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

描述（由申请人提供）：人类暴露于危险的遗传毒素是不可避免的，因为DNA损伤剂在我们的环境和细胞中无处不在。由细胞代谢、环境来源或疾病相关的细胞缺陷引起的DNA损伤剂和其他遗传毒素导致细胞死亡（例如，神经变性）、基因突变、基因重排以及在许多情况下癌症、疾病和衰老表型的发作。此外，许多外源性暴露如化疗和放疗依赖于诱导肿瘤细胞遗传毒性来介导治疗反应。此外，有效和准确地修复自发或诱导的DNA损伤的能力取决于细胞DNA修复能力。因此，直接在人类细胞中量化DNA损伤和核基因组损伤修复速率的能力在从流行病学到药物开发的应用中至关重要。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遗传毒性，并在不需要识别特定DNA修复基因缺陷的情况下量化DNA修复能力，我们提出了下一代DNA损伤检测和定量技术的发展。开发“芯片上的DNA修复”技术的该提案结合使用基于琼脂糖的微孔阵列、空间编码的细胞识别、具有遗传定义的DNA修复状态的人类肿瘤细胞系和细胞外基质蛋白来优化，验证并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在单个阵列或芯片上的遗传毒素暴露之后（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，微孔直径为10-50 μ m，深度为20-50 μ m，适用于重力捕获各种大小的单个细胞。微孔阵列的有效性将使用辐射和小分子抑制剂进行验证。此外，将使用DNA修复基因表达存在明确缺陷的人肿瘤细胞系的同基因组和遗传毒性应激后，评价微孔阵列分析细胞DNA修复能力的灵敏度。迭代分析和微孔表征将为最终确定一套用于生产和分销的24孔微孔阵列提供信息。目标2中描述的研究涉及微孔阵列的添加剂，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，提供关于细胞损伤后体内修复能力的关键数据。这一技术进步通过客观的定量分析为药物发现、遗传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大到提供96孔能力，用于空间识别和定量的最终用户软件，以及用于专门细胞生长和附着的微孔添加剂选项。 公共卫生相关性：我们描述了一种新的方法，提供了强大的，高通量的DNA损伤和修复分析，利用重力捕获的单细胞到微孔阵列。DNA损伤水平通过单细胞凝胶电泳从形态学上揭示。Microwell阵列能够同时在多个实验条件下进行全自动DNA损伤和DNA修复测量。这一技术进步通过客观的定量分析为药物发现、遗传毒性测试和环境健康研究的新策略打开了大门。

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.