SBIR Phase II: A novel multicolor cell line engineering platform that enables high-throughput microscopy-based screening of living cells for drug discovery

小型企业创新研究(SBIR)第二阶段项目的更广泛影响/商业潜力是开发新工具，以了解在疾病中中断的细胞机械的动态行为。解开细胞生理学的动态方面，可能是治疗上的靶向，需要新的技术来描述整个信号通路对针对单个通路节点的药物干预的反应。生理上相关的活细胞模型的可用性将是实现这一方法的关键，该模型与可视化和量化与疾病相关的信号转导途径和细胞机制的时空调控相兼容。以这种方式监测关键癌症信号通路的多个方面的能力，代表着一个宝贵的机会，可以识别有效的和选择性的“不可用药”靶标的治疗抑制剂，例如RAS蛋白，它是30%以上癌症的关键驱动因素。通过开发强大且可扩展的高通量活细胞分析平台，这项技术可能会减少确定药物化学物质的作用机制和非靶点效应的时间和成本，从而提供快速、经济高效地识别安全有效的治疗方法的新能力。这项SBIR第二阶段项目将开发一个强大而灵活的平台，用于快速生成精密工程的多色荧光细胞系和相关的高通量基于显微镜的分析。这一平台与开发此类细胞系和检测的行业标准方法形成了鲜明对比，这些方法冗长且缺乏灵活性。该项目包括四个部分的优化和执行：1)生成一组与快速、可靠的稳定报告基因整合相兼容的细胞系；2)提供一个包含大约25个RAS/MAPK途径的多色报告基因的文库；3)快速生成和验证一个包含大约100个有效的稳定报告细胞株的文库，该文库表达所有与治疗相关的RAS/MAPK途径的突变和异构体；以及4)384孔板试验开发和使用工具化合物筛选这些RAS/MAPK报告细胞。该项目旨在展示该平台通过监测与活细胞中关键的高价值肿瘤学靶点相关的以前无法获得的生物学的多个方面，快速准确定位复合作用机制和潜在的非靶点效应的能力。在该项目过程中建立的标准化平台将允许快速扩展到更多的临床相关信号通路。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase II project is the development of new tools to understand the dynamic behavior of cellular machinery that is disrupted in disease. Unraveling the dynamic aspects of cellular physiology that may be targeted therapeutically requires new technologies capable of profiling the response of entire signaling pathways to pharmacological intervention targeted at single pathway nodes. The availability of physiologically relevant live-cell models that are compatible with visualizing and quantifying the spatiotemporal regulation of disease-relevant signal transduction pathways and cellular machinery will be key to enabling this approach. The ability to monitor multiple facets of key cancer signaling pathways in this way represents a valuable opportunity to identify potent and selective therapeutic inhibitors of "undruggable" targets, such as the Ras protein, which is a crucial driver of more than 30% of cancers. By enabling development of a robust and scalable high-throughput live-cell assay platform, this technology may reduce the time and cost to pinpoint the mechanism of action and off-target effects of pharmaceutical chemicals, thus delivering new capabilities to rapidly and cost-effectively identify safe and effective therapeutics.This SBIR Phase II project will develop a robust and flexible platform for rapid generation of precision-engineered, multicolor fluorescent cell lines and associated high-throughput microscopy-based assays. This platform contrasts with industry standard methods for developing such cell lines and assays, which are lengthy and inflexible. The project comprises optimization and execution of four components: 1) Generation of a panel of cell lines compatible with rapid, reliable stable reporter integration; 2) Delivery of a library of approximately 25 multicolor reporters of the Ras/MAPK pathway; 3) Rapid generation and validation of a library of approximately 100 validated stable reporter cell lines expressing all therapeutically relevant mutations and isoforms of the Ras/MAPK pathway; and 4) 384-well plate assay development and screening of these Ras/MAPK reporter cells using tool compounds. The project aims to demonstrate the capability of the platform to rapidly pinpoint compound mechanism of action and potential off-target effects by monitoring multiple facets of previously inaccessible biology associated with a critical, high-value oncology target in live cells. The standardized platform established in the course of this project will allow rapid expansion to additional clinically relevant signaling pathways.