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

项目总结/摘要 与合成阿片类药物有关的过量死亡人数急剧增加。芬太尼导致许多人过量服用 死亡然而，羟考酮是美国最常用的阿片类药物之一。当前体外试验 和体内模型，旨在研究阿片类药物使用障碍（OUD）的病理生理学， 治疗靶点是有用的，但需要另外的模型系统。我们的初步数据和服务 使用单细胞测序的临床前研究已经揭示，每种阿片类药物可能具有独特的分子生物学特性。 概况和作用机制。这些发现强调了需要额外的模型来评估药物 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 在诱导多能干细胞（iPSC）中整合基因表达和染色质可及性的框架- 衍生的脑类器官。这项研究的目的是为药物作用机制提供新的见解 单细胞分辨率。我们的研究策略包括使用单细胞测序技术， iPSC衍生的3D脑类器官使用两种常用处方鉴定OUD的分子特征 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： OUD患者和健康对照在单细胞水平。目的2，我们建议重建转录 通过应用新的网络生物学方法来区分脑类器官中不同细胞类型的调节子 潜在的候选人，以检测嵌入在大数据海洋中的有意义的生物信息， 解释生物表型特性的新的调节机制。这些方法可以帮助 为实验验证提出机械假说。目标3，我们将研究基因和途径 从目标1和目标2中确定了它们作为OUD治疗的新型药物靶点的潜在用途，或 预防，通过使用适当的iPS衍生的CNS细胞类型和脑进行功能基因组研究 我们的研究结果将提高对药物作用机制的一般理解， 潜在的病理生理学负责阿片类药物成瘾的药物依赖的方式，从而打开新的 发现治疗OUD的新治疗靶点的途径。总而言之，这一提议是基于 广泛的初步数据，以及数十年使用药物作为“分子探针”的经验， 基因组和其他组学机制。因此，拟议的研究对以下方面具有重大意义： 分子机制，从而了解OUD的病理生理学以及发现新的 用于OUD治疗和/或预防的治疗剂。如果成功，我们的研究方法可以 可推广至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.