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

阿片类药物导致的神经病理事件的加重和艾滋病毒转录的改变导致艾滋病毒 相关的中枢神经系统功能障碍的报道也很多。尽管持续多年的抑制性抗逆转录病毒治疗 (ART)，潜伏的艾滋病毒持续存在，并在与阿片类药物使用障碍有关的许多相同的大脑区域找到避难所 (OUD)表明艾滋病毒与脑细胞中的阿片类药物之间的相互作用。然而，我们在这方面存在着相当大的差距。 人类抗逆转录病毒治疗对HIV感染脑内细胞反应和病毒持久性的影响 或相关的模型器官。这项行政补充建议旨在产生新的地形 来自父母奖的数据集和证据显示了海马体的单细胞分辨率，这是一个大脑区域 在非人灵长类动物(NHP)中，以易感染艾滋病毒而闻名。这些数据将提供一个 史无前例的多种模式的细胞格局，可以被利用来制定限制病毒的策略 坚持不懈，恢复和保持艾滋病毒携带者的最佳大脑健康。在我们已出版的和初步的 我们开发了创新的单细胞方法，包括单细胞异构体RNA测序 (ScISOr-Seq)，它使数千个多腺化RNA的单细胞长读RNA测序成为可能 单细胞的数量。这些新的测序和计算方法与ScATAC-Seq一起用于 染色质的可及性，将允许绘制细胞基因表达的图谱，开放染色质区域，异构体 以及跨海马单细胞的SIV检测。最近的文献支持艾滋病病毒存在于 大脑，更具体地说，存在于海马体内的小胶质细胞和星形胶质细胞。重要的是，这 大脑区域也参与了OUD的联想学习过程。此外，我们之前对啮齿动物的研究 通过建立区域和细胞特异性的海马体，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，以及这些分布是如何 受性、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染色体 集成基因组学，我们在这个补充应用程序中建议生成多维单细胞 数据集，并定义在有和没有SIV感染NHP的受控模型中这些观察结果的相关性 记录的慢性吗啡暴露的不同阿片类药物与父母研究评估的比较 羟考酮。我们还建议验证在选择基因中观察到的单细胞表达的任何变化 原位杂交。我们建议在以下具体目标下实现这些目标：目标1 SIV感染受控环境下海马区全基因组单细胞转录图谱的研究 NHP监测抗逆转录病毒药物，并长期接触吗啡。目标2.建立全基因组范围的单细胞 在目标1中检查的组的海马体的表观基因组图谱。

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.