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

描述(由申请人提供)：人类不可避免地接触到危险的基因毒素，因为DNA破坏剂在我们的环境和细胞内都是无处不在的。细胞代谢、环境来源或与疾病相关的细胞缺陷产生的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，在许多情况下，还会导致癌症、疾病和衰老表型的发生。此外，许多外源性暴露，如化疗和放射治疗，依赖于诱导肿瘤细胞的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的DNA修复能力。因此，量化DNA损伤和直接在人类细胞中修复核基因组损伤的速度的能力在从流行病学到药物开发的各种应用中都是至关重要的。为了满足研究界的这一技术需求，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损伤检测和量化技术的发展。这项建议旨在开发“DNA芯片修复”技术，结合使用基于琼脂糖基的微孔阵列、空间编码的细胞识别、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来优化、验证和商业化一系列空间编码的微孔阵列，这些微孔阵列将用作工具来量化DNA损伤，并在单个阵列或芯片上暴露基因毒素(DNA芯片修复)后测量细胞DNA修复能力。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，其微孔直径为10-50 5米，深度为20-50 5米，适合于对不同大小的单个单元进行重力捕获。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，微孔阵列用于分析细胞DNA修复能力的敏感性将使用具有DNA修复基因表达明确缺陷和随后的遗传毒性应激的人类肿瘤细胞系的等基因小组进行评估。迭代分析和微井特征将有助于最终确定一套24井微井阵列，用于生产和分配。AIM 2中描述的研究涉及微孔阵列中的添加剂，这些添加剂将促进细胞生长和附着，从而提供基线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。这一技术进步为通过客观、定量的分析为药物发现、遗传毒性测试和环境健康研究打开了新战略的大门。该项目的第二阶段将扩大到提供96孔的能力、用于空间识别和量化的终端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。 与公共卫生相关：我们描述了一种新的方法，通过利用重力捕获单个细胞进入微孔阵列，提供强大的、高通量的DNA损伤和修复分析。DNA损伤程度通过单细胞凝胶电泳法进行形态观察。微孔阵列可以同时对多种实验条件进行全自动DNA损伤和DNA修复测量。这一技术进步为通过客观、定量的分析为药物发现、遗传毒性测试和环境健康研究打开了新战略的大门。

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.