DNA Repair-on-a-Chip: Spatially Encoded Microwell Arrays

DNA 芯片修复：空间编码微孔阵列

• 批准号: 8250928
• 负责人: Bevin P. Engelward
• 金额: $ 31.88万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8250928
• 关键词: Address; Adhesions; Aging; Biological Assay; Caliber; Cell Death; Cell Line; Cell-Matrix Junction; Cells; Comet Assay; Communities; Computer software; Cytoskeleton; DNA Damage; DNA Repair; DNA Repair Gene; Data; Defect; Detection; Development; Disease; Environment; Environmental Health; Epidemiology; Exposure to; Extracellular Matrix Proteins; Force of Gravity; Gel; Gene Expression; Gene Mutation; Gene Rearrangement; Genome; Genotoxic Stress; Glass; Growth; Human; Joints; Life; Ligands; Malignant Neoplasms; Measurement; Measures; Mediating; Metabolism; Methodology; Mutagenicity Tests; Mutagens; Nerve Degeneration; Nuclear; Outcome; Pharmacologic Substance; Phase; Phenotype; Plastics; Positioning Attribute; Production; Proteins; Radiation; Research; Research Personnel; Sepharose; Series; Source; Surface; Technology; Therapeutic; Tumor Cell Line; base; cell growth; cell type; chemotherapy; design; drug development; drug discovery; expectation; exposed human population; genotoxicity; in vivo; inhibitor/antagonist; neoplastic cell; new technology; next generation; repaired; response; small molecule; tool

DESCRIPTION (provided by applicant): Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures such as chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generation in DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M in depth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will be expanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment. PUBLIC HEALTH RELEVANCE: We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.

描述（由申请人提供）：人类接触危险的基因毒素是不可避免的，因为 DNA 损伤剂在我们的环境和细胞内无处不在。由细胞代谢、环境来源或疾病相关细胞缺陷产生的 DNA 损伤剂和其他基因毒素会导致细胞死亡（例如神经变性）、基因突变、基因重排，并在许多情况下导致癌症、疾病和衰老表型的发生。此外，许多外源性暴露（例如化疗和放射治疗）依赖于诱导肿瘤细胞遗传毒性来介导治疗反应。此外，有效且准确地修复自发或诱发的DNA损伤的能力取决于细胞DNA修复能力。因此，直接量化人类细胞中 DNA 损伤和核基因组损伤修复率的能力对于从流行病学到药物开发等应用至关重要。为了满足研究界的这一技术需求，为了更好地表征新开发药物的基因毒性，并量化 DNA 修复能力而不需要识别特定的 DNA 修复基因缺陷，我们建议开发下一代 DNA 损伤检测和量化技术。该提案旨在开发“芯片上的 DNA 修复”技术，结合使用基于琼脂糖的微孔阵列、空间编码细胞识别、具有基因定义的 DNA 修复状态的人类肿瘤细胞系和细胞外基质蛋白，以优化、验证和商业化一系列空间编码微孔阵列，这些阵列将作为量化 DNA 损伤和测量基线细胞 DNA 修复能力的工具 以及单个阵列或芯片上的基因毒素暴露后（芯片上的 DNA 修复）。目标1中描述的研究涉及开发一系列24孔空间编码微孔阵列，微孔直径范围为10-50 5M，深度为20-50 5M，适合重力捕获各种尺寸的单细胞。微孔阵列的功效将使用辐射和小分子抑制剂进行验证。此外，将使用具有确定的 DNA 修复基因表达缺陷和遗传毒性应激后的人类肿瘤细胞系同基因组来评估微孔阵列分析细胞 DNA 修复能力的灵敏度。迭代分析和微孔表征将有助于最终确定一组用于生产和分销的 24 孔微孔阵列。目标 2 中描述的研究涉及微孔阵列中的添加剂，这些添加剂将增强细胞生长和附着，提供基线 DNA 损伤的最佳分析，最重要的是，提供有关细胞体内损伤后修复能力的关键数据。这一技术进步为通过客观、定量分析进行药物发现、基因毒性测试和环境健康研究的新策略打开了大门。该项目的第二阶段将扩展到提供用于空间识别和定量的 96 孔功能的最终用户软件以及用于专门细胞生长和附着的微孔添加剂选项。 公共健康相关性：我们描述了一种新方法，该方法通过利用微孔阵列中单细胞的重力捕获来提供稳健、高通量的 DNA 损伤和修复分析。 DNA 损伤水平通过单细胞凝胶电泳从形态学角度揭示。 Microwell 阵列可同时在多个实验条件下进行全自动 DNA 损伤和 DNA 修复测量。这一技术进步为通过客观、定量分析进行药物发现、基因毒性测试和环境健康研究的新策略打开了大门。