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.

项目总结/文摘