The Next-Generation Developmental and Reproductive Toxicology (DART) Assay using High-Content Analysis of Genetically Diverse C. elegans Populations

使用遗传多样性线虫种群高内涵分析进行下一代发育和生殖毒理学 (DART) 测定

• 批准号: 10738193
• 负责人: Evan Hegarty
• 金额: $ 99.67万
• 依托单位: VIVOVERSE, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738193
• 关键词: Adolescent; Adult; Age; Animal Model; Animal Testing; Animal Testing Alternatives; Animal Welfare; Animals; Biological Assay; Biological Sciences; Body measure procedure; Caenorhabditis elegans; Case Study; Categories; Cell Line; Chemicals; Classification; Collection; Communities; Data; Databases; Development; Devices; Dimensions; Dose; Embryo; Embryonic Development; Ensure; Environmental Risk Factor; Ethics; Exposure to; Fishes; Genetic; Genetic Predisposition to Disease; Genetic Variation; Geography; Goals; Guidelines; Hazardous Chemicals; Health; Human; Image; Image Analysis; Immobilization; In Vitro; Inbreeding; Industry; Intestines; Invertebrates; Laboratory culture; Length; Life; Mammals; Mercury; Methods; Microfluidics; Microscopic; Modeling; Modernization; Monitor; Nematoda; Organ; Organism; Oryctolagus cuniculus; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharyngeal structure; Phase; Phenotype; Poison; Population; Procedures; Protocols documentation; Publishing; Qualifying; Rattus; Reproducibility; Reproductive system; Resolution; Risk; Risk Assessment; Safety; Soil; Source; Spearman Rank Correlation Coefficient; Statutes and Laws; Study models; Surveys; System; Technology; Testing; Time; Toxic Environmental Substances; Toxic effect; Toxicity Tests; Toxicology; Variant; Vertebrates; Visualization; Vulnerable Populations; Weight; Whole Organism; age related; analysis pipeline; automated analysis; cell motility; cost; cost effective; developmental toxicity; dosage; environmental justice; falls; high resolution imaging; high throughput screening; imaging platform; improved; in silico; in utero; in vivo; in vivo evaluation; inorganic phosphate; manufacture; microphysiology system; model organism; mutant; new chemical entity; next generation; novel; novel strategies; pressure; reproductive; reproductive toxicity; response; safety testing; social; toxicant; treatment response

1 Modern toxicology assessment of chemicals is under pressure from both scientific and social sources. Traditional 2 study models using small numbers of highly inbred mammals fail to reflect the wide genetic, geographic, and 3 demographic variation underlying differing population-specific responses to environmental toxicants and drugs. 4 Secondly, long-standing public pressure to reduce the use of animals in safety testing has resulted in regulatory 5 directives to ban the use of animal studies in approvals of new chemical entities and existing chemical product 6 re-registrations by 2035. This pressure along with continual advances in life science technology has led to the 7 development of new approach methods (NAMs) including in vitro, ethical in vivo, and in silico methods. 8 Environmental justice, especially for vulnerable fence line communities at much greater risk of environmental 9 exposure to chemicals, highlights the need to include vulnerable populations in toxicology studies. Modeling 10 population variation in toxic responses at the necessary throughputs is not feasible using outbred in vivo 11 mammalian models, and in vitro methods are unsuitable for assays requiring complete organisms such as 12 developmental and reproductive toxicity (DART). Using its novel vivoChip device, vivoVerse proposes a NAM 13 for DART testing based on the microscopic, soil-dwelling nematode, Caenorhabditis elegans. C. elegans has a 14 simple culturing protocol, ability to produce 300 progenies per adult, conserved toxicology pathways with 15 humans, intact germline with tractable in utero embryogenesis, is a non-sentient invertebrate with a 3-day life 16 cycle that is not subject to animal welfare legislation, and has well-characterized panels of several hundred 17 naturally occurring strains with diverse genetic backgrounds, making it a highly suitable small animal model for 18 DART assays. The vivoChip is a microfluidic-based imaging platform uniquely facilitating high-throughput 19 toxicology assays with C. elegans, using high-resolution imaging to quantify relevant phenotypic endpoints in 20 ~1,000 animals per chip. In Aim 1, we will develop a new vivoChip specifically for DART testing that allows rapid 21 immobilization of ~1,500 C. elegans of widely varying sizes. We will establish an AI/ML-assisted pipeline for 22 automated analysis of high-resolution, on-chip images of in utero, body, and organ phenotypes relevant to DART. 23 In Aim 2, we will develop a GLP-qualified DART assay that surveys 12 genetically diverse strains and 24 demonstrate strain and age-specific sensitivity for two reference chemicals. In Aim 3, we will compare DART 25 assessments with our panel of 12 strains and two sensitized mutants, with published data for chemicals of 26 significance to stakeholders, to demonstrate the value of our assay. With a more sensitive DART assay using 27 high-content readouts of in utero effects from twelve genetically diverse backgrounds, we expect to improve the 28 known safety prediction accuracies of C. elegans when compared to higher mammalian models. Armed with 29 such data, we will present our case study to the regulatory agencies for formal risk analysis, and in due course, 30 full acceptance of C. elegans as an alternative animal model for DART, making an impact on many industries.

