Nuclear receptor regulation of epigenetic mechanisms regulating HIV CNS latency

表观遗传机制的核受体调节调节HIV中枢神经系统潜伏期

• 批准号: 10747002
• 负责人: Paula M Cannon
• 金额: $ 74.52万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747002
• 关键词: ATAC-seq; Acetylation; Agonist; Antiinflammatory Effect; Astrocytes; Biological Assay; Biological Models; Brain; Cell Maturation; Cell model; Cells; Central Nervous System; Central Nervous System Infections; Characteristics; Chromatin Structure; Clinical; Coculture Techniques; Collaborations; Complement; Complex; Defect; Drug Targeting; Environment; Enzymes; Epigenetic Process; Evaluation; Event; Exhibits; FDA approved; Family; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; HIV; HIV Infections; HIV-1; HIV-associated neurocognitive disorder; Heterogeneity; Histones; Human; IL13RA1 gene; Individual; Induced pluripotent stem cell derived neurons; Infection; Inflammation; Inflammatory Response; Intervention; Knock-out; Laboratories; Lead; Ligands; Maps; Measures; Mediating; Methods; Microglia; Modeling; Molecular Cloning; Monitor; Mus; NR4A2 gene; Neurocognitive; Neurons; Neuropathogenesis; Nuclear; Nuclear Receptors; Outcome; Patients; Pharmaceutical Preparations; Phosphorylation; Physiology; Play; Promoter Regions; RNA; RXR; Refractory; Regulation; Resolution; Role; Sampling; Symptoms; System; T-Lymphocyte; Testing; Therapeutic; Transformed Cell Line; Transgenic Mice; United States; Variant; Vesicular stomatitis Indiana virus; Viral; Virus; Virus Replication; Work; antiretroviral therapy; chromatin immunoprecipitation; cohort; design; drug candidate; epigenetic regulation; epigenetic silencing; epigenomics; experimental study; genetic manipulation; histone methylation; histone modification; humanized mouse; induced pluripotent stem cell; knock-down; member; mouse model; neuroinflammation; neurotoxic; novel; novel therapeutic intervention; pharmacologic; pre-clinical; programs; promoter; reactivation from latency; receptor; recruit; response; single cell sequencing; single cell technology; single-cell RNA sequencing; transcriptome; translatable strategy

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.

背景这项提案题为“核受体调节HIV CNS的表观遗传机制 潜伏期”是为了响应RFA-MH-22-280：调节HIV CNS潜伏期的表观遗传机制和 使用新的单细胞技术的神经发病机制”。小胶质细胞是HIV感染的主要靶细胞 在中枢神经系统（CNS），并形成一个水库的感染，持续尽管抗病毒治疗， 导致HIV相关的神经认知障碍（HAND）。 我们的目标。使用小胶质细胞模型，我们先前证明了Nurr 1核受体，工作 与配体激活的核受体结合，在调节HIV潜伏期中起关键作用。我们 在iPSC衍生的人小胶质细胞（iMG）中证实了这些观察结果，这提供了最多的信息 原代细胞模型来研究艾滋病毒和这个重要的靶细胞之间的相互作用。在这里，我们将测试 假设Nurr 1、RXR和RAR核受体的激动剂导致HIV沉默，表明 CNS中HIV感染的新治疗策略。支撑这项工作的是两个关键技术 这一进展使得在人类小胶质细胞中研究艾滋病毒成为可能。首先，我们现在有能力研究艾滋病毒的潜伏期， 使用iMG单独或与iPSC衍生的神经元和星形胶质细胞组合-第一个可用的模型系统 不依赖于转化的细胞系。第二，与坎农实验室合作，我们建立了一个 基于新的NOG-hIL 34转基因小鼠的HIV在小胶质细胞中潜伏的人源化小鼠模型， 我们证明了 人源化小鼠支持人小胶质细胞的潜伏HIV感染。我们已经能够识别出潜伏感染者 从人源化小鼠回收的细胞。此外，我们应用单细胞RNA测序（scRNA-Seq）， 证实所述小胶质细胞具有与参照人小胶质细胞匹配的转录特征， 确定了与活动性HIV感染相关的反应性小胶质细胞亚群。 唯一支持完全人小胶质细胞成熟的人源化小鼠模型。 使用这些模型， 我们将应用高灵敏度的染色质免疫沉淀（ChIP）、单细胞测序和表观基因组学 分析以确定核受体与HIV启动子区域的相互作用，以及表观遗传学的机制。 在HIV LTR上沉默。 我们将如何推进这一领域？许多临床有效的，大脑渗透药物，调节核 受体是可用的，其中许多现在是FDA批准的，并将使用iPSC衍生的 细胞模型和人源化小鼠模型中对HIV表达和潜伏期的影响， 完整的大脑我们正在进行这些技术上具有挑战性的研究，因为只有在 真实的人类小胶质细胞的背景和更好地反映HIV感染的CNS的环境， 潜在的生理学被准确地表示。我们相信这些研究将使我们能够获得令人信服的 这一重要治疗策略的概念验证，以靶向HIV CNS潜伏期和神经发病机制。