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

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.