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

人体细胞突变分析作为体内致突变性评估的体外替代方案

• 批准号: 10155923
• 负责人: James Todd Auman
• 金额: $ 24.99万
• 依托单位: INTEGRATED LABORATORY SYSTEMS, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-17 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10155923
• 关键词: Amendment; Ames Assay; Animal Testing; Animals; Biological Assay; Biological Sciences; CD34 gene; Cell Culture Techniques; Cell Line; Cells; Chemicals; Chromosome abnormality; Client; Comet Assay; Congenital Abnormality; Cosmetics; DNA Damage; DNA Sequence Alteration; Data; Development; Dose; European Union; Event; Family suidae; Food Additives; Gene Mutation; Genes; Genetic; Genome; Hazard Identification; Heritability; Human; Human Cell Line; Human Genome; In Vitro; Knowledge; Malignant Neoplasms; Mammalian Cell; Measures; Methodology; Methods; Mutagenicity Tests; Mutation; Nature; Pharmacologic Substance; Phase; Poison; Public Health; Reaction Time; Risk Assessment; Rodent; Safety; Sales; Scheme; Small Business Innovation Research Grant; System; Techniques; Technology; Testing; Time; Toxicity Tests; Toxicogenetics; Toxicology; Transgenic Organisms; Ursidae Family; Validation; base; carcinogenesis; cost; daughter cell; drug candidate; environmental chemical; experimental study; follow-up; genotoxicity; human disease; improved; in vitro testing; in vivo; innovation; mutation assay; nervous system disorder; next generation sequencing; novel; novel strategies; novel therapeutics; programs; response; safety assessment; screening; stem cells; transmission process; validation studies

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.

项目概要 新药候选物和环境化学品安全评估的重大转变 监管毒理学实践的彻底变革正在进行中，这种转变包括减少，或者在某些情况下。 通过实施更高通量的测试方案，消除传统的动物毒性测试 使用人体细胞系统的知识有关新化学品、食品添加剂和 破坏人类基因组并导致突变的药物对公众健康仍然至关重要。 是细胞基因组中的可遗传变化，是诱发癌症、出生缺陷和癌症的关键事件 筛选化学物质是否有可能在人类相关检测中引起突变。 改善公众健康的有效策略 缺乏人体细胞致突变性生物测定是一个主要问题。 监管机构用于危险识别和评估的遗传毒理学测试组中的数据缺口 我们的新方法最初是为了识别内部的遗传亚克隆而开发的。 癌症，允许对这些非常罕见的事件的致突变性进行分析评估和精确量化（1 100,000 - 1,000,000）这些方法可以与人体细胞结合作为遗传毒理学测定 可以取代 50 年前的克隆选择技术来评估突变。 创新和“游戏规则改变者”，无需集成到明确定义的基于人类细胞的系统中，并且仔细 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。 我的申请是开发一种结合人体细胞和双工测序的新方法（NAM） 作为体内突变测定的体外替代方法，我们将通过进行“原理证明”来实现这一目标。 正如具体目标 1 中所述，我们将使用成熟的人类细胞系进行实验。 使用原型双链测序确定在多个位点诱导突变的时间过程 在具体目标 2 中，我们将确定诱导突变的剂量反应。 使用双链测序对两种原型诱变化合物进行多位点测序，完成 I 期 SBIR。 将导致基于人类细胞的突变测定的开发，该测定可用作细菌的后续研究 突变测定并作为 NAM 来减少对当前啮齿类动物体内基因突变测定的依赖。 还将支持 II 期应用来验证该测定并调整这些方法来量化突变 人类 CD34+ 细胞和 HepaRG™ 细胞。