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

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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.