Long-term microglia-targeted endogenous retrovirus-like particle (ERVLP) delivery of Cas12f editor to cure HIV

长期小胶质细胞靶向内源性逆转录病毒样颗粒 (ERVLP) 递送 Cas12f 编辑器以治愈 HIV

• 批准号: 10686078
• 负责人: Wenhui Hu
• 金额: $ 3.98万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10686078
• 关键词: Astrocytes; Blood Vessels; Brain; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Cre lox recombination system; Devices; Disease; Endogenous Retroviruses; FDA approved; Genome; HIV; HIV-1; HIV-associated neurocognitive disorder; In Vitro; Injectable; Injections; Link; Macrophage; Mediating; Messenger RNA; Microglia; Mission; Mus; Myeloid Cells; Neurons; Outcome; Peptides; Phase I Clinical Trials; Proviruses; Public Health; Reporter; Research; Serotyping; Substance Use Disorder; System; Technology; Therapeutic; Tissues; Transgenes; Transgenic Mice; United States National Institutes of Health; Virus; antiretroviral therapy; blood-brain barrier crossing; blood-brain barrier penetration; clinical application; exosome; gene therapy; genome editing; high reward; high risk; in vivo; neuroAIDS; neuroinflammation; novel; novel therapeutics; particle; syncytin; targeted treatment; therapeutic development; tool

Summary Latently infected brain myeloid cells including microglia (MG) and perivascular macrophages can serve as HIV reservoirs, contributing to NeuroHIV persistence, chronic neuroinflammation and HIV-associated neurocognitive disorders (HAND). Strategies aimed at eliminating HIV reservoirs are highly promising to cure HIV, even in the presence of effective anti-retroviral therapy. Extensive studies including FDA-approved phase I clinical trial have demonstrated the therapeutic potential of CRISPR/Cas genome editing to cure HIV. However, a major barrier to the clinical application is the lack of effective and specific delivery to the targeted disease-relevant tissues and/or cells in vivo, particularly in NeuroHIV. The overall objective of this proposal is to develop AAV-mediated stealth cargo delivery of miniature Cas12f genome editor to the HIV cellular reservoir in the brain. We will utilize novel PEG10-mediated endogenous retrovirus-like particle (ERVLP) technology that relies on endogenous PEG10 and syncytin-A for Cargo(Cas12f) mRNA transfer into MG. This approach will harness the benefits of the most promising AAV gene therapy. Several AAV serotypes such as AAV1, 2, 5, 6 can transduce MG (AAV-M) with >80% efficiency in vitro and in vivo, but cannot cross the blood-brain barrier (BBB). In contrast, the currently available BBB-penetrating AAV serotypes (AAV-B) such as AAV9, PhP.B, PhP.eB, F, B10 or B22 have low efficiency in transducing MG both in vitro and in vivo. Therefore, novel AAV serotypes that effectively cross the BBB and transduce MG (AAV-BM) are urgently needed. We hypothesize that AAV-B can offer a one-time injectable systemic delivery of stealth cargo (cDNA) into astrocytes and/or neurons thatin turn serve as relay stationsfor sustained mRNA/sgRNA transfer to MG. This stealth AAV cargo will also include a designer exosome transfer into cells (EXOtic) device via CD63 linked with MG-specific peptide (CD63M). We expect that PEG10-mediated ERVLP and CD63M-mediated EXOtic will synergistically boost the endogenous spreading of HIV eradicator to MG. To accomplish this, we will first use the Cre-LoxP system for proof of concept that MG-targeted exosome-enveloped ERVLP system (Exo-ERVLP) via AAV-B can efficiently deliver Cargo(Cre)-mRNA in vivo from transduced astrocytes/neurons to non-transduced MG in LoxP-STOP- LoxP (LSL)-tdTomato reporter mice (Aim 1). Then, we will assess MG-targeting and genome editing efficiency of multiplexed Cas12f mRNA/sgRNA sustained delivery in LSL-tdTomato mice and HIV Tg26 transgenic mice (Aim 2). Finally, we will explore the therapeutic potential of Exo-ERVLP AAV-B-Cas12f systemic injection in HIV Tg26 transgenic mice (Aim 3). This high-risk high-reward proposal brings together several advancing technologies and established teams with complementary expertise. The all-in-one multiplexed Cas12f/sgRNA transgene is delivered via AAV-B, PEG10 cargo and CD63M EXOtic for sustained targeting and HIV eradication. The expected positive outcomes will offer a novel tool to systemically deliver CRISPR/Cas editor to MG, and provide new avenues for therapeutics development for multiple MG-related diseases.

概括 潜在感染的脑髓样细胞在内 水库，有助于神经凝血持续性，慢性神经炎症和HIV相关 神经认知障碍（手）。旨在消除艾滋病毒水库的策略非常有希望治愈 HIV，即使在有效的抗逆转录病毒疗法的情况下也是如此。广泛的研究，包括FDA批准的阶段 I临床试验证明了CRISPR/CASPR基因组编辑对HIV的治疗潜力。 但是，临床应用的主要障碍是缺乏对目标的有效和特定的交付 与疾病相关的组织和/或体内细胞，特别是在神经hiv中。该提议的总体目标是 为了开发AAV介导的隐形货物传递，将微型CAS12F基因组编辑器传递到HIV Cellular Reservoir 大脑。我们将利用新型的PEG10介导的内源性逆转录病毒样粒子（ERVLP）技术 依靠内源性PEG10和Syncytin-A用于货物（CAS12F）mRNA转移到mg中。这种方法会 利用最有希望的AAV基因疗法的好处。几种AAV血清型，例如AAV1、2、5、6 可以在体外和体内转导MG（AAV-M），但不能越过血脑屏障 （BBB）。相比之下，当前可用的BBB穿透AAV血清型（AAV-B），例如AAV9，PHP.B， PHP.EB，F，B10或B22在体外和体内转导MG的效率较低。因此，新颖的AAV 迫切需要有效地穿越BBB并转导MG（AAV-BM）的血清型。我们假设 AAV-B可以一次性地注射全身性货物（cDNA）到星形胶质细胞和/或 转弯的神经元可作为继电器站，用于持续的mRNA/sgrNA转移至mg。这款隐形AAV货物 还将通过与MG特异性肽相关的CD63包括设计器外泌体转移到细胞（异国）设备 （CD63M）。我们预计PEG10介导的ERVLP和CD63M介导的外来都将协同增强 HIV根除剂的内源性扩散到mg。为此，我们将首先使用Cre-loxp系统 通过AAV-B有效的概念证明，通过AAV-B可以有效 从转导的星形胶质细胞/神经元到LOXP-Stop-中未转导的毫克的体内货物（CRE） - mRNA LOXP（LSL）-TDTOMATO Reporter小鼠（AIM 1）。然后，我们将评估靶标和基因组编辑效率 在LSL-TDTOMATO小鼠和HIV TG26转基因小鼠中，多路复用的Cas12f mRNA/SGRNA持续递送 （目标2）。最后，我们将探索Exo-vlp aav-b-cas12f全身注入的治疗潜力 HIV TG26转基因小鼠（AIM 3）。这个高风险的高回报提案汇集了几个前进 具有互补专业知识的技术和建立团队。多功能的CAS12F/sgrna 转基因通过AAV-B，PEG10货物和CD63M进行持续靶向和HIV传递 根除。预期的积极结果将提供一种新颖的工具，可以系统地提供CRISPR/CAS编辑器 到MG，并为多种MG相关疾病的治疗性开发提供新的途径。