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

项目摘要 新药候选物和环境化学品的安全性评估的重大转变， 彻底改变毒理学监管实践的工作正在进行中。这种转变包括减少，或在某些情况下， 通过实施更高通量的测试方案，消除传统的动物毒性测试 使用人类细胞系统。关于新化学品、食品添加剂和 药物破坏人类基因组和导致突变仍然对公共卫生至关重要。突变 是细胞基因组中的可遗传变化，是诱发癌症、出生缺陷和 神经系统疾病在人类相关检测中筛选可能导致突变的化学物质， 改善公共卫生的有效战略。在人体细胞中缺乏致突变性生物测定是一个主要的问题， 监管机构用于危害识别的遗传毒理学测试组合存在数据缺口， 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，允许对这些非常罕见的事件进行致突变性分析评估和精确定量（1 100，000 - 1，000，000）。这些方法可以与人细胞整合作为遗传毒理学测定， 可以替代50年。旧的克隆选择技术来评估突变虽然双链测序是高度 创新和“游戏规则改变者”，没有整合到一个定义明确的基于人类细胞的系统中， 验证研究，其在监管遗传毒理学中的应用将受到限制。本SBIR阶段的重点 I应用是开发结合人类细胞和双链体测序的新方法学（NAM） 作为体内突变测定的体外替代方法。我们将通过进行“原则证明”来实现这一点。 使用成熟的人类细胞系进行实验，如两个具体目标所述。具体目标1： 使用原型双链测序确定多个基因座诱导突变的时间过程 致突变化合物在具体目标2中，我们将确定在以下条件下诱导突变的剂量反应： 使用双链体测序对两种原型诱变化合物进行多重基因座。完成第一阶段SBIR 将导致开发一种基于人类细胞的突变检测方法，可用作细菌 突变测定和作为NAM以减少对啮齿动物中当前体内基因突变测定的依赖。该数据 还将支持第二阶段应用，以验证该检测方法，并调整这些方法以量化 人CD 34+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.