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化学品的现代毒理学评估面临着来自科学和社会两方面的压力。传统型 使用少量高度近亲繁殖的哺乳动物的研究模型未能反映广泛的遗传、地理和 3不同人群对环境毒物和药物反应的人口统计学差异。 其次，长期存在的公众压力要求减少使用动物进行安全测试，这导致了监管 5禁止在批准新的化学实体和现有化学产品时使用动物研究的指示 到2035年，6个重新注册。这种压力加上生命科学技术的不断进步，导致了 7发展新的方法(NAMS)，包括体外方法、体内伦理方法和硅胶方法。 8环境正义，特别是对于环境风险更大的脆弱围栏社区 9暴露于化学品，突出了将易受伤害人群纳入毒理学研究的必要性。建模 10在必要的吞吐能力下的毒性反应的群体变异是不可能使用在体外交的 11哺乳动物模型，体外方法不适合于需要完整生物的分析，如 12发育和生殖毒性(DART)。VivoVerse利用其新的vivoChip器件，提出了一种NAM 13用于基于微小的土壤线虫--秀丽线虫的DART测试。线虫有一种 14个简单的培养方案，每个成虫能产生300个后代，保守的毒理学途径 15个人类，完整的生殖系，在子宫胚胎发育中容易驯服，是一种无意识的无脊椎动物，只有3天的生命 16个周期，不受动物福利立法的约束，并有数百个具有良好特征的小组 17个具有不同遗传背景的自然产生的菌株，使其成为非常适合用于 18种DART试验。VivoChip是一种基于微流控技术的成像平台，其独特之处在于可实现高通量 19种线虫的毒理学检测，使用高分辨率成像来量化相关的表型终点 每片20~1000只动物。在目标1中，我们将开发一种新的活体芯片，专门用于DART测试，允许快速 21固定约1,500个大小不一的线虫。我们将建立一个AI/ML辅助的管道，用于 22自动分析与DART相关的高分辨率、芯片上的子宫、身体和器官表型图像。 23在目标2中，我们将开发一种符合GLP条件的DART方法，该方法调查了12个不同的基因菌株和 24显示对两种参考化学品的菌株和特定年龄的敏感性。在目标3中，我们将比较DART 25与我们的12个菌株和两个致敏突变体组成的小组进行的评估，以及公布的关于化学物质的数据 26对利益相关者的意义，以证明我们的分析的价值。使用更灵敏的DART检测 来自12个不同遗传背景的27个高含量的宫内效应读数，我们希望改善 与高等哺乳动物模型相比，线虫的已知安全预测准确率为28。全副武装 29这类数据，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，并在适当时候， 30完全接受线虫作为DART的替代动物模型，对许多行业产生影响。