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

项目摘要/摘要 糖尿病在美国和世界范围内的患病率正在急剧上升，而新的药物 能够保护或恢复分泌胰岛素的胰岛β细胞的功能是迫切需要的。为 几十年来，糖尿病药物的发现工作一直受到两件事的阻碍：1)缺乏充足的人类 用于药物筛选的β细胞，以及2)基于抗体的检测胰岛素的困难和昂贵 分泌物，β细胞功能的关键指标。诱导多能细胞来源的β细胞的出现 干细胞(IPSC)作为再生医学解决方案(RMS)的先驱，开启了一个容易 可用于高通量筛查(HTS)的Beta细胞。与此同时，在布罗德研究所，一种新的荧光素酶- 胰岛素融合蛋白已经被开发出来，它具有准确报告胰岛素的能力 永生化的啮齿动物β细胞系和原代人类胰岛的分泌。我们建议联合 这两项技术，从而产生了一个急需的筛查工具，由IPSC来源的人组成 表达荧光素酶-胰岛素分泌报告的类β细胞。RMS将设计IPSC生产线，其中 报告融合蛋白在人类胰岛素启动子的控制下表达，从而随后被 在葡萄糖和其他促分泌剂的作用下被裂解和分泌。在随后的第二阶段项目中，我们 将开发利用这些细胞进行胰岛素分泌的中高通量分析的方法。这个 最终产品，由用于化合物筛选的报告细胞板材组成，将代表一种 糖尿病药物发现的突破性平台，将是RMS当前和未来的高需求 顾客。

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.