Development of a Genotoxicity Assay using Duplex Sequencing

A RAPIDACCURATE AND MECHANISTICALLY INFORMATIVE GENOTOXICITY ASSAY USINGDUPLEX SEQUENCING Genotoxicity testingthe evaluation of chemicals for the potential to induce DNA mutations increasing human cancer riskis a crucial part of development of drugscosmetics and other productsBut the available battery of mutagenicity assays forces frustrating trade offs between performance and convenienceAnd while existing tests can detect mutagenesismost provide no insight into mechanism of actionA more convenient accuraterapidand mechanistically informative assay is needed to speed drug and other chemical development and better detect threats to human healthThis SBIR grant application proposes to develop an end to end laboratory kit and software based genotoxicity testing product using Duplex Sequencingthe most accurate DNA sequencing technology in existenceBecause Duplex Sequencing can detect ultra low frequency mutations at levels below one in ten millionit can identify a chemical as mutagenic within weeks of exposureBecause it relies on a simple workflow that only requires extracted DNA as an inputit can be deployed far more widely than existing in vivo assaysAnd because it assesses not just mutation frequency but also the identities of the specific mutations inducedDuplex Sequencing will be the only genotoxicity test that reveals chemicalstrinucleotidemutational signatureswhich can implicate specific mutagenic mechanismsthus enabling strategic redesign of a mutagenic but otherwise promising drugWe have generated strong proof of principle resultsbut substantial work is needed to translate our experimental assay into an off the shelf product that customers can easily adoptIn this proposal we will optimize and validate a user friendly Duplex Sequencing genotoxicity assay in preparation for commercializationIn Phase I we will construct Duplex Sequencing panels for measurement of genotoxicity in ratsmice and humansand evaluate their performanceIn Phase II we will optimize the assay s protocols and reagentsbuild and deploy cloud based software allowing biologists to easily render Duplex Sequencing outputs into interpretable resultsand validate our assay in animal and human cell line models over diverse mutagensinclusive of assessing their mutational signaturesGenotoxicity testing is a vital discipline that has relied on antiquated techniques for too longThe speedaccuracy and information content of Duplex Sequencing promises a radical improvement to the status quoWe expect this convenient and easy to implement test to find rapid uptake among drug companies searching for betterearlier genotoxicity readoutsin CROs excited for a broadly applicable assay they can offer their customers and among academicslooking to push the envelope of their fields Project Narrative Although multiple assays for mutagenicity testing existnone is sufficiently fastaccurate and simple enough for widespread useThis 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 linesThis assay will also provide the field a new capabilityby measuring trinucleotide mutation signaturesit will be the first genotoxicity assay able to routinely inform on mutagenic mechanism of action