艾滋病毒和大麻素背景下的纳米技术研究。 项目总结（摘要）： 虽然大麻用于医疗目的已经显示出治疗某些疾病的巨大希望， 在医疗条件下，大麻素滥用在神经认知和行为方面产生显著损害， 这些作用在HIV感染患者中加剧。研究表明，即使在 HIV-1抑制联合抗逆转录病毒疗法（cART），HIV-1达特是在大脑中产生的， 前病毒DNA，并作为潜伏感染和炎性小体发展的病原体 介导的神经炎症。CRISPR/Cas9基因编辑技术已被 我们和其他人证明，它能有效地切除整合到宿主基因组中的HIV基因组。我们 初步研究使用了 最近发现的有效的代谢稳定的内源性大麻素类似物 AMG 315证明，这种合成大麻素通过抑制 NLRP 3炎性小体与HIV感染因此，我们假设消除HIV-1达特基因 在CNS细胞中使用达特特异性CRISPR/Cas9和用CB 1特异性稳定的CRISPR/Cas9抑制炎性小体 内源性大麻素类似物AMG 315可以消除活动性HIV感染/诱导永久潜伏期， 神经退行性变。 然而，AMG-315和CRISPR对大脑是不可穿透的， 预防HIV感染、炎性小体激活和随后的神经变性所必需的量。 为了克服这一点，我们将使用我们的专利磁电纳米粒子（MENP）技术， 脂质体以递送CRISPR/Cas9和AMG 315，并用于按需控释。用于持续 为了释放CRISPR并保护CRISPR免受溶酶体降解，将与MENP结合的CRISPR和AMG 315结合。 包封在脂质体中。因此，在具体目标1中，我们将开发、表征和评估 使用基于MENP的药物递送Tat特异性CRISPR Cas9/gRNA和AMG 315穿过体外BBB 切除HIV-1达特基因并减弱大麻素和塔特诱导的炎性体的递送方法， 分别在具体目标#2中，我们将研究MENP纳米制剂的体内治疗功效。 含有CRISPR的小鼠和AMG 315，使用强力霉素诱导的HIV-1达特转基因小鼠（i达特）作为 HIV/neuroAIDS和大麻素给药动物模型。在具体目标-3中，我们将验证效果 这些纳米制剂对体内神经元可塑性和神经认知功能的影响。成功完成 这项拨款的一半将在预防HIV-1达特和大麻素介导的 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 用稳定的内源性大麻素配体AMG 315和达特特异性靶向脑中炎性体的概念 使用基于MENP的技术的CRISPR/Cas9是对RFA-DA-20-026的响应，并且将对以下研究有用： 抑制HIV复制、NLRP 3炎性体活性和治疗大麻素诱导神经元 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.