Development of a Genotoxicity Assay using Duplex Sequencing

一种快速、准确、机械信息丰富的遗传毒性检测方法 双链测序 遗传毒性测试，评估化学物质诱发DNA突变的可能性增加 人类的癌症风险，是开发药品、化妆品等产品的重要组成部分。但是 可用的诱变性分析电池迫使性能和性能之间的权衡令人沮丧 方便。虽然现有的测试可以检测到突变，但大多数测试都没有提供对机制的洞察 行动的一部分。为了加快速度，需要一种更方便、准确、快速和机械信息的检测方法 药物和其他化学品的开发和更好地检测对人类健康的威胁。 这项SBIR拨款申请建议开发端到端实验室套件和基于软件的 遗传毒性检测产品采用最准确的DNA测序技术--双链测序 存在着。因为双链测序可以检测到以下水平的超低频突变 十万分之一，它可以在暴露后几周内识别出一种化学物质具有致突变性。因为它依赖于 在只需要提取的DNA作为输入的简单工作流上，它可以部署得更广泛 而不是现有的活体检测方法。因为它不仅评估突变频率，还评估身份 在诱发的特定突变中，双重测序将是唯一揭示 化学物质的三核苷酸“突变特征”，这可能意味着特定的突变机制， 从而能够对一种诱变但在其他方面很有前途的药物进行战略重新设计。 我们已经产生了强有力的原则证明结果，但还需要大量的工作来将我们的 实验化验成客户可以轻松采用的现成产品。在这份提案中，我们 将优化和验证用户友好的双重测序遗传毒性测试，以准备 商业化。在第一阶段，我们将建造双向测序板，用于测量 对大鼠、小鼠和人类的遗传毒性，并评估它们的表现。在第二阶段，我们将优化 测试的协议和试剂，构建和部署基于云的软件，使生物学家能够轻松 将双链测序输出转化为可解释的结果，并在动物和 人类细胞系对不同诱变剂的模型，包括评估它们的突变特征。 基因毒性测试是一门重要的学科，长期以来一直依赖过时的技术。速度， 双链测序的准确性和信息量有望从根本上改善现状 现状。我们希望这项方便且易于实施的测试能在制药公司中迅速普及。 为了寻找更好的、更早的遗传毒性读数，他们在为广泛适用的检测而兴奋的CRO中 可以为他们的客户和学术界提供服务，希望突破他们领域的极限。

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 assay’s 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.