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

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力是开发新的工具来研究细胞机制的动态行为，特别是在药物发现中的应用。揭示细胞生理学的动态方面，可能是靶向治疗需要一个新的范式筛选疾病生物学在活细胞。与疾病相关的信号转导通路和细胞机制的时空调节的可视化和量化相兼容的生理相关活细胞模型将是这一方法的关键组成部分。与现有的终点细胞和生化分析相比，这些模型能够提供更好的机制见解，并且与用于临床前药物发现研究和毒理学测试的动物模型相比，它们的吞吐量更高，成本更低，因此能够提高潜在治疗方法的优先级。这些属性除了是基础细胞生物学研究的宝贵能力外，还代表了新药物发现、药物再利用倡议和有效治疗组合的确定方面的重要进展。这个SBIR一期项目建议开发一种新的报告细胞系工程平台，该平台将能够快速生成适合动态活细胞高含量筛选（LC-HCS）的多色荧光细胞系。使用传统的抗生素选择方法生成稳定的细胞系是一个漫长而不灵活的过程，不适合高效地生成lc - hcs兼容细胞。目的是通过实施一种新的策略来解决这些缺点，在转染后的4-6周内，在特定位点以明确的、生理相关的和化学计量平衡的表达水平传递细胞系，这些细胞系包含可诱导控制的多种细胞标记的荧光报告。这将通过使用基因组编辑策略来实现，该策略将产生一组细胞系，这些细胞系可以快速配置以报告多达4种荧光标记的任何所需组合。通过该SBIR项目产生的多色报告细胞系将为临床相关活细胞的下一代药物发现提供有价值的新工具，并为人类疾病提供宝贵的见解。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is the development of new tools to study the dynamic behavior of cellular machinery, especially for applications in drug discovery. Unraveling the dynamic aspects of cellular physiology that may be targeted therapeutically requires a new paradigm for screening disease biology in living cells. 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 a key component of this approach. Such models will enable improved prioritization of potential therapeutics by virtue of their ability to deliver enhanced mechanistic insights compared to existing end-point cell-based and biochemical assays, and their increased throughput and reduced cost compared with animal models used in preclinical drug discovery research and toxicology testing. These attributes represent important advances for de novo drug discovery, drug repurposing initiatives and the identification of productive therapeutic combinations, in addition to being valuable capabilities for basic cell biology research.This SBIR Phase I project proposes to develop a novel reporter cell line engineering platform that will enable the rapid generation of multicolor fluorescent cell lines suitable for kinetic live-cell high-content screening (LC-HCS). Stable cell line generation using traditional antibiotic-selection methods is a lengthy and inflexible process ill-suited for the efficient generation of LC-HCS-compatible cells. The aim is to address these shortcomings by implementing a novel strategy to deliver cell lines that incorporate fluorescent reporters of multiple cellular markers under inducible control, and at well-defined, physiologically relevant, and stoichiometrically balanced expression levels at a specified locus within 4-6 weeks of transfection. This will be accomplished through the use of a genome editing strategy that will yield a panel of cell lines that can be quickly configured to report on any desired combination of up to 4 fluorescent markers. The multicolor reporter cell lines generated through this SBIR project will represent valuable new tools for the next generation of drug discovery in clinically relevant living cells and contribute invaluable insights into human disease.