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: 10523246
• 负责人: Wenhui Hu
• 金额: $ 62.78万
• 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 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10523246
• 关键词: Astrocytes; Blood - brain barrier anatomy; Brain; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Devices; Disease; Endogenous Retroviruses; FDA approved; Genome; HIV; HIV-1; HIV-associated neurocognitive disorder; In Vitro; Injectable; Injections; Link; 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; Time; Tissues; Transgenes; Transgenic Mice; United States National Institutes of Health; Virus; antiretroviral therapy; clinical application; exosome; gene therapy; genome editing; high reward; high risk; in vivo; macrophage; 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 潜伏感染的脑髓细胞，包括小胶质细胞 (MG) 和血管周围巨噬细胞，可充当 HIV 病毒 储存库，导致 NeuroHIV 持续存在、慢性神经炎症和 HIV 相关的 神经认知障碍（HAND）。旨在消除艾滋病毒储存库的策略非常有希望治愈 HIV，即使存在有效的抗逆转录病毒治疗。广泛的研究，包括 FDA 批准的阶段 我的临床试验证明了 CRISPR/Cas 基因组编辑治疗 HIV 的治疗潜力。 然而，临床应用的一个主要障碍是缺乏有效和特异性的靶向递送 体内疾病相关组织和/或细胞，特别是 NeuroHIV。该提案的总体目标是 开发 AAV 介导的微型 Cas12f 基因组编辑器隐形货物递送至 HIV 细胞储存库 大脑。我们将利用新型 PEG10 介导的内源性逆转录病毒样颗粒 (ERVLP) 技术 依赖内源性 PEG10 和合胞素-A 将 Cargo (Cas12f) mRNA 转移到 MG 中。这种方法将 利用最有前途的 AAV 基因疗法的优势。多种 AAV 血清型，例如 AAV1、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 穿透型 AAV 血清型（AAV-B）如 AAV9、PhP.B、 PhP.eB、F、B10 或 B22 在体外和体内转导 MG 的效率均较低。因此，新型 AAV 迫切需要有效穿过 BBB 并转导 MG (AAV-BM) 的血清型。我们假设 AAV-B 可以将隐形货物 (cDNA) 一次性注射全身递送到星形胶质细胞和/或 神经元反过来充当持续 mRNA/sgRNA 转移至 MG 的中继站。这架隐形 AAV 货物 还将包括通过与 MG 特异性肽连接的 CD63 设计的外泌体转移至细胞 (EXOtic) 装置 （CD63M）。我们预计 PEG10 介导的 ERVLP 和 CD63M 介导的 EXOtic 将协同促进 HIV根除剂内源性传播至MG。为了实现这一目标，我们将首先使用 Cre-LoxP 系统 概念证明，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/神经元体内递送至非转导的 MG LoxP (LSL)-tdTomato 报告小鼠（目标 1）。然后，我们将评估 MG 靶向和基因组编辑效率 LSL-tdTomato 小鼠和 HIV Tg26 转基因小鼠中多重 Cas12f mRNA/sgRNA 的持续递送 （目标 2）。最后，我们将探讨 Exo-ERVLP AAV-B-Cas12f 全身注射的治疗潜力 HIV Tg26 转基因小鼠（目标 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成熟团队。多合一多重 Cas12f/sgRNA 转基因通过 AAV-B、PEG10 货物和 CD63M EXOtic 传递，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 编辑器提供新的工具 MG，并为多种 MG 相关疾病的治疗开发提供新途径。