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

项目摘要/摘要。 根治艾滋病毒的方法需要安全和有效地清除所有储存库中具有复制能力的病毒， 包括大脑。中枢神经系统储存库的特征和新治愈策略的经验测试需要 与生理相关的艾滋病毒在大脑中持续存在的动物模型。在这里，我们建议将主要 我们团队在Shiv NHP模型和AAV提供的CRISPR/Cas9编辑方面的进步，以描绘关键 根据中枢神经系统储存库的特点，确定以CRISPR为基础的脑内根除的疗效。 传播/创立者(TF)SHIV编码最小适应的TF HIV-1 env，代表着一项重大进步 在生物学上相关的NHP模型中。TFSHIV已经在恒河猴身上显示出强大的复制能力， 反映人类HIV-1感染的病毒动力学、细胞嗜性和发病机制。此外，他们最近还 显示出忠实地概括了病毒与宿主的相互作用，坚持通过抑制艺术，并反弹到 与HIV-1类似的动力学和克隆性。在这里，我们将使用一种新颖的、遗传条形码的TFSHV模型 中枢神经系统的致病机制和持久性，基于Tf SHIV.D.191859(SHIV.D)，它编码一个D分支Tf HIV- 1Env是CCR5嗜性的，在CD4T细胞和单核细胞来源的巨噬细胞中有效复制，以及 展示了一致的中枢神经系统复制、发病机制和持久性。使用此条形码的TF SHIV.D模型 CNS持久性，我们将测试一个新型的一体式AAV9介导的CRISPR/Cas9基因编辑系统。最近 首次在非人类灵长类动物中对感染SIV的恒河猴进行研究，证明了该药的耐受性和有效性 这种方法。AAV9-CRISPR-Cas9在组织中广泛分布，导致切割和 切除SIV基因组，并大幅缩小组织中前病毒储存库的大小。值得注意的是， AAV在中枢神经系统内分布均匀，导致前病毒在脑区的切除。在此应用程序中， 我们将利用巨噬细胞嗜好条形码SHIV.D模型和AAV递送的CRISPR/Cas9的进展 编辑以深入了解中枢神经系统的神经发病机制和持久性。我们的假设是通过(I)刻画 SHIV.D在关键中枢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)测试全局和髓系靶向CRISPR方法的效果 在体内减少中枢神经系统储存库方面，我们将推进从大脑中根除艾滋病毒的前景。如果这个 假设得到肯定，对HIV治疗领域的意义将是实质性的，因为它将改善我们的 了解中枢神经系统的储存库，建立艾滋病毒在大脑中的致病机制和持久性的稳健模型， 并提供了关于基于CRISPR的有前景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.