Cell-type specific molecular and functional analyses to target dorsal horn pain circuitry in mice and non-human primates

针对小鼠和非人类灵长类动物背角疼痛回路的细胞类型特异性分子和功能分析

• 批准号: 10863324
• 负责人: Andreas Robert Pfenning
• 金额: $ 49.91万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10863324
• 关键词: ATAC-seq; Acute; Affect; Afferent Neurons; Atlases; Automobile Driving; Bioinformatics; Biological Models; Cell Nucleus; Cell model; Cells; Cholecystokinin; Chromatin; Chronic; Clinical; Communities; Computer Analysis; Data; Data Set; Engineering; Epigenetic Process; Esthesia; Future; Gene Expression; Genes; Genetic; Goals; Hand; High Prevalence; Horns; Human; In Situ Hybridization; Individual; Inflammatory; Injury; Knowledge; Laboratories; Ligands; Macaca; Macaca mulatta; Machine Learning; Maps; Measures; Mediating; Methods; Modeling; Molecular; Movement; Mus; Nerve; Neurons; Neuropathy; Pain; Pain management; Parvalbumins; Patients; Persistent pain; Phylogenetic Analysis; Population; Positioning Attribute; Posterior Horn Cells; Process; Regulatory Element; Research; Site; Specificity; Spinal; Technology; Therapeutic; Time; Tissues; Touch sensation; Transgenic Mice; Translating; Vertebral column; Viral Vector; Work; behavior test; candidate identification; cell type; chronic pain patient; design; dorsal horn; excitatory neuron; experimental study; genetic manipulation; human data; improved; inhibitory neuron; insight; integration site; machine learning algorithm; mechanical allodynia; neurotransmission; nonhuman primate; novel; novel therapeutics; pain patient; protein kinase C gamma; receptor; recombinase; screening; searchable database; single cell sequencing; single nucleus RNA-sequencing; somatosensory; targeted treatment; therapeutic candidate; therapeutic target; tool; transcriptomics; virus genetics

Project Summary Persistent pain remains a major clinical problem because of its high prevalence and the lack of adequate treatment options. The spinal dorsal horn is a major site for the integration of somatosensory information and for circuit-based transformations that underlie persistent pain. The overall goal our work is to develop novel pain therapies that target the dorsal horn circuitry for mechanical allodynia. We use cell type-specific targeting of chemogenic receptors to identify spinal dorsal horn neurons that mediate mechanical allodynia without affecting acute sensation in mice. Mice are a commonly used model system for these studies because of their general similarity to humans, the ease of genetic manipulations and the large number of tools available. However, targeting spinal dorsal horn neurons for therapeutic purposes requires an understanding of the cellular and molecular conservation between species. Rhesus macaque are phylogenetically more similar to humans and with re-engineered tools can serve as a proof-of- principle model system. To this end, we analyzed the molecular and cellular organization of the macaque dorsal horn by single cell transcriptomics and in situ hybridization methods. We compared these data to mouse and human data to generate a harmonized atlas of dorsal horn cell types across species. Here we will 1) Identify the epigenetic landscape of human, macaque and mouse dorsal horn neurons using single nucleus RNA- and ATAC-Seq data together with machine learning-assisted bioinformatic approaches to identify regulatory elements (REs) that drive robust cell type-specific gene expression across species. We will 2) screen candidate REs for cell-type specificity in mice and subsequently macaque and then use the cell type-specific REs to systematically generate a functional map of the dorsal horn across species. Studies proposed here will generate large-scale searchable databases for single cell transcriptomics and open chromatin regions in mice, macaque and human. Importantly, we will have the knowledge and viral vectors needed to pursue macaque proof-of-principle studies of novel therapies for mechanical allodynia.

项目概要 持续性疼痛仍然是一个主要的临床问题，因为其发病率高且缺乏治疗措施。 足够的治疗选择。脊髓背角是整合的主要部位 体感信息以及持续疼痛背后基于电路的转换。 我们工作的总体目标是开发针对背角电路的新型疼痛疗法 用于机械性异常性疼痛。我们使用化学受体的细胞类型特异性靶向来识别 介导机械异常性疼痛而不影响急性感觉的脊髓背角神经元 在小鼠中。小鼠是这些研究中常用的模型系统，因为它们具有一般性 与人类的相似性、基因操作的简便性以及大量可用的工具。 然而，以脊髓背角神经元为治疗目的需要 了解物种之间的细胞和分子保守性。恒河猴是 在系统发育上与人类更加相似，并且通过重新设计的工具可以作为证据 原理模型系统。为此，我们分析了细胞的分子和细胞组织。 通过单细胞转录组学和原位杂交方法研究猕猴背角。我们 将这些数据与小鼠和人类数据进行比较，生成统一的背角图谱 跨物种的细胞类型。在这里我们将 1) 识别人类、猕猴的表观遗传景观 和小鼠背角神经元，使用单核 RNA 和 ATAC-Seq 数据以及 机器学习辅助生物信息方法来识别调控元件（RE） 驱动跨物种的强大细胞类型特异性基因表达。我们将 2) 筛选候选 RE 用于小鼠和随后猕猴的细胞类型特异性，然后使用细胞类型特异性 RE 系统地生成跨物种背角的功能图。提出的研究 这里将生成用于单细胞转录组学的大规模可搜索数据库并开放 小鼠、猕猴和人类的染色质区域。重要的是，我们将拥有知识和 进行猕猴新疗法原理验证研究所需的病毒载体 机械性异常性疼痛。