1 现代化学品毒理学评估面临来自科学和社会的压力。传统的 2 使用少量高度近交哺乳动物的研究模型未能反映广泛的遗传、地理和 3 人口统计差异是不同人群对环境毒物和药物的不同反应的基础。 4 其次，长期以来公众要求减少在安全测试中使用动物的压力导致监管部门 5项指令禁止在批准新化学实体和现有化学产品时使用动物研究 到 2035 年将有 6 次重新注册。这种压力以及生命科学技术的不断进步导致了 7 开发新方法 (NAM)，包括体外方法、伦理体内方法和计算机方法。 8 环境正义，特别是对于面临更大环境风险的脆弱栅栏社区 9 接触化学品凸显了将弱势群体纳入毒理学研究的必要性。造型 10 在必要的通量下毒性反应的群体变异在体内使用远交是不可行的 11 哺乳动物模型和体外方法不适合需要完整生物体的测定，例如 12 发育和生殖毒性（DART）。利用其新颖的 vivoChip 器件，vivoVerse 提出了 NAM 13 用于基于微观土壤线虫秀丽隐杆线虫的 DART 测试。线虫有一个 14 个简单的培养方案，每个成年个体可产生 300 个后代，具有保守的毒理学途径 15 个人，完整的种系，在子宫胚胎发生过程中易于处理，是一种无知觉的无脊椎动物，寿命只有 3 天 16 个周期不受动物福利立法的约束，并且具有数百个特征明确的小组 17种天然存在的品系，具有不同的遗传背景，使其成为非常适合的小动物模型 18 次 DART 检测。 vivoChip 是一种基于微流体的成像平台，独特地促进高通量 使用高分辨率成像对线虫进行 19 项毒理学测定，以量化相关表型终点 每个芯片 20 ~ 1,000 只动物。在目标1中，我们将开发一种专门用于DART测试的新型vivoChip，可以快速 21 固定约 1,500 条大小差异很大的秀丽隐杆线虫。我们将建立一个人工智能/机器学习辅助管道 22 对与 DART 相关的子宫内、身体和器官表型的高分辨率片上图像进行自动分析。 23 在目标 2 中，我们将开发一种符合 GLP 要求的 DART 检测方法，可调查 12 种遗传多样性菌株并 图 24 展示了两种参考化学品的应变和年龄特异性敏感性。在目标 3 中，我们将比较 DART 我们对 12 个菌株和两个致敏突变体进行了 25 次评估，并公布了以下化学物质的数据 26 对利益相关者具有重要意义，以证明我们的测定的价值。使用更灵敏的 DART 检测 来自 12 种不同遗传背景的 27 个子宫内效应的高内涵读数，我们希望能够改善 与高等哺乳动物模型相比，线虫的 28 个已知安全预测精度。武装有 29这样的数据，我们将向监管机构提交我们的案例研究以进行正式的风险分析，并在适当的时候， 30 全面接受线虫作为 DART 的替代动物模型，对许多行业产生影响。