Functionally Validated Lentiviral siRNA Libraries

描述（由申请人提供）：尽管人类基因组计划最近完成，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和将此信息整合到基于细胞的操作模型中。不幸的是，这是目前具有挑战性的，主要是由于缺乏合适的工具集，以快速描绘基因功能基因组。RNA干扰（RNAi）已被证明是一种非常有效和通用的工具，可以特异性地降低靶基因的表达，从而在哺乳动物细胞中进行功能丧失的遗传筛选。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，并且该技术存在重大限制。为了解决这些问题，并扩大以前的计划资助，我们开发了一种新的实验平台来构建功能验证（FV）的shRNA文库。在I期资助下，我们最初建议在生物安全的慢病毒载体中开发FV人shRNA文库，靶向5000个药学上易于处理的基因家族，这些基因家族适合于小分子药物发现研究。与我们在Fred哈钦森癌症研究中心（FHCRC）和斯克里普斯研究所（TSRI）的生物学顾问合作，我们随后将在一个简单的基于细胞培养的模型中验证该文库，以确定负责癌细胞中TGF-β生长限制作用的基因。在第二阶段，我们将扩展该计划，包括开发全基因组功能验证的人类和小鼠siRNA文库。我们建议使用我们的新的HT RNAi资源来描绘癌细胞增殖失调的过程，并将这些知识应用到转化研究计划中。例如，我们将使用我们新开发的FV siRNA文库技术来鉴定癌细胞中的合成致死相互作用。使用FV siRNA文库的遗传筛选具有极大地简化基因操作和发现的潜力，用于许多生物医学应用，例如基因功能的验证、探测基因之间的相互作用和动物模型的建立。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，这些试剂具有相当大的前景，以确定新的目标，治疗干预，和发展日益相关的范例，药物发现。因此，我们预见这些工具集将显著提高进行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、具有成本效益的替代品。拟议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：一套功能验证的全基因组人类和小鼠慢病毒siRNA文库，旨在高通量发现新的药物靶点。我们建议应用这种强大的技术来确定新的抗癌药物靶点。所开发的技术将显著提高与多种人类疾病机制的分子解剖、新药开发相关的转化研究的效率，因此对改善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.