CometChip: Enabling Translation of DNA Damage and Repair Assays

描述（由申请人提供）：DNA损伤促进癌症、衰老、神经系统疾病和遗传性疾病。暴露于DNA损伤是不可避免的，因为DNA损伤剂在我们的环境和细胞中无处不在。尽管DNA损伤的重要性，但技术障碍限制了DNA损伤的常规测量。我们最近与材料科学家和工程师合作开发了“彗星芯片”，这是一种测量DNA损伤的新技术，其基础是公认的彗星试验。在这里，我们建议通过进行必要的研究来利用我们的技术进行翻译，以便将这项技术应用于人类DNA修复能力的研究。我们已经证明，彗星芯片是有效的测量DNA损伤和DNA修复人类白色血细胞在体外，并已开发的图像分析软件，使样品可以分析在一个自动化的方式，这大大提高了吞吐量，使测定更强大。我们对CometChip在广泛应用中的潜在效用感到兴奋。特别令人感兴趣的是识别出生时DNA修复水平低于平均水平的人的可能性，因为这些人希望采取特别预防措施来预防癌症，并且可能需要特别考虑药物。为了优化我们识别DNA修复缺陷患者的平台，我们首先需要使用携带已知DNA修复缺陷的细胞系。理想情况下，我们希望比较除了单个DNA修复基因表达受到抑制之外基因相同的细胞。为了实现这一点，我们敲低了DNA修复基因的表达，以创建具有特定缺陷的等基因细胞系。在这里，我们建议完成一组10个敲除细胞系的创建，其中五个主要DNA修复途径中的每一个的两个关键基因被敲除。我们建议优化CometChip的条件和参数，以揭示这些缺陷的存在。重要的是，将对该测定进行优化，以分析与群体研究相容的原代人细胞。我们的假设是，通过改变DNA损伤的质量和数量，以及分析条件，我们将能够同时识别至少四种主要DNA修复途径的缺陷，并揭示DNA修复途径之间平衡的变化。由此产生的数据将揭示我们的基因如何影响对环境暴露的易感性，以及暴露如何影响人们的DNA损伤水平。我们预计，拟议中的CometChip平台将在工业、学术、医学和公共卫生领域得到广泛应用，从而使基础研究中的工具和理解的应用能够直接造福公众。 公共卫生相关性：我们建议开发一个高通量DNA损伤分析平台，用于研究人类细胞中的DNA损伤。我们将开发的新功能将有许多应用，包括药物发现和遗传毒性测试（对制药行业很重要），检测人和细胞之间的DNA修复缺陷（对个性化医疗很重要），以及检测暴露于环境危害的人中DNA损伤水平的增加（对公共卫生很重要）。总的来说，拟议的研究将大大加快一项非常有价值的技术的转化。

DESCRIPTION (provided by applicant): DNA damage promotes cancer, aging, neurological disorders and heritable diseases. Exposure to DNA damage is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. Despite the importance of DNA damage, technological obstacles limit routine measurements of DNA damage. We have recently collaborated with materials scientists and engineers to develop the "CometChip", a new technology for measuring DNA damage that has its basis in the well-accepted Comet assay. Here, we propose to leverage our technology toward translation by performing studies that are essential in order to enable the application of this technology to studies of DNA repair capacity in people. We have demonstrated that the CometChip is effective for measuring both DNA damage and DNA repair in human white blood cells in vitro and have developed image analysis software so that samples can be analyzed in an automated fashion, which greatly increases throughput and makes the assay more robust. We are excited about the potential utility of the CometChip for a broad range of applications. Of particular interest is the possibility of identifying people who are born with lower than average levels of DNA repair, since these people would want to take special precautions to prevent cancer and may need special consideration with regard to pharmaceuticals. In order to optimize our platform for identifying people with DNA repair deficits, we first need to work with cell lines that carry known DNA repair deficiencies. Ideally, we would like to compare cells that are the same genetically with the exception of suppressed expression of a single DNA repair gene. To accomplish this, we have knocked down expression of DNA repair genes to create isogenic cell lines harbouring specific deficiencies. Here we propose to complete creation of a set of 10 knock down cell lines in which two of the key genes for each of five major DNA repair pathways are knocked down. We propose to optimize conditions and parameters of the CometChip to reveal the presence of these defects. Importantly, the assay will be optimized for analysis of primary human cells compatible with population studies. Our hypothesis is that by varying the quality and quantity of the DNA damage, as well as the conditions of analysis, we will be able to identify deficiencies in at least four major DNA repair pathways in parallel and reveal variations in the balance among DNA repair pathways. Resulting data will shed new light on how our genes affect susceptibility to environmental exposures, and in turn, how exposures can affect the levels of DNA damage in people. We anticipate that the proposed CometChip platform will have broad applications in industry, academia, medicine and public health, thus enabling the application of tools and understanding from basic research to directly benefit the public. PUBLIC HEALTH RELEVANCE: We propose to develop a high throughput DNA damage analysis platform for studies of DNA damage in human cells. The new capabilities that we will develop will have many applications, including drug discovery and genotoxicity testing (important for the pharmaceutical industry), detection of DNA repair deficiencies among people and among cells (important for personalized medicine), and detection of increased levels of DNA damage among people exposed to environmental hazards (important for public health). Taken together, the proposed studies will greatly accelerate translation of a highly valuable technology.