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: 11003833
• 负责人: Wenhui Hu
• 金额: $ 55.27万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/11003833
• 关键词: Long; term; microglia; targeted; endogenous

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.

总结 潜伏感染的脑髓样细胞，包括小胶质细胞（MG）和血管周围巨噬细胞可以作为艾滋病毒 水库，有助于NeuroHIV的持久性，慢性神经炎症和HIV相关的 神经认知障碍（HAND）。旨在消除艾滋病病毒储存库的战略非常有希望治愈 艾滋病毒，即使在有效的抗逆转录病毒治疗的存在。广泛的研究，包括FDA批准的阶段 临床试验已经证明了CRISPR/Cas基因组编辑治疗HIV的治疗潜力。 然而，临床应用的主要障碍是缺乏有效和特异性的靶向递送。 疾病相关组织和/或细胞，特别是NeuroHIV。本建议的总体目标是 开发AAV介导的隐形货物递送微型Cas 12 f基因组编辑器到HIV细胞库， 大脑我们将利用新型PEG 10介导的内源性逆转录病毒样颗粒（ERVLP）技术， 依赖于内源性PEG 10和合胞素-A将Cargo（Cas 12 f）mRNA转移到MG中。这种方法将 利用最有前途的AAV基因疗法的好处。几种AAV血清型，如AAV 1、2、5、6 可以在体外和体内以>80%的效率转导MG（AAV-M），但不能穿过血脑屏障 （BBB）。相比之下，目前可用的BBB穿透性AAV血清型（AAV-B）如AAV 9、PhP.B、 PhP、eB、F、B10或B22在体内外转导MG的效率均较低。因此，新型AAV 迫切需要有效地穿过BBB和HMG的血清型（AAV-BM）。我们假设 AAV-B可以提供隐形货物（cDNA）到星形胶质细胞中的一次性可注射全身递送，和/或 神经元反过来充当持续mRNA/sgRNA转移到MG的中继站。这架隐形飞机的货物 还将包括通过与MG特异性肽连接的CD 63将设计的外泌体转移到细胞（EXOtic）装置 （CD63M）。我们预期PEG 10介导的ERVLP和CD 63 M介导的EXOtic将协同促进ERVLP的表达。 HIV根除剂对MG的内源性传播。为了实现这一点，我们将首先使用Cre-LoxP系统， 通过AAV-B的MG靶向外泌体包膜ERVLP系统（Exo-ERVLP）可以有效地 在LoxP-STOP中将Cargo（Cre）-mRNA从转导的星形胶质细胞/神经元体内递送至未转导的MG。 LoxP（LSL）-tdTomato报告基因小鼠（Aim 1）。然后，我们将评估MG靶向和基因组编辑效率 在LSL-tdTomato小鼠和HIV Tg 26转基因小鼠中的多重Cas 12 f mRNA/sgRNA持续递送 (Aim 2）。最后，我们将探索Exo-ERVLP AAV-B-Cas 12 f全身注射在肿瘤中的治疗潜力。 HIV Tg 26转基因小鼠（Aim 3）。这个高风险高回报的提案汇集了几个先进的 技术和具有互补专业知识的团队。一体化多重Cas 12 f/sgRNA 通过AAV-B、PEG 10货物和CD 63 M EXOtic递送转基因，用于持续靶向和HIV 根除预期的积极成果将为系统地提供CRISPR/Cas编辑器提供一种新的工具 为多种MG相关疾病的治疗药物开发提供了新的途径。