Nuclear receptor regulation of epigenetic mechanisms regulating HIV CNS latency

背景资料。这项题为“核受体调控HIV-CNS表观遗传机制”的提案 潜伏期“是对RFA-MH-22-280：调节HIV CNS潜伏期和 使用新的单细胞技术的神经发病机制“。小胶质细胞是HIV感染的主要目标 在中枢神经系统(CNS)中，并形成感染的蓄水池，尽管接受了抗病毒治疗和 导致艾滋病毒相关的神经认知障碍(手)。 我们的目标。使用小胶质细胞模型，我们之前证明了Nurr1核受体，工作 与配体激活的核受体一起，在HIV潜伏期的调节中起着关键作用。我们 在IPSC来源的人小胶质细胞(IMG)中证实了这些观察结果，这提供了最丰富的信息 建立原代细胞模型，研究HIV与这一重要靶细胞的相互作用。在这里，我们将测试 假设Nurr1，RXR和RAR核受体激动剂导致HIV沉默，这表明 一种治疗中枢神经系统艾滋病毒感染的新策略。这项工作的基础是两项关键技术 人类小胶质细胞中艾滋病毒研究的进展。首先，我们现在有能力研究艾滋病毒的潜伏期 单独使用IMG或与IPSC来源的神经元和星形胶质细胞联合使用--这是第一个可用的模型系统 不依赖于转化的细胞系。第二，与大炮实验室合作，我们建立了一个 基于NOG-hIL34转基因小鼠的人源化小鼠小胶质细胞HIV潜伏模型 我们演示了 人源化小鼠支持人类小胶质细胞潜在的HIV感染。我们已经能够识别潜伏感染的 从人源化的小鼠身上恢复了细胞。此外，我们还将单细胞RNA测序(scRNA-Seq)应用于 确认小胶质细胞具有与参考人类小胶质细胞匹配的转录特征，并进一步 确定了与活动性艾滋病毒感染相关的反应性小胶质细胞亚群。 唯一支持人类小胶质细胞完全成熟的人源化小鼠模型。 使用这些模型， 我们将应用高灵敏的染色质免疫沉淀(芯片)和单细胞测序和表观基因组学 分析确定核受体与HIV启动子区域的相互作用，以及表观遗传机制 在HIV LTR上保持沉默。 我们将如何推进这一领域？许多临床有效的、脑穿透的药物，调节核 受体现已上市，其中许多现已获得FDA批准，并将使用IPSC衍生的 细胞模型和人源化小鼠模型对HIV表达和潜伏期的影响 完好无损的大脑。我们正在进行这些具有技术挑战性的研究，因为只有在 真实的人类小胶质细胞的背景和更好地反映艾滋病毒感染的中枢神经系统的环境， 基本的生理学被准确地表达出来。我们相信这些研究将使我们获得令人信服的 针对HIV中枢神经系统潜伏期和神经发病机制的这一重要治疗策略的概念验证。

Background. This proposal entitled “"Nuclear receptor regulation of epigenetic mechanisms regulating HIV CNS latency" is submitted in response to RFA-MH-22-280: Epigenetic Mechanisms Regulating HIV CNS Latency and Neuropathogenesis Using Novel Single Cell Technologies”. Microglial cells are the main target of HIV infection in the central nervous system (CNS) and form a reservoir of infection that persists despite antiviral treatment and contributes to HIV-associated neurocognitive disorder (HAND). Our goal. Using microglia cellular models, we demonstrated previously that the Nurr1 nuclear receptor, working in conjunction with ligand-activated nuclear receptors, plays a critical role in the regulation of HIV latency. We confirmed these observations in iPSC-derived human microglia (iMG), which provide the most informative primary cell model to study the interaction between HIV and this important target cell. Here, we will test the hypothesis that agonists of the Nurr1, RXR and RAR nuclear receptors lead to HIV silencing, suggesting a novel therapeutic strategy for HIV infections in the CNS. Underpinning this work are two key technical advances allowing the study of HIV in human microglia. First, we now have the capability to study HIV latency using iMG alone or in combination with iPSC-derived neurons and astrocytes - the first available model systems that do not rely on transformed cell lines. Second, in collaboration with the Cannon laboratory, we established a humanized mouse model of HIV latency in microglia based on the new NOG-hIL34 transgenic mouse, which is We demonstrated that the humanized mice support latent HIV infection of human microglia. We have been able to identify latently infected cells recovered from the humanized mice. In addition, we applied single cell RNA sequencing (scRNA-Seq) to confirm that the microglia have transcriptional characteristics that match reference human microglia and further identified subsets of reactive microglial cells that are associated with active HIV infections. the only humanized mouse model to support full human microglial cell maturation. Using these models, we will apply highly sensitive chromatin immunoprecipitation (ChIP) and single cell sequencing and epigenomics analyses to define nuclear receptor interactions with the HIV promoter region, and the mechanism of epigenetic silencing on the HIV LTR. How will we advance the field? Numerous clinically-effective, brain-penetrant drugs that regulate the nuclear receptors are available, many of which are now FDA approved, and will be evaluated using the iPSC derived cell models and in the humanized mouse model for their impact on HIV expression and latency in the context of an intact brain. We are undertaking these technically challenging studies because it is only by working within the context of authentic human microglial cells and an environment that better reflects the HIV-infected CNS, that the underlying physiology is accurately represented. We believe these studies will allow us to obtain a convincing proof-of-concept for this important therapeutic strategy to target HIV CNS Latency and Neuropathogenesis.