Novel macrophage-tropic transmitted/founder SHIV model of CNS persistence to evaluate CRISPR/Cas9 gene editing

项目概要/摘要。 根除艾滋病毒治疗需要安全有效地清除所有宿主中的复制能力病毒， 包括大脑。CNS储库的表征和新型治疗策略的经验性测试需要 HIV在大脑中持续存在的生理学相关动物模型。在这里，我们建议将主要 我们团队在SHIV NHP模型和AAV递送的CRISPR/Cas9编辑方面的进展， 研究CNS储库的特征，并确定基于CRISPR的根除在脑中的功效。 编码最低适应性TF HIV-1 Env的Transmitted/founder（TF）SHIV代表了一项重大进展， 生物学相关的NHP模型。TF SHIV在恒河猴中表现出稳健的复制， 病毒动力学、细胞嗜性和发病机制反映了人类HIV-1感染。此外，他们最近被 显示忠实地再现病毒-宿主相互作用，通过抑制性ART持续存在， 与HIV-1相似的动力学和克隆性。在这里，我们将采用一种新的，基因条形码TF SHIV模型， CNS发病机制和持久性，基于TF SHIV.D.191859（SHIV.D），其编码进化枝D TF HIV-1。 1 Env是CCR 5-嗜性的，在CD 4 T细胞和单核细胞衍生的巨噬细胞中有效复制，和 证明了一致的CNS复制、发病机制和持久性。使用这个带条形码的TF SHIV.D模型 CNS持久性，我们将测试一种新型的全合一AAV 9介导的CRISPR/Cas9基因编辑系统。最近的一 在SIV感染的恒河猴中进行的首次非人灵长类动物研究证实了 这种方法。AAV 9-CRISPR-Cas9广泛分布在组织中，导致切割和细胞分裂。 SIV基因组的切除和跨组织的前病毒储库的大小的显著减小。值得注意的是， AAV在CNS内分布良好，导致前病毒穿过脑区域被切除。在本申请中， 我们将利用嗜巨噬细胞条形码SHIV.D模型和AAV递送的CRISPR/Cas9 编辑以深入了解CNS神经发病机制和持久性。我们的假设是，通过（i）表征 SHIV.D在关键CNS细胞和组织中的持久性，（ii）优化AAV递送的CRISPR-Cas9方法， SHIV.D在大脑中的持久性，以及（iii）测试全局和骨髓靶向CRISPR方法的效果 在减少体内CNS储库方面，我们将推进从大脑中根除艾滋病毒的前景。如果这 假设得到肯定，对艾滋病治疗领域的意义将是实质性的，因为它将提高我们的 了解中枢神经系统的水库，开发一个强大的模型，艾滋病毒的发病机制和持久性在大脑中， 并提供关于基于CRISPR的治疗策略的安全性和有效性的关键临床前数据。

Project Summary/Abstract. An eradicative HIV cure requires safe and effective clearance of replication competent virus from all reservoirs, including the brain. Characterization of the CNS reservoir and empiric testing of novel cure strategies require physiologically relevant animal models of HIV persistence in the brain. Here, we propose to integrate major advances from our groups in SHIV NHP models and AAV-delivered CRISPR/Cas9 editing to delineate key features of the CNS reservoir and determine the efficacy of CRISPR-based eradication in the brain. Transmitted/founder (TF) SHIVs, which encode minimally adapted TF HIV-1 Envs, represent a major advance in biologically relevant NHP models. TF SHIVs have demonstrated robust replication in rhesus macaques, with viral kinetics, cell tropism, and pathogenesis that mirror HIV-1 infection of humans. Further, they recently been shown to faithfully recapitulate virus – host interactions, persist through suppressive ART, and rebound with similar kinetics and clonality as HIV-1. Here, we will employ a novel, genetically barcoded TF SHIV model of CNS pathogenesis and persistence, based on TF SHIV.D.191859 (SHIV.D), which encodes a clade D TF HIV- 1 Env that is CCR5-tropic, efficiently replicates in CD4 T cells and monocyte-derived macrophages, and demonstrates consistent CNS replication, pathogenesis and persistence. Using this barcoded TF SHIV.D model CNS persistence, we will test a novel all-in-one AAV9-mediated CRISPR/Cas9 gene editing system. A recent first-in nonhuman-primate study of SIV-infected rhesus macaques demonstrated the tolerability and efficacy of this approach. The AAV9-CRISPR-Cas9 was broadly distributed across tissues, leading to cleavage and excision of the SIV genome and substantial reductions in the size of the proviral reservoir across tissues. Notably, the AAV was well distributed within CNS resulting in excision of provirus across brain regions. In this application, we will leverage advances in the macrophage-tropic barcoded SHIV.D model and AAV-delivered CRISPR/Cas9 editing to gain insight on CNS neuropathogenesis and persistence. Our hypothesis is that by (i) characterizing SHIV.D persistence in key CNS cells and tissues, (ii) optimizing AAV-delivered CRISPR-Cas9 approaches for SHIV.D persistence in the brain, and (iii) testing the effects of global and myeloid-targeting CRISPR approaches on CNS reservoir reduction in vivo, we will advance prospects for eradicating HIV from the brain. If this hypothesis is affirmed, the significance to HIV cure field would be substantial, since it would improve our understanding of the CNS reservoir, develop a robust model for HIV pathogenesis and persistence in the brain, and provide key pre-clinical data on the safety and efficacy of a promising CRISPR-based cure strategy.