Single cell multi-omics of iPSC-derived brain organoids from patients with opioid use disorder: synthetic opioids as molecular probes

项目摘要/摘要 与合成阿片类药物有关的过量死亡人数急剧增加。芬太尼导致了其中许多过量服药 死亡。然而，羟考酮是美国处方最多的阿片类药物之一。目前的体外检测方法 以及体内模型，旨在研究阿片类药物使用障碍(OUD)的病理生理学并发现潜在的 治疗靶点是有用的，但需要额外的模型系统。我们的初步数据和数据 使用单细胞测序的临床前研究表明，每种阿片类药物可能都有独特的分子 简况和行动机制。这些发现突显了需要更多的模型来评估药物 在单细胞水平上的大脑活动。我们的研究团队结合了成瘾医学的专业知识， 药物基因组学和生物信息学，开发计算和实验所需的专业知识 在诱导多能干细胞中整合基因表达和染色质可及性的框架- 衍生的脑有机化合物。这项拟议的研究的目标是为药物作用提供新的机制洞察力。 在单细胞分辨率下。我们的研究策略涉及使用单细胞测序技术和 IPSC衍生的3D脑有机化合物使用两种常用处方识别OUD的分子签名 合成阿片类药物：羟考酮和芬太尼作为分子探针。目标1，我们将定义分子特征 对合成阿片类药物的反应：羟考酮和芬太尼暴露于IPSC衍生的前脑有机体 单细胞水平的OUD患者和健康对照组。目的2，我们建议重建转录 应用新的网络生物学方法确定大脑有机体中不同细胞类型的调节因子 潜在的候选者，以检测嵌入在大数据海洋中的有意义的生物信息并发现 解释生物表型特性的新的调控机制。这些方法可能会有所帮助 开发用于实验验证的机械假说。目标3，我们将研究基因和途径 从目标1和目标2中确定，关于它们作为OUD治疗的新药物靶点的潜在用途 通过使用适当的iPS来源的CNS细胞类型和脑进行功能基因组研究进行预防 有机化合物我们的发现将增进对药物作用机制(S)的一般理解和 以药物依赖的方式导致阿片成瘾的潜在病理生理学，从而打开了新的 寻找治疗黑色素瘤的新靶点的途径。总而言之，这项提议的基础是 广泛的初步数据，以及数十年将药物用作基础研究的分子探针的经验 基因组和其他基因组机制。因此，拟议的研究对以下方面具有重要影响 分子机制导致了解OUD的病理生理以及新的发现 用于治疗和/或预防的治疗剂。如果成功，我们的研究方法可能是 可推广到其他阿片类药物和其他物质使用障碍。

Project Summary/Abstract Synthetic opioid-involved overdose deaths have increased sharply. Fentanyl is driving many of those overdose deaths. However, oxycodone is one of the most prescribed opioid medications in the US. Current in vitro assays and in vivo models designed to study the pathophysiology of opioid use disorder (OUD) and to discover potential therapeutic targets are useful, but there is a need for additional model systems. Our preliminary data and serval preclinical study using single-cell sequencing have revealed that each opioid agent might have unique molecular profiles and mechanisms of action. Those findings highlight the need for additional models to evaluate drug action in the brain at the single-cell level. Our research team combines expertise in addiction medicine, pharmacogenomics, and bioinformatics, expertise required to develop a computational and experimental framework to integrate gene expression and chromatin accessibility in induced pluripotent stem cell (iPSC)- derived brain organoids. The goal of the proposed study is to provide novel mechanistic insight into drug action at single-cell resolution. Our research strategy involves the use of single-cell sequencing technology and iPSC-derived 3D brain organoids to identify molecular signatures for OUD using two commonly prescribed synthetic opioids: oxycodone and fentanyl as molecular probes. Aim 1, we will define molecular characteristics of response to synthetic opioids: oxycodone and fentanyl exposure of iPSC-derived forebrain organoids from both OUD patients and healthy controls at the single-cell level. Aim 2, we propose to reconstruct transcriptional regulons in different cell types in the brain organoids by applying novel network biology approaches to prioritize potential candidates, to detect meaningful biological information embedded in the sea of Big Data and to uncover novel regulatory mechanisms that explain the properties of biological phenotypes. These approaches could help to develop mechanistic hypothesis for experimental validation. Aim 3, we will study genes and pathways identified from Aim 1 and Aim 2 with regard to their potential use as novel drug targets for OUD treatment or prevention, by pursuing functional genomic studies using appropriate iPS-derived CNS cell types and brain organoids Our findings will enhance the general understanding of drug mechanism(s) of action and the underlying pathophysiology responsible for opioid addiction in a drug-dependent fashion, thus opening new avenues to discover novel therapeutic targets for the treatment of OUD. In summary, this proposal is based on extensive preliminary data, and decades of experience in using drugs as “molecular probes” for underlying genomic and other omic mechanisms. As a result, the proposed studies have significant implications for molecular mechanisms leading to understanding of the pathophysiology of OUD as well as the discovery of novel therapeutic agents for OUD treatment and/or prevention. If successful, our research approaches could be generalizable to other opioids and to other substance use disorders.