Functionally Validated Lentiviral siRNA Libraries

描述（由申请人提供）：尽管最近完成了人类基因组计划，但表面上更困难的后基因组挑战将是所有人类基因的功能注释，并将这些信息整合到可操作的基于细胞的模型中。不幸的是，目前这是具有挑战性的，主要是因为缺乏合适的工具集来快速描述基因功能。RNA干扰（RNAi）已被证明是一种非常有效和通用的工具，可以特异性地降低目标基因的表达，从而允许在哺乳动物细胞中进行功能缺失的遗传筛选。尽管取得了这些成功，但高通量（HT） RNAi筛选在技术上具有挑战性，并且存在显著的局限性。为了解决这些问题，并扩大之前的项目资金，我们开发了一个新的实验平台来构建功能验证（FV） shRNA库。在第一阶段的资金支持下，我们初步建议以生物安全的慢病毒载体开发FV人类shRNA文库，目标是5000个可用于小分子药物发现研究的药学上可处理的基因家族。随后，我们将与Fred Hutchinson癌症研究中心（FHCRC）和Scripps研究所（TSRI）的生物学顾问合作，在一个简单的基于细胞培养的模型中验证这个文库，以确定在癌细胞中负责TGF-¿生长限制作用的基因。在第二阶段，我们将扩展该计划，包括全基因组功能验证的人类和小鼠siRNA文库的开发。我们建议利用我们的新HT RNAi资源来描述癌细胞中不受管制的增殖过程，并将这些知识应用于转化研究项目。例如，我们将使用我们新开发的FV siRNA文库技术来识别癌细胞中合成的致命相互作用。利用FV siRNA文库进行遗传筛选有可能极大地简化基因操作和发现的许多生物医学应用，如基因功能的验证，探测基因之间的相互作用，以及建立动物模型。此外，我们设想对人类疾病机制的分子解剖产生重大影响。例如，这些试剂在确定治疗干预的新靶点和开发越来越相关的药物发现范例方面具有相当大的前景。因此，我们预计这些工具集将显著提高HT RNAi筛选的效率、经济性和易用性，并将为基础研究人员提供现有市售试剂的首选、成本效益高的替代方案。该项目的最终目标是开发并商业化一种新的强大的研究工具：一套功能有效的全基因组人类和小鼠慢病毒siRNA文库，用于高通量发现新的药物靶点。我们建议应用这一强大的技术来识别新的抗癌药物靶点。所开发的技术将大大提高与各种人类疾病机制的分子解剖和新药物开发有关的转化研究的效率，因此对改进药物发现研究具有重大意义。

DESCRIPTION (provided by applicant): Despite the recent completion of the human genome project, an ostensibly more difficult post-genomic challenge will be the functional annotation of all human genes and integration of this information into an operational cell-based model. Unfortunately, this is at present challenging, primarily due to the absence of suitable toolsets to rapidly delineate gene function en masse. RNA interference (RNAi) has proven to be an extremely potent and versatile tool to specifically reduce expression of targeted genes, allowing for loss-of- function genetic screens in mammalian cells. Despite these successes, high-throughput (HT) RNAi screening is technically challenging and significant limitations in the technology exist. To address these issues, and to expand on previous program funding, we have developed a novel experimental platform to construct functionally validated (FV) shRNA libraries. Under Phase I funding, we initially propose to develop a FV human shRNA library in biosafe lentiviral vectors targeting 5000 pharmaceutically tractable gene families that are amenable to small molecule drug discovery research. In collaboration with our biology consultants at the Fred Hutchinson Cancer Research Center (FHCRC) and the Scripps Research Institute (TSRI), we will subsequently validate this library in a simple cell culture-based model, to identify genes responsible for the growth limiting actions of TGF-¿ in cancer cells. In Phase II, we will extend this program to include the development of genome-wide functionally validated human and mouse siRNA libraries. We propose to use our novel HT RNAi resource to delineate the processes which underlie deregulated proliferation in cancer cells, and to apply this knowledge into translational research programs. For example, we will use our newly developed FV siRNA library technology to identify synthetic lethal interactions in cancer cells. Genetic screens with FV siRNA libraries have the potential to greatly simplify gene manipulation and discovery for many biomedical applications, such as validation of gene function, probing interactions between genes, and the establishment of animal models. Moreover, we envisage a major impact on the molecular dissection of human disease mechanisms. For example, these reagents harbor considerable promise to identify new targets for therapeutic intervention, and the development of increasingly relevant paradigms for drug discovery. As a result, we foresee that these toolsets will significantly improve the efficiency, economy and ease of performing HT RNAi screens, and will provide basic researchers with preferred, cost-effective alternatives to existing commercially available reagents. The ultimate goal of the proposed project is to develop and make commercially available a new powerful research tool: a set of functionally validated genome-wide human and mouse lentiviral siRNA libraries designed for high-throughput discovery of novel drug targets. We propose to apply this powerful technology to identify novel anti- cancer drug targets. The developed technologies will significantly improve the efficiency of translational researches related to molecular dissection of diverse human disease mechanisms, development of new pharmaceuticals and therefore, have major implications for improving drug discovery research.