Development of a rapid and inexpensive luciferase-based high throughput screening assay to identify compounds that alter pancreatic β cell function

开发快速且廉价的基于荧光素酶的高通量筛选测定法，以识别改变胰腺β细胞功能的化合物

• 批准号: 9254620
• 负责人: Erik J Forsberg
• 金额: $ 22.5万
• 依托单位: REGENERATIVE MEDICAL SOLUTIONS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-16 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9254620
• 关键词: Antibodies; Beta Cell; Biological Assay; Biotechnology; Cell Line; Cell physiology; Cells; Chimeric Proteins; Cleaved cell; Codon Nucleotides; DNA; DNA cassette; Detection; Development; Diabetes Mellitus; Drug Industry; Drug toxicity; Engineering; Enzyme-Linked Immunosorbent Assay; Female; Future; Gene Expression; Genes; Genome; Glucose; Hormones; Human; Institutes; Insulin; Karyotype; Luciferases; Measurement; Measures; Medical; Methodology; Methods; Mus; Neomycin; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Preclinical Drug Evaluation; Prevalence; Proinsulin; Proteins; Protocols documentation; Reporter; Reporter Genes; Reporting; Research Personnel; Resistance; Rodent; Sequence Analysis; Site; Source; Southern Blotting; Structure of beta Cell of islet; Technology; Testing; Toxicity Tests; Transfection; Transgenes; Transgenic Organisms; United States; abstracting; base; cell behavior; cellular engineering; diabetic patient; drug discovery; functional restoration; gene function; high throughput analysis; high throughput screening; induced pluripotent stem cell; insulin secretion; islet; male; meetings; novel; novel therapeutics; pluripotency; promoter; regenerative; response; screening; stem cell differentiation; tool

Project Summary/Abstract The prevalence of diabetes in the United States and world-wide is increasing dramatically, and new drugs that can protect or restore the function of insulin-producing pancreatic beta cells are urgently needed. For decades, diabetes drug discovery efforts have been hampered by two things: 1) the lack of abundant human beta cells for drug screening, and 2) the difficultly and expense of antibody-based assays to measure insulin secretion, the key indicator of beta cell function. The advent of beta cells derived from induced pluripotent stem cells (iPSC), as pioneered by Regenerative Medical Solutions (RMS), has opened an era of readily available beta cells for high-throughput screening (HTS). In parallel, at the Broad Institute, a novel luciferase- insulin fusion protein has been developed which has the demonstrated ability to accurately report insulin secretion from both immortalized rodent beta cell lines and primary human islets. We propose to combine these two technologies and thereby generate a much-needed screening tool consisting of iPSC-derived human beta-like cells that express the luciferase-insulin secretion reporter. RMS will engineer iPSC lines in which the reporter fusion protein is expressed under control of the human insulin promoter, such that it is subsequently cleaved and secreted in response to glucose and other secretagogues. In the subsequent Phase II project, we will develop methods to utilize these cells for medium- and high-throughput analysis of insulin secretion. The ultimate product, consisting of plates of reporter cells arrayed for compound screening, will represent a breakthrough platform for diabetes drug discovery and will be in high demand among RMS’s current and future customers.

项目概要/摘要 在美国和世界范围内，糖尿病的患病率正在急剧增加，新药 迫切需要能够保护或恢复产生胰岛素的胰腺β细胞的功能。为了 几十年来，糖尿病药物的发现工作受到两件事的阻碍：1）缺乏充足的人类资源 用于药物筛选的 β 细胞，以及 2) 基于抗体的胰岛素检测方法的难度和费用 分泌，β细胞功能的关键指标。诱导多能性β细胞的出现 再生医学解决方案 (RMS) 开创的干细胞 (iPSC) 开启了一个易于使用的时代 可用于高通量筛选 (HTS) 的 β 细胞。与此同时，在布罗德研究所，一种新型荧光素酶- 胰岛素融合蛋白已被开发出来，具有准确报告胰岛素的能力 永生化啮齿动物β细胞系和原代人类胰岛的分泌。我们建议结合 这两项技术，从而产生了急需的筛选工具，由 iPSC 衍生的人类 表达荧光素酶-胰岛素分泌报告基因的β样细胞。 RMS 将设计 iPSC 生产线，其中 报告融合蛋白在人胰岛素启动子的控制下表达，因此随后 响应葡萄糖和其他促分泌剂而裂解和分泌。在后续的二期工程中，我们 将开发利用这些细胞进行胰岛素分泌的中通量和高通量分析的方法。这 最终产品由排列用于化合物筛选的报告细胞板组成，将代表 糖尿病药物发现的突破性平台，RMS 当前和未来的需求量很大 顾客。