CometChip: Enabling Translation of DNA Damage and Repair Assays

描述(申请人提供)：DNA损伤会导致癌症、衰老、神经紊乱和遗传性疾病。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内都是无处不在的。尽管DNA损伤很重要，但技术障碍限制了对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，其基础是广为接受的彗星测试。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，以使这项技术能够应用于人类DNA修复能力的研究。我们已经证明，CometChip在体外测量人类白细胞的DNA损伤和DNA修复是有效的，并开发了图像分析软件，使样本可以以自动化的方式进行分析，这大大提高了吞吐量，使分析更加稳健。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，有可能识别出出生时DNA修复水平低于平均水平的人，因为这些人希望采取特殊预防措施来预防癌症，并可能需要在药物方面进行特殊考虑。为了优化我们识别DNA修复缺陷患者的平台，我们首先需要与携带已知DNA修复缺陷的细胞系合作。理想情况下，我们希望比较基因上相同的细胞，只是单个DNA修复基因的表达受到抑制。为了实现这一点，我们下调了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个击倒细胞系的创建，其中五个主要DNA修复途径中每一个的关键基因都被击倒。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，该分析方法将被优化，以分析与群体研究兼容的原代人类细胞。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，我们将能够并行识别至少四个主要DNA修复途径的缺陷，并揭示DNA修复途径之间平衡的变化。由此产生的数据将为我们提供新的线索，了解我们的基因如何影响对环境暴露的敏感性，进而了解暴露如何影响人的DNA损伤水平。我们预计，拟议的CometChip平台将在工业界、学术界、医学界和公共卫生领域有广泛的应用，从而使基础研究的工具和理解的应用直接惠及公众。 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，用于研究人类细胞的DNA损伤。我们将开发的新能力将有许多应用，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(对个性化药物很重要)，以及检测暴露在环境危害中的人的DNA损伤水平增加(对公共健康很重要)。综上所述，拟议中的研究将极大地加速这项极具价值的技术的翻译。

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.