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:
- 金额:$ 28.46万
- 依托单位:
- 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:
- 关键词:3-DimensionalABCB1 geneAddressAffinityAllelesAnimal ModelArchitectureAssessment toolBiological AssayBiomimeticsCRISPR/Cas technologyCYP2B6 geneCYP2C19 geneCYP2D6 geneCYP3A4 geneCaucasiansCause of DeathCell LineCellsCessation of lifeChemicalsClassificationClustered Regularly Interspaced Short Palindromic RepeatsDiffusionDisease modelDoxycyclineDrug toxicityEngineeringEnzymesEpigenetic ProcessEthnic PopulationFailureGenesGenetic EngineeringGenetic PolymorphismGenetic VariationGoalsGuide RNAHepG2HepaticHepatocyteHepatotoxicityHospitalizationHumanHuman EngineeringHydrogelsImageIn VitroIndividualIntestinesLaboratoriesLentivirusLinkLiverMarketingMeasuresMediator of activation proteinMetabolic BiotransformationMetabolismMicrofluidicsModelingMolecularMorbidity - disease rateNatureNutrientOrganoidsOxygenPathogenicityPatientsPerfusionPharmaceutical PreparationsProteinsProtocols documentationReproducibilitySmall Business Innovation Research GrantStainsSystemTechnologyTestingTimeTissue ModelTissuesToxic effectToxicity TestsUnited States Food and Drug AdministrationVariantWorkbioprintingcost estimatedrug candidatedrug developmentdrug efficacydrug induced liver injurydrug metabolismdrug withdrawalfeasibility testingfetalhepatoma cellhigh throughput screeningimprovedin vivoindividual patientindividual variationinduced pluripotent stem cellinsightinterestmanufacturing scale-upmatrigelmortalityoverexpressionpre-clinicalresponsesafety 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.
项目总结/摘要
非预期药物不良反应(ADR)包括药物性肝损伤(DILI)是第4位
此外,个体的DILI是药物滥用的主要原因之一。
退出市场,难以使用常规体外肝毒性试验预测,
临床前动物模型。由于DILI和药物失败之间的关键联系,迫切需要
在药物开发的早期阶段,通过研究
DILI的主要致病机制,如药物代谢酶(DME)的遗传变异
和药物转运蛋白。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/灌注板上的类器官(HLO)
携带CRISPR/Cas9协同激活介体(CRISPR-SAM)、诱导型Csy 4和多重
指导RNA(gRNA),并概括不同种族群体中的药物代谢差和超快的药物代谢者。使用
柱/灌注板上的正常和工程化HLO,以及高通量,高含量,
HLO成像分析,我们建议破译细胞和分子机制的基础上,
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表达可以重演超快
不同种族群体中可能对解决ADR至关重要的药物代谢者;(ii)代谢-
使用正常和工程化HLO与模型化合物可以建立诱导的肝毒性;
和(iii)柱/灌注板上HLO的高通量、高含量分析可用于
鉴定DILI,其又可以提高体内化合物肝毒性可预测性。
拟议工作的具体目标是:(1)创建基因工程HLO,其中包含
多西环素诱导的CRISPR-SAM用于多个肝脏基因的过表达以模拟超快
(2)用模型化合物验证正常和工程化的HLO,
代谢,并导致人体肝脏中的毒性细胞反应。
尽管几种人肝细胞/组织模型包括原代肝细胞(ATCC),
工程化肝癌细胞系(来自Hera BioLabs的HepG 2-HepG 4细胞组)、肝球状体(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),
和微流控肝芯片(来自Emulate的Liver-Chip)是可商购的,这些体外肝
模型已用于评估化合物对正常药物代谢者的一般肝毒性
并且在他们的测定中不能容易地模拟受以下疾病影响最大的不良和超快药物代谢者
DILI。因此,将工程化HLO应用于柱/灌注板上作为安全的方法具有很大的潜力。
评估工具。基因工程改造的人类器官可用于表达任何内源性的类器官。
通过使用向导RNA的组合,在细胞中检测感兴趣的蛋白质用于疾病建模。有一个
基因工程人类类器官的巨大潜力可用于整合遗传多样性
用于毒性测试和疾病建模。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Pranav Joshi其他文献
Pranav Joshi的其他文献
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{{ truncateString('Pranav Joshi', 18)}}的其他基金
Pillar and perfusion well plate platforms for reproducible organoid culture from iPSC
用于从 iPSC 进行可重复类器官培养的支柱和灌注孔板平台
- 批准号:
10080406 - 财政年份:2020
- 资助金额:
$ 28.46万 - 项目类别:
Pillar and perfusion well plate platforms for reproducible organoid culture from iPSC
用于从 iPSC 进行可重复类器官培养的支柱和灌注孔板平台
- 批准号:
10210319 - 财政年份:2020
- 资助金额:
$ 28.46万 - 项目类别:
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