Gene-edited liver organoids for predictive hepatotoxicity

用于预测肝毒性的基因编辑肝脏类器官

• 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
• 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

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.

项目摘要/摘要 包括药物性肝损伤(DILI)在内的意外药物不良反应(ADR)排在第4位 在美国居死因之首。此外，帝力在个人中也是吸毒的主要原因之一 退出市场，很难用常规的体外肝毒性试验和 临床前动物模型。由于帝力和药物失败之间的关键联系，迫切需要 为了改进药物开发早期阶段的人体肝毒性测试，通过调查 DILI的主要致病机制，如药物代谢酶(DMES)的遗传变异 和毒品运输商。为了满足这一需求，我们建议开发基因工程人肝。 利用基因编辑的诱导多能干细胞(IPSC)系在柱状/灌注板上植入有机化合物(HLOS) 携带CRISPR/Cas9协同激活介质(CRISPR-SAM)、可诱导的Csy4和多路传输 引导RNA(GRNA)，并概括不同民族的不良和超快药物代谢物。vbl.使用 柱/灌注板上的普通和工程HLOS，连同高通量、高含量、 HLO成像分析，我们建议破译背后的细胞和分子机制 候选药物和化学品的毒性，并评估帝力潜力。 我们的核心假设是：(I)Dme和药物转运蛋白的过度表达可以概括为超快 不同种族人群中可能对解决不良反应至关重要的药物代谢物；(2)新陈代谢- 诱导肝毒性可用正常和工程HLOS与模型化合物建立； 和(Iii)柱/灌注板上的高通量、高含量的HLOS分析可用于 确定DILI，这反过来可以提高体内复合肝毒性的可预测性。 拟议工作的具体目标是：(1)创造包含以下内容的基因工程HLOS 多西环素诱导的CRISPR-SAM用于多个肝脏基因的过度表达以建立超快模型 代谢剂；(2)用模型化合物验证正常和工程HLOS，它们经历 并导致人体肝脏的毒性细胞反应。 虽然几种人类肝细胞/组织模型包括原代肝细胞(ATCC)， 工程肝癌细胞系(来自Hera BioLabs的HepG2-CYP细胞组)，肝脏球体(3D Insight 来自InSphero的肝微组织)、生物打印的肝组织(来自Organovo的Exvive人肝组织)、 而微流控肝脏芯片(来自EMPATE的肝脏芯片)可以在商业上买到，这些体外肝脏 已有模型用于评估化合物对正常药物代谢物的一般肝毒性。 在他们的化验中不能很容易地模拟糟糕的和超快的药物代谢物，这些人受到的影响最大 帝力。因此，在柱/灌注板上应用工程HLOS作为一种安全措施具有很大的潜力 评估工具。经过基因工程的人类有机类物质可以用来表达任何内源性的 细胞中感兴趣的蛋白质，用于疾病建模，使用组合的引导RNA。有一个 基因工程人类有机化合物用于整合遗传多样性的巨大潜力 用于毒性测试和疾病建模。