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

摘要 潜伏感染的脑髓系细胞包括小胶质细胞(MG)和血管周围巨噬细胞可作为HIV 蓄水池，有助于神经艾滋病毒的持久性、慢性神经炎和艾滋病毒相关 神经认知障碍(手)。旨在消除艾滋病毒携带者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情况下也是如此。广泛的研究，包括FDA批准的阶段 我的临床试验证明了CRISPR/Cas基因组编辑在治愈HIV方面的治疗潜力。 然而，临床应用的一个主要障碍是缺乏有效和特定的靶向递送 体内与疾病相关的组织和/或细胞，特别是在神经艾滋病毒中。这项提议的总体目标是 开发AAV介导的将微型Cas12f基因组编辑器隐形运送到HIV细胞储存库的研究 大脑。我们将利用PEG10介导的内源性逆转录病毒样颗粒(ERVLP)新技术 依赖内源性PEG10和合胞素-A进行货物(Cas12f)mRNA转移到MG。这一方法将 利用最有希望的AAV基因疗法的好处。几种AAV血清型，如AAV1、2、5、6 可在体内外以80%的效率转导MG(AAV-M)，但不能跨越血脑屏障 (Bbb)。相比之下，目前可用的穿透血脑屏障的AAV血清型(AAV-B)，如AAV9，PhP.B， 无论在体内外，PhP.eB、F、B10或B22对MG的转导效率都较低。因此，新的AAV 迫切需要有效跨越血脑屏障和转导MG的血清型(AAV-BM)。我们假设 AAV-B可以提供一次性系统注射隐形货物(CDNAs)到星形胶质细胞和/或 神经元反过来作为中继站，将持续的mRNA/sgRNA转移到MG。这批隐形的AAV货物 还将包括一种通过与MG特异性多肽连接的CD63将外周小体转移到细胞(外来)的装置 (CD63M)。我们预计PEG10介导的ERVLP和CD63M介导的外来基因将协同促进 HIV清除剂对MG的内源性传播。为此，我们将首先使用CRE-loxP系统 经AAV-B的MG靶向外切小体包膜ERVLP系统(Exo-ERVLP)可以有效的概念证明 转导星形胶质细胞/神经元向非转导MG体内递送Cre-mRNA LoxP(LSL)-td番茄报告小鼠(目标1)。然后，我们将评估MG靶向和基因组编辑效率 复合Cas12f mRNA/sgRNA在LSL-td番茄小鼠和HIV TG26转基因小鼠中的持续传递 (目标2)。最后，我们将探讨Exo-ERVLP AAV-B-Cas12f系统注射对小鼠的治疗潜力。 HIV TG26转基因小鼠(目标3)。这项高风险、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。一体式多路复用Cas12f/sgRNA 转基因通过AAV-B、PEG10货物和CD63M外源基因传递，用于持续靶向和HIV 根除。预期的积极结果将为系统地交付CRISPR/CAS编辑提供一种新的工具 为治疗多种重症肌无力相关疾病提供了新的途径。

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.