SBIR-STTR Award

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.

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.