DNA repair deficient cells for analysis

描述（由申请人提供）：I期的成功完成导致了一组人类细胞系的开发，每个细胞系都缺乏11种DNA糖基酶中的一种。靶mRNA损耗高达95%，靶蛋白水平和酶活性相应降低。为了扩大背景多样性，同样的shRNA慢病毒也被用于在不同的肿瘤背景（包括胶质瘤和乳腺癌细胞系）中建立平行细胞系板，在不同的肿瘤细胞背景中显示相似的靶mRNA消耗。DNA糖基酶的基因表达敲低例证了DNA修复缺陷对人类转录组的影响。作为一组DNA修复缺陷细胞系的深远潜力的一个例子，我们表明DNA糖基化酶缺陷调节转录组和表观基因组，暗示一些DNA糖基化酶参与甲基化维持和基因组表达多样性。此外，通过结合DNA糖基化酶和BRCA1敲低，我们已经开始研究PARP抑制剂在BRCA1敲低肿瘤系中的有效性对DNA糖基化酶的要求。该项目的第二阶段将利用第一阶段优化的成功工作流程范例，对缺乏所有已知DNA修复基因的等基因人类细胞系进行开发、功能表征、细胞库和转录组分析。这些基因包括参与碱基切除修复、损伤直接逆转、错配切除修复、核苷酸切除修复、同源重组、非同源末端连接、核苷酸池调节、DNA聚合酶、编辑和加工核酸酶、Rad6途径、染色质结构、疾病中有缺陷的DNA修复基因和保守的DNA损伤反应基因。Aim 1中描述的研究包括制备表达慢病毒的shRNA，转导和生成三种不同的人类肿瘤细胞敲低板，用于所有已知的DNA修复基因（>150），然后对敲低细胞系进行mRNA表达表征（qRT-PCR），并优化放大和逐步表征，为细胞系分布做准备（cell Banking）。在目标2中，细胞系将通过蛋白质表达谱和基因毒素挑战来验证预期的DNA修复功能缺陷。最后（目标3），将进行全基因组转录谱，以定量内源性DNA修复能力变化介导的转录重编程，并在适当的情况下，在特定的基因毒性应激下进行。由于DNA修复能力会自发地影响基本细胞功能，并对基因毒性应激作出反应，改变转录和表观遗传景观，并指示细胞对应激的反应，因此，开发跨多种背景的完整的等基因DNA修复缺陷细胞系将为基因和药物发现提供有价值的平台。抑制剂特异性的验证和基因/药物合成致死性组合的反应生物标志物和新靶点的鉴定。这组细胞系的现成可用性将允许学术界和制药科学家研究肿瘤基因组不稳定性的分子病因学，并将其用于肿瘤学研究。我们预计对细胞系和与全球转录组相关的信息的强劲市场需求。

DESCRIPTION (provided by applicant): Successful completion of Phase I led to the development a panel of human cell lines, each deficient in one of the eleven DNA glycosylase enzymes. Depletion of target mRNA was as high as 95%, with corresponding depletion of target protein levels and enzymatic activity. To expand background diversity, the same shRNA lentiviruses were also used to develop parallel cell line panels in diferent tumor backgrounds, including glioma and breast cancer cell lines, demonstrating similar target mRNA depletion across different tumor cell backgrounds. Gene expression knockdown of the DNA glycosylases exemplify the impact of DNA repair defects on the human transcriptome. As an example of the far reaching potential for a panel of DNA repair deficient cell lines, we show that DNA glycosylase deficiency modulated both the transcriptome and epigenome, implicating some DNA glycoylases in methylation maintenance and genome expression diversity. Further, by combining both DNA glycosylase and BRCA1 knockdown, we have begun to investigate the requirement for DNA glycosylases in the effectiveness of PARP inhibitors in a BRCA1 knockdown tumor line. Phase II of the project wil utilize the successful work-flow paradigm optimized in Phase I for the development, functional characterization, cell banking and transcriptome analysis of isogenic human cel lines deficient in all known DNA repair genes. These include genes involved in Base Excision Repair, Direct Reversal of Damage, Mismatch Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Non- homologous End-Joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA Repair genes defective in diseases and conserved DNA Damage Response genes. The studies described in Aim 1 involve the preparation of the shRNA expressing lentiviruses, transduction and generation of three different human tumor cell knockdown panels for all known DNA repair genes (>150), followed by the mRNA expression characterization (qRT-PCR) of the knockdown cell lines and optimized scale-up and step-wise characterization to prepare for cell line distribution (Cell Banking). In aim 2, the cell lines will be validated for the expected DNA repair functional deficiency by protein expression profiling and genotoxin challenge. Finally (Aim 3), whole-genome transcriptional profiles will be conducted to quantitate transcriptional reprogramming mediated by changes in endogenous DNA repair capacity and where appropriate, following specific genotoxic stress. With the expectation that DNA repair capacity impacts basic cellular functions both spontaneously and in response to genotoxic stress, alters the transcriptional and epigenetic landscape and dictates the cellular response to stress, the development of a complete panel of isogenic DNA repair deficient cell lines across multiple backgrounds will be a valuable platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations. The ready availability of this panel of cell lines will permit both academic and pharmaceutical scientists to study the molecular etiology of tumor genomic instability and to exploit it in oncology research. We envision robust market demand for the cell lines and information that relates to the global transcriptome. PUBLIC HEALTH RELEVANCE: In this Phase II proposal we plan to utilize the successful work-flow paradigm optimized in Phase I for the cell-line development and transcriptome analysis of isogenic human cells lines deficient in all known DNA repair genes. These highly characterized and annotated isogenic cell lines will form the basis for a platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations.