A RAPID, ACCURATE AND MECHANISTICALLY INFORMATIVE GENOTOXICITY ASSAY USING DUPLEX SEQUENCING Genotoxicity testing, the evaluation of chemicals for the potential to induce DNA mutations increasing human cancer risk, is a crucial part of development of drugs, cosmetics and other products. But the available battery of mutagenicity assays forces frustrating trade-offs between performance and convenience. And while existing tests can detect mutagenesis, most provide no insight into mechanism of action. A more convenient accurate, rapid, and mechanistically-informative assay is needed to speed drug and other chemical development and better detect threats to human health. This SBIR grant application proposes to develop an end-to-end laboratory kit and software-based genotoxicity testing product using Duplex Sequencing, the most accurate DNA sequencing technology in existence. Because Duplex Sequencing can detect ultra-low frequency mutations at levels below one-in-ten-million, it can identify a chemical as mutagenic within weeks of exposure. Because it relies on a simple workflow that only requires extracted DNA as an input, it can be deployed far more widely than existing in vivo assays. And because it assesses not just mutation frequency but also the identities of the specific mutations induced, Duplex Sequencing will be the only genotoxicity test that reveals chemicals trinucleotide mutational signatures, which can implicate specific mutagenic mechanisms, thus enabling strategic redesign of a mutagenic but otherwise promising drug. We have generated strong proof-of-principle results, but substantial work is needed to translate our experimental assay into an off-the-shelf product that customers can easily adopt. In this proposal we will optimize and validate a user-friendly Duplex Sequencing genotoxicity assay in preparation for commercialization. In Phase I we will construct Duplex Sequencing panels for measurement of genotoxicity in rats, mice and humans, and evaluate their performance. In Phase II we will optimize the assays protocols and reagents, build and deploy cloud-based software allowing biologists to easily render Duplex Sequencing outputs into interpretable results, and validate our assay in animal and human cell line models over diverse mutagens, inclusive of assessing their mutational signatures. Genotoxicity testing is a vital discipline that has relied on antiquated techniques for too long. The speed, accuracy and information content of Duplex Sequencing promises a radical improvement to the status quo. We expect this convenient and easy-to-implement test to find rapid uptake among drug companies searching for better, earlier genotoxicity readouts, in CROs excited for a broadly applicable assay they can offer their customers and among academics, looking to push the envelope of their fields.
Public Health Relevance Statement: Project Narrative Although multiple assays for mutagenicity testing exist, none is sufficiently fast, accurate and simple enough for widespread use. This project proposes to use Duplex Sequencing to develop a convenient and robustly performing kit and software genotoxicity testing product that can directly measure the frequency and identity of mutations arising in mutagen-treated model organisms and cell lines. This assay will also provide the field a new capability: by measuring trinucleotide mutation signatures, it will be the first genotoxicity assay able to routinely inform on mutagenic mechanism of action.
Project Terms: Adopted; Aftercare; Animal Model; Animals; Applications Grants; base; Bioinformatics; Biological Assay; cancer risk; Carcinogens; Cell Line; Cells; Chemicals; cloud based; commercialization; Complex; Computer software; Cosmetics; Data; Databases; design; Development; Discipline; DNA; DNA Damage; DNA Sequence Alteration; DNA sequencing; drug development; drug standard; drug testing; Ensure; Evaluation; experimental study; Frequencies; Funding; genotoxicity; Health; Human; Human Biology; Human Cell Line; Human Genome; improved; in vivo; Individual; Induced Mutation; insight; Intention; Laboratories; Letters; Libraries; Link; Measurement; Measures; Mediating; Modeling; mouse genome; Mus; Mutagenesis; Mutagenicity Tests; Mutagens; Mutation; novel therapeutics; Organism; Output; Performance; Pharmaceutical Preparations; Phase; Preparation; Protocols documentation; rat genome; Rattus; Reagent; Rodent; Running; Sales; Sampling; Small Business Innovation Research Grant; Speed; Techniques; Technology; Test Result; Testing; Tissues; Toxicology; Transgenic Organisms; Translating; tumor growth; uptake; user-friendly; Validation; validation studies; Work
