Functional Dissection of Signaling Pathways

描述(申请人提供)：后基因组时代的最大挑战是揭示每一个人类基因的功能。开发新的高通量功能基因组学工具对于在全基因组范围内发现基因的功能并将其产物连接到信号通路至关重要。最近基于小干扰RNA(SiRNA)的基因敲除技术的引入，有望给基因功能分析带来革命性的变化。引入可选择的路径特异性报告系统将极大地补充siRNA方法，该方法旨在对正常和疾病影响的路径进行功能剖析。拟议计划的目标是开发一套安全的慢病毒转录报告载体，并使其在商业上可用，用于最关键的疾病相关信号转导途径。这些途径特异的报告载体，与基因特异性siRNA或全基因组siRNA文库相结合，将使研究人员能够研究途径的机制，并确定参与人类疾病发病机制的变化。具有全基因组siRNA文库和路径特异性报告载体的全面遗传筛选可以识别其失活导致抑制与疾病相关的变化的靶基因。该计划的第一阶段将包括以下技术的开发：(I)下一代基于慢病毒的安全报告结构，用于检测p53途径的变化；(Ii)慢病毒siRNA表达载体，设计用于沉默p53途径的几个已知成分。这些载体的功能性能将在正常(人胚胎成纤维细胞，HEF)和疾病(宫颈癌细胞系HeLa)模型系统中进行测试。该计划的第二阶段将扩展到开发一套全面的慢病毒报告载体并将其商业化，该载体带有β-半乳糖苷酶、荧光素酶和荧光记者，涵盖与广泛的人类疾病相关的大约30个主要信号转导途径。开发的双色荧光报告载体和全基因组siRNA文库包括所有已知的基因，将允许在活细胞的背景下全球搜索新的途径特异性成分和疾病相关基因。这项已建立的技术将与克利夫兰临床基金会合作，用于确定药物开发的目标，目的是在一组人类癌症模型中恢复被抑制的p53途径。拟议的研究和产品开发计划的预期结果将是一套商业可用的试剂盒，包括用于高通量基因功能分析的慢病毒报告载体和siRNA文库、定制的全基因组功能筛选服务和几个经过验证的抗癌药物靶标。

DESCRIPTION (provided by applicant): The greatest challenge of the post genomic era is to uncover the functions of every individual human gene. Development of novel high throughput functional genomics tools is critical for a genome-wide discovery of a gene's functions and for linking their products into signaling pathways. The recent introduction of gene knockdown technology, based on small interfering RNAs (siRNA), promises to revolutionize gene functional analysis. Introduction of selectable pathwayspecific reporter systems would significantly complement the siRNA approach that is aimed at functional dissection of normal and disease-affected pathways. The goal of the proposed program is to develop and make commercially available a set of safe lentiviral transcriptional reporter vectors for the most critical disease-related signal transduction pathways. These pathway-specific reporter vectors, in combination with gene-specific siRNAs or genome-wide siRNA libraries, will allow researchers to study the mechanisms of pathways and identify changes in the mechanisms involved in the pathogenesis of human diseases. Comprehensive genetic screens with genome-wide siRNA libraries and pathwayspecific reporter vectors could identify target genes whose inactivation leads to suppression of disease-related changes. Phase I of the program will cover the development of technology for construction of (i) next-generation safe lentiviral-based reporter constructs for detection of changes within p53 pathway, and (ii) lentiviral siRNA-expressing vectors designed for silencing of several known components of the p53 pathway. Functional performance of these vectors will be tested in normal (human embryonic fibroblasts, HEFs) and disease (cervical carcinoma cell line HeLa) model systems. The Phase II of the program will be extended towards development and commercialization of a comprehensive set of lentiviral reporter vectors with beta-galactosidase, luciferase and fluorescent reporters, covering about 30 major signal transduction pathways associated with a wide range of human diseases. The developed dual color fluorescent reporter vectors and genome-wide siRNA libraries, comprising all known genes, will allow a global search for novel pathway-specific components and disease-associated genes in the context of living cells. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development aimed at the restoration of suppressed p53 pathway in a set of human carcinoma models. The anticipated outcomes of the proposed research and product development program will be a commercially available set of kits comprising of lentiviral reporter vectors and siRNA libraries for high-throughput gene functional analysis, custom genome-wide functional screen service and several validated anticancer drug targets.