Integrative Single Cell isoform and chromatin accessibility Mapping of Chronic Opioid Exposure in Cognitive Brain Areas in HIV

阿片类药物导致神经病理学事件的恶化和 HIV 转录的改变，从而导致 HIV 相关的中枢神经系统功能障碍已有充分报道。尽管多年来持续进行抑制性抗逆转录病毒治疗 （ART），潜伏的艾滋病毒持续存在，并在涉及阿片类药物使用障碍的许多相同大脑区域中找到庇护所 （OUD）表明艾滋病毒和阿片类药物在脑细胞中存在相互作用。但我们的差距还很大 关于 OUD 如何影响人类 ART 中感染 HIV 的大脑中的细胞反应和病毒持久性的知识 或相关模型有机体。该行政补充提案旨在产生新的地形 来自家长奖励的数据集和海马体（大脑区域）单细胞分辨率的证据 因在非人类灵长类动物 (NHP) 中偏好 HIV 持续存在和 OUD 而闻名。这些数据将提供 前所未有的多种模式的细胞景观，可用于制定限制病毒的策略 坚持并恢复和保持艾滋病毒感染者的最佳大脑健康。在我们发布的和初步的 我们开发了创新的单细胞方法，包括单细胞同工型 RNA 测序 (ScISOr-Seq)，可对数千个多腺苷酸化 RNA 进行单细胞长读长 RNA 测序 单细胞。这些新颖的测序和计算方法与 ScATAC-Seq 相结合，可用于 染色质可及性，将允许绘制细胞基因表达、开放染色质区域、亚型的图谱 以及海马单细胞中 SIV 的检测。最近的文献支持艾滋病毒的存在 大脑，更具体地说是海马体内的小胶质细胞和星形胶质细胞。重要的是，这 大脑区域也参与 OUD 的联想学习过程。此外，我们之前对啮齿动物的研究 海马体通过建立区域和细胞特异性为拟议的研究奠定了基础 阿片肽和受体以及相关信号分子的分布，以及这些分布如何 受到性、压力和阿片类药物相关学习的影响。使用新的尖端单细胞多组学 整合基因组学，我们在此补充申请中建议生成多维单细胞 数据集并定义受控 SIV 感染 NHP 模型中此类观察结果的相关性，无论是否有 记录的慢性吗啡暴露的不同阿片类药物，用于与母研究评估进行比较 羟考酮。我们还建议使用以下方法验证在选定基因的单细胞表达中观察到的任何变化 原位杂交。我们建议通过以下具体目标来实现这些目标： 目标 1. 一代 在 SIV 感染的受控环境中海马全基因组单细胞转录组谱的研究 NHP 接受 ART 监测并长期接触吗啡。目标 2. 建立全基因组单细胞 目标 1 中检查的各组海马的表观基因组图谱。

Opioid driven exacerbations of neuropathological events and alterations in HIV transcription contributing to HIV associated CNS dysfunction are well-reported. Despite years of continuous suppressive antiretroviral therapy (ART), latent HIV persists and finds sanctuary in many of the same brain regions involved in opioid use disorder (OUD) suggesting interactions between HIV and opioids in brain cells. However, there is a sizeable gap in our knowledge on how OUD impacts cellular responses and viral persistence in HIV-infected brain on ART in humans or relevant model organsims. This administrative supplement proposal seeks to generate new topographical data sets from the parent award and evidence at single cell resolution across the hippocampus, a brain region known for predilection for HIV persistence and OUD in non-human primate (NHP). These data will provide an unprecedented cellular landscape of multiple modalities that can be harnessed to develop strategies to limit viral persistence and restore and retain optimal brain health in people living with HIV. In our published and preliminary work we have developed innovative single-cell approaches including Single-cell isoform RNA sequencing (ScISOr-Seq), which enables single-cell long-read RNA sequencing of polyadenylated RNAs across thousands of single cells. In concert these novel sequencing and computational methods, along with ScATAC-Seq for chromatin accessibility, will permit the mapping of cellular gene expression, open chromatin regions, isoforms and the detection of SIV across single-cells of hippocampus. Recent literature supports the presence of HIV in the brain and more specifically in microglia and astrocytes present within the hippocampus. Importantly, this brain region is also involved in associative learning processes for OUD. Moreover, our prior studies in rodent hippocampus have laid the groundwork for the proposed studies by establishing the regional and cell-specific distributions of opioid peptides and receptors as well as related signaling molecules, and how these distributions are impacted by sex, stress and opioid-associated learning. Using new cutting-edge single-cell mulitomic integrated genomics, we propose in this supplemental application to generate multi-dimensional single-cell data sets and define the relevance of such observations in a controlled SIV infected NHP model with and without a different opioid of documented chronic morphine exposure for comparison with the parent study evaluating oxycodone. We also propose to validate any changes observed in single-cell expression in select genes using in situ hybridization. We propose to achieve these goals under the following specific aims: Aim 1. Generation of genome-wide single-cell transcriptomic profiles in the hippocampus in a controlled setting of SIV infection of NHPs monitored on ART and exposed to chronic morphine. Aim 2. Establish genome-wide single-cell epigenomic profiles of the hippocampus in the groups examined in aim 1.