Mutational profiling in human cells as an in vitro alternative to in vivo mutagenicity assessments

项目摘要 对新药候选药物和环境化学品的安全性评估发生重大转变，这些化学品可以 正在对监管毒理学的做法进行革命性的改革。这一转变包括减少，或者在某些情况下 通过实施更高吞吐量的测试计划，消除传统的动物毒性测试 使用人类细胞系统。了解新化学品、食品添加剂的潜力，以及 破坏人类基因组并导致突变的药物仍然对公共健康至关重要。突变 是细胞基因组中可遗传的变化，是诱发癌症、出生缺陷和 神经系统疾病。筛选化学物质在与人类相关的检测中引起突变的可能性 改善公共卫生的有效战略。在人类细胞中缺乏诱变性生物测试是一个主要的 监管机构用于危险识别的遗传毒理学测试电池中的数据缺口 定量风险评估。我们的新方法，最初是为了识别基因亚克隆 癌症，使得对突变的分析评估和对这些非常罕见的事件的精确量化(1 在100,000-1,000,000)。这些方法可以与人类细胞集成，作为一种遗传毒理学测试 可以更换50年。评估突变的古老克隆选择技术。尽管双重测序在很大程度上 创新，是游戏规则的改变者，没有集成到定义明确的基于人体细胞的系统中，而且小心翼翼 验证研究，其在调节性遗传毒理学中的应用将受到限制。本SBIR阶段的重点 我的应用是开发一种新的方法(NAM)，将人类细胞和双向测序相结合 作为体内突变检测的体外替代物。我们将通过进行“原则证明”来实现这一点。 使用已建立的人类细胞系的实验，如两个具体目标所述。在具体目标1中，我们将 用双链测序法确定多个基因座突变诱导的时间进程 致突变化合物。在特定的目标2中，我们将确定诱发突变的剂量反应 对两种典型诱变化合物进行双链测序的多基因座研究。完成第一阶段SBIR 将导致开发一种基于人类细胞的突变试验，可用于细菌的后续研究 突变分析和作为一种NAM，以减少对当前啮齿动物体内基因突变分析的依赖。此数据 还将支持第二阶段应用程序，以验证此测试并调整这些方法以量化 人CD34+细胞和HepaRg™细胞。

Project Summary A significant shift in the safety assessment of new drug candidates and environmental chemicals that can revolutionize the practice of regulatory toxicology is ongoing. This shift includes a reduction, or in some cases elimination, of traditional toxicity testing in animals with the implementation of higher-throughput testing schemes using human cell systems. Knowledge regarding the potential of new chemicals, food additives, and pharmaceuticals to damage the human genome and cause mutations remains critical to public health. Mutations are heritable changes in the cellular genome and are key events in the induction of cancer, birth defects, and neurological diseases. Screening chemicals for their potential to cause mutation in a human relevant assay offers an effective strategy for improving public health. The lack of a mutagenicity bioassay in human cells is a major data gap in the genetic toxicology test battery used by regulatory agencies for hazard identification and quantitative risk assessments. Our novel methods, initially developed to identify genetic subclones within cancers, permits analytical assessment of mutagenicity and precise quantification of these very rare events (1 in 100,000 - 1,000,000). These methods can be integrated with human cells as a genetic toxicology assay that can replace 50 yr. old clonal selection techniques to assess mutation. Although duplex sequencing is highly innovative and a “game changer”, without integration into a well-defined human cell-based system and careful validation studies, its application in regulatory Genetic Toxicology will be limited. The focus of this SBIR Phase I application is to develop a New Approach Methodologies (NAM) combining human cells and duplex sequencing as an in vitro alternative to in vivo mutation assays. We will accomplish this by conducting “proof of principle” experiments using a well-established human cell line, as outlined in two specific aims. In Specific Aim 1, we will determine the time course for the induction of mutations at multiple loci using duplex sequencing for a prototypic mutagenic compound. In Specific Aim 2, we will determine the dose response for the induction of mutations at multiple loci using duplex sequencing for two prototypic mutagenic compounds. Completion of this Phase I SBIR will lead to development of a human cell-based mutation assay that can be used as a follow up to bacterial mutation assays and as a NAM to reduce reliance on current in vivo gene mutation assays in rodents. This data will also support a Phase II application to validate this assay and adapt these methods to quantify mutation in human CD34+ cells and HepaRG™ cells.