Gene-edited liver organoids for predictive hepatotoxicity

用于预测肝毒性的基因编辑肝脏类器官

基本信息

批准号：
10758179
负责人：
Pranav Joshi
金额：
$ 28.46万
依托单位：
BIOPRINTING LABORATORIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10758179
关键词：
3-Dimensional ABCB1 gene Address Affinity Alleles Animal Model Architecture Assessment tool Biological Assay Biomimetics CRISPR/Cas technology CYP2B6 gene CYP2C19 gene CYP2D6 gene CYP3A4 gene Caucasians Cause of Death Cell Line Cells Cessation of life Chemicals Classification Clustered Regularly Interspaced Short Palindromic Repeats Diffusion Disease model Doxycycline Drug toxicity Engineering Enzymes Epigenetic Process Ethnic Population Failure Genes Genetic Engineering Genetic Polymorphism Genetic Variation Goals Guide RNA HepG2 Hepatic Hepatocyte Hepatotoxicity Hospitalization Human Human Engineering Hydrogels Image In Vitro Individual Intestines Laboratories Lentivirus Link Liver Marketing Measures Mediator of activation protein Metabolic Biotransformation Metabolism Microfluidics Modeling Molecular Morbidity - disease rate Nature Nutrient Organoids Oxygen Pathogenicity Patients Perfusion Pharmaceutical Preparations Proteins Protocols documentation Reproducibility Small Business Innovation Research Grant Stains System Technology Testing Time Tissue Model Tissues Toxic effect Toxicity Tests United States Food and Drug Administration Variant Work bioprinting cost estimate drug candidate drug development drug efficacy drug induced liver injury drug metabolism drug withdrawal feasibility testing fetal hepatoma cell high throughput screening improved in vivo individual patient individual variation induced pluripotent stem cell insight interest manufacturing scale-up matrigel mortality overexpression pre-clinical response safety assessment

项目摘要

Project Summary/Abstract Unexpected adverse drug responses (ADRs) including drug-induced liver injury (DILI) are the 4th leading cause of death in the U.S. In addition, DILI in individuals is one of the major reasons for drug withdrawal from the market and is difficult to predict using conventional in vitro hepatotoxicity tests and preclinical animal models. Due to the critical link between DILI and drug failure, there is an urgent need for improved human hepatotoxicity testing in the early stage of drug development by investigating the major pathogenic mechanisms of DILI, such as genetic variations in drug metabolizing enzymes (DMEs) and drug transporters. To address this need, we propose to develop genetically engineered human liver organoids (HLOs) on a pillar/perfusion plate using gene-edited, induced pluripotent stem cell (iPSC) lines carrying CRISPR/Cas9 synergistic activation mediator (CRISPR-SAM), inducible Csy4, and multiplexed guide RNA (gRNA), and recapitulate poor and ultrafast drug metabolizers in different ethnic groups. Using normal and engineered HLOs on the pillar/perfusion plate, together with high-throughput, high-content, HLO imaging assays, we propose to decipher the cellular and molecular mechanisms underlying the toxicity of drug candidates and chemicals and assess DILI potential. Our core hypotheses are: (i) overexpression of DMEs and drug transporters can recapitulate ultrafast drug metabolizers in different ethnic groups that may be critical in addressing ADRs; (ii) metabolism- induced hepatotoxicity can be established using normal and engineered HLOs with model compounds; and (iii) high-throughput, high-content analysis of HLOs on the pillar/perfusion plate can be used to identify DILI, which in turn can improve predictability of compound hepatotoxicity in vivo. The specific aims of the proposed work are to: (1) create genetically engineered HLOs containing doxycycline-inducible, CRISPR-SAM for overexpression of multiple hepatic genes to model ultrafast metabolizers; (2) validate normal and engineered HLOs with model compounds, which undergo metabolism and lead to toxic cellular responses in the human liver. Although several human hepatic cell/tissue models including primary hepatocytes (ATCC), engineered hepatoma cell lines (HepG2-CYP cell panel from Hera BioLabs), liver spheroids (3D InSight liver microtissues from InSphero), bioprinted liver tissues (ExVive human liver tissues from Organovo), and microfluidic liver chips (Liver-Chip from Emulate) are commercially available, these in vitro liver models have been used for assessing general hepatotoxicity of compounds for normal drug metabolizers and cannot easily simulate poor and ultrafast drug metabolizers in their assays who suffer the most from DILI. Thus, there is great potential to apply engineered HLOs on the pillar/perfusion plate as a safety assessment tool. Genetically engineered human organoids can be used to express any endogenous proteins of interest in the cells for disease modeling by using a combination of guide RNAs. There is a great potential for genetically engineered human organoids to be used to incorporate genetic diversity into toxicity testing and for disease modeling.

项目概要/摘要包括药物性肝损伤（DILI）在内的意外药物不良反应（ADR）位列第四。是美国主要死亡原因。此外，个人体内 DILI 是吸毒的主要原因之一从市场上撤出，并且使用传统的体外肝毒性测试很难预测临床前动物模型。由于 DILI 与药物失败之间的关键联系，迫切需要通过调查改善药物开发早期阶段的人类肝毒性测试 DILI的主要致病机制，例如药物代谢酶（DME）的遗传变异和药物转运体。为了满足这一需求，我们建议开发基因工程人类肝脏使用基因编辑的诱导多能干细胞 (iPSC) 系在柱/灌注板上制备类器官 (HLO) 携带 CRISPR/Cas9 协同激活介质 (CRISPR-SAM)、诱导型 Csy4 和多重指导RNA（gRNA），并概括了不同种族群体中不良和超快的药物代谢者。使用柱/灌注板上的正常和工程 HLO，以及高通量、高含量、 HLO 成像分析，我们建议破译其背后的细胞和分子机制候选药物和化学品的毒性并评估 DILI 潜力。我们的核心假设是：（i）DME 和药物转运蛋白的过度表达可以重现超快不同种族群体的药物代谢者对于解决 ADR 可能至关重要； (ii) 新陈代谢- 可以使用正常的和工程化的 HLO 与模型化合物来确定诱导的肝毒性； (iii) 对柱/灌注板上的 HLO 进行高通量、高内涵分析，可用于识别 DILI，进而可以提高化合物体内肝毒性的可预测性。拟议工作的具体目标是：（1）创建包含多西环素诱导型 CRISPR-SAM 用于过度表达多个肝基因以建立超快模型代谢者； (2) 使用模型化合物验证正常和工程化的 HLO，这些化合物经过代谢并导致人类肝脏中的毒性细胞反应。尽管包括原代肝细胞（ATCC）在内的几种人类肝细胞/组织模型，工程肝癌细胞系（来自 Hera BioLabs 的 HepG2-CYP 细胞组）、肝球体 (3D InSight 来自 InSphero 的肝微组织）、生物打印肝组织（来自 Organovo 的 ExVive 人类肝组织）、和微流控肝脏芯片（来自 Emulate 的 Liver-Chip）已市售，这些体外肝脏模型已用于评估化合物对正常药物代谢者的一般肝毒性并且不能轻易地在他们的检测中模拟不良和超快的药物代谢者，他们遭受的痛苦最大帝力。因此，在柱/灌注板上应用工程 HLO 作为一种安全措施具有巨大的潜力。评估工具。基因工程人类类器官可用于表达任何内源性通过使用指导 RNA 的组合，对细胞中感兴趣的蛋白质进行疾病建模。有一个基因工程人类类器官在整合遗传多样性方面具有巨大潜力进入毒性测试和疾病建模。