In vivo Transgenic Reporter for Hepatobiliary Toxicity

肝胆毒性的体内转基因报告基因

• 批准号: 6840566
• 负责人: Seth William Kullman
• 金额: $ 13.86万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6840566
• 关键词: bioimaging /biomedical imaging; blood chemistry; cholestasis; confocal scanning microscopy; fluorescent dye /probe; gene induction /repression; genetically modified animals; hepatotoxin; high throughput technology; histochemistry /cytochemistry; histopathology; liver imaging /visualization /scanning; liver toxic disorder; medaka; noninvasive diagnosis; protein glutamine gamma glutamyltransferase; reporter genes; technology /technique development; toxicant screening

DESCRIPTION (provided by applicant): Recent advances in combinatorial chemistry and high throughput screening practices have dramatically increased the number of new chemical entities for pharmaceutical discovery. The pharmaceutical industry is now faced with specific challenges associated with lead candidate selection and preclinical development through the Research and Development pipeline. To meet the demands of drug safety assessment, there is an increasing demand for development of novel high throughput in-vitro and in-vivo models for assessing toxicity of lead drug candidates. To date however, few validated, predictive, rapid throughput in vivo toxicity assays have been established. Herein we propose to develop a novel transgenic fluorescent reporter for prediction of hepatobiliary toxicity during preclinical development of chemotherapeutic drugs. Two specific aims are proposed: (1) Construct a cell specific transgenic see-through medaka to extend noninvasive detection of acute and chronic biliary toxicity/injury, through induction of a gamma-glutamyl transpeptidase driven fluorescent reporter gene. (2) Produce verified hepatic injury in adult see-through medaka by exposure to reference hepatotoxins known to induce canalicular membrane damage; bile duct epithelia cell damage and cholestasis with resultant expression of gamma-glutamyl transpeptidase. Once toxicity is produced, we shall verify linkage between reporter gene expression and biliary cell injury by serial, noninvasive imaging of liver in the transparent fish followed by clinical chemistry and targeted hepatic histological analysis. The see-through medaka planned for use in this study is a unique animal model, which we believe has enormous potential for drug screening and assessment of toxicity. This fish is transparent with principal internal organs visible through the body wall in in all life stages. With the see-through medaka, structural and functional changes at molecular, cellular, tissue and organ/system levels may be imaged noninvasively, leading to greatly enhanced identification and analysis of in vivo toxicity following pharmaceutical exposure. The compressed life cycle of the medaka, when coupled with its transparent features, makes the see-through medaka particularly well suited to study both acute and chronic toxicity, mutagenesis and carcinogenesis. Effort in the proposed research will focus on the development of a non-invasive reporter assay for screening hepatotoxicity during preclinical evaluation of leading chemotherapy drugs. We envision that this model can be employed as both a rapid screen for optimization of the lead molecule series selection (i.e. acute toxicity studies) as well as for more long-term chronic evaluations.

描述(由申请人提供)： 组合化学和高通量筛选实践的最新进展极大地增加了用于药物发现的新化学实体的数量。制药业现在面临着与通过研究和开发流水线选择主要候选者和临床前开发相关的具体挑战。为了满足药物安全性评价的需要，人们越来越需要开发新的高通量的体外和体内模型来评价候选先导药物的毒性。然而，到目前为止，几乎没有经过验证的、可预测的、快速的体内毒性分析被建立。在此，我们建议开发一种新的转基因荧光报告，用于在化疗药物的临床前开发过程中预测肝胆毒性。我们提出了两个特定的目标：(1)构建细胞特异性的转基因透明青竹，通过诱导γ-谷氨酰转肽酶驱动的荧光报告基因，扩大对急性和慢性胆毒性/损伤的非侵入性检测。(2)通过暴露于已知可引起小管膜损伤的参比肝毒素、胆管上皮细胞损伤和胆汁淤积，从而导致γ-谷氨酰转肽酶的表达，从而对成年透明青竹造成经证实的肝脏损伤。一旦产生毒性，我们将通过对透明鱼肝脏进行一系列非侵入性成像，然后进行临床化学和有针对性的肝脏组织学分析，来验证报告基因表达和胆管细胞损伤之间的联系。计划在这项研究中使用的透明青竹是一种独特的动物模型，我们相信它在药物筛选和毒性评估方面具有巨大的潜力。这种鱼是透明的，通过体壁可以看到所有生命阶段的主要内脏。有了透视式青竹，分子、细胞、组织和器官/系统水平的结构和功能变化可以非侵入性地成像，从而大大加强了对药物暴露后体内毒性的识别和分析。青竹压缩的生命周期，再加上其透明的特征，使透明青竹特别适合研究急性和慢性毒性、诱变和致癌。在拟议的研究中，工作重点将集中在开发一种非侵入性报告试验，用于在主要化疗药物的临床前评估期间筛选肝毒性。我们设想，该模型既可用于优化铅分子系列选择(即急性毒性研究)的快速筛选，也可用于更长期的慢性评估。