Functionally Validated Lentiviral siRNA libraries

功能验证的慢病毒 siRNA 文库

基本信息

批准号：
7802615
负责人：
ALEX CHENCHIK
金额：
$ 83.91万
依托单位：
CELLECTA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-06 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7802615
关键词：
Address Agar Anchorage-Independent Growth Animals Antineoplastic Agents Apoptosis Bar Codes Bioinformatics Biological Biological Assay Biological Markers Breast Breast Cancer Cell Breast Cancer Genetics Cancer cell line Cell Line Cell Survival Cell model Cells Characteristics Clinical Collaborations Collection Communities Computer software Custom Cytostatics Data Data Analyses Databases Development Disease Disease model Drug Delivery Systems Epithelial Cells Fred Hutchinson Cancer Research Center Gene Expression Profile Generations Genes Genetic Screening Genome Genomics Goals Grant Growth Human Implant In Vitro Individual Information Networks Knowledge Lentivirus Vector Libraries Link Literature Malignant Epithelial Cell Malignant Neoplasms Mammary Neoplasms Mammary gland Maps Mining Modeling Molecular Molecular Profiling Molecular Target Mus Mutation Nude Mice Pathology Pathway interactions Patients Performance Pharmaceutical Preparations Phase Phenotype Primary carcinoma of the liver cells Process Protocols documentation Publications Published Comment Publishing RNA Interference Reagent Reporter Research Research Institute Research Personnel Resources Screening procedure Sequence Analysis Services Signal Pathway Signal Transduction Small Business Innovation Research Grant Small Interfering RNA Software Tools Specificity System Technology Testing Tetanus Helper Peptide The Sun Therapeutic Therapeutic Intervention Validation Xenograft Model anticancer research base cancer cell cancer stem cell cancer therapy cost cytotoxic design drug development drug discovery experience functional genomics genome wide association study genome-wide high throughput technology human disease improved in vivo innovation knowledge base malignant breast neoplasm mouse genome mouse model new therapeutic target novel novel therapeutics performance tests phase 1 study phase 2 study phase 3 study programs public health relevance research study scale up small hairpin RNA technology validation therapeutic target tool tumorigenesis tumorigenic validation studies

项目摘要

DESCRIPTION (provided by applicant): Despite rapid advances in elucidating the molecular basis of human diseases, an ostensibly more difficult post-genomic challenge is the functional annotation of disease-specific signaling pathways and integration of this information into the development of novel drugs. RNA interference (RNAi) now makes it possible to use large-scale functional genomic strategies for target identification. Unfortunately, while RNAi has opened many potential avenues for improving the drug discovery process, these avenues remain only potential opportunities until we develop robust RNAi screening technologies, as well as experimental and bioinformatics tools for data validation and integration of this information into operational cell-based models. To address these issues, in Phase I, we developed second generation functionally validated (FV) human druggable genome lentiviral 15K shRNA libraries, and we have demonstrated their utility for deciphering cell- signaling pathways. The ultimate goal of the Phase II studies is to develop and establish a cost-effective novel functional genomics platform to facilitate the discovery of therapeutic molecular targets en masse. Specifically, we propose to scale-up development of and commercialize a comprehensive set of human and mouse genome- wide FV shRNA libraries. These libraries will have improved performance and be designed for cost-effective pooled-format screening and identification of effectors by high-throughput (HT) sequencing. As supporting tools, we will develop protocols, reagents and software tools for in vitro and in vivo screening hit validation and therapeutic target prioritization. To test the performance of our functional genomics platform, we propose to use our novel RNAi resource to delineate the processes that underlie tumorigenesis in breast epithelial cells. We will perform synthetic lethality screens in a unique panel of isogenic human mammary epithelial cell (HMEC) lines that comprise the most relevant breast cancer genetic alterations. Furthermore, we will validate the results of our in vitro screens in xenograft models using both fully transformed HMECs and common breast cancer cell lines. Our findings will then be combined with data collected from scientific publications and presented in a publicly available knowledge base, with the ultimate goal of developing models of signaling pathways that specifically control the proliferation and survival of breast cancer cells. These developed RNAi screening, validation and software tools will be commercialized as products and custom services to provide the research community with highly modular, cost-effective approaches for studies aimed at understanding and integrating dynamic changes in signal transduction networks and ultimately delineating disease-specific phenotypes. As a result, we foresee that these toolsets will significantly improve the efficiency, economy and ease of elucidating and modeling disease-specific signal transduction networks and provide basic researchers with preferred, cost-effective alternatives to existing commercially available reagents and software. The proposed RNAi screening and bioinformatics strategies harbor considerable potential to systematically identify new anti-cancer targets for therapeutic intervention and to facilitate the development of highly specific drugs, biomarkers and novel therapeutic concepts. PUBLIC HEALTH RELEVANCE: The ultimate goal of the Phase II project is to develop and make commercially available a novel orthogonal functional genomics platform to facilitate discovery and validation of therapeutic molecular targets en masse. As a first step, we propose to develop and make commercially available a set of second generation of functionally-validated genome-wide human and mouse 65K pooled shRNA lentiviral libraries with improved performance and optimized design for cost-effective genetic screens. As a confirmation tool, we will develop protocols for high-throughput in-vitro and ex-vivo validation of drug target candidates identified in the screen with pooled shRNA sublibraries. From a bioinformatics viewpoint, we will make software tools for integration of RNAi screening data with transcriptome profiling and molecular network information mined from scientific literature. The proposed functional genomics platform will be applied and validated for the discovery of novel cancer therapeutic targets in a unique collection of isogenic human mammary epithelial cell (HMEC) lines, comprising the most common breast cancer genetic alterations. As a result of these studies we will reconstruct synthetic lethality pathways and make publicly available breast cancer knowledge database. These developed RNAi screening, validation and software tools will be commercialized as products and custom services to provide the research community with highly modular, cost-effective approaches for studies aimed at understanding and integrating dynamic changes in signal transduction networks and ultimately delineating disease-specific phenotypes. As a result, we foresee that these toolsets will significantly improve the efficiency, economy and ease of elucidating and modeling disease-specific signal transduction networks and provide basic researchers with preferred, cost-effective alternatives to existing commercially available reagents and software. The proposed RNAi screening and bioinformatics strategies harbor considerable potential to systematically identify new anti-cancer targets for therapeutic intervention and to facilitate the development of highly specific drugs, biomarkers and novel therapeutic concepts.

描述（由申请人提供）：尽管阐明人类疾病的分子基础方面取得了迅速的进步，但表面上更困难的基因组挑战是疾病特异性信号通路的功能注释以及将此信息整合到新药的发展中。 RNA干扰（RNAi）现在使使用大规模功能基因组策略进行靶标识别。不幸的是，尽管RNAi开设了许多潜在的途径来改善药物发现过程，但这些途径仍然是潜在的机会，直到我们开发了强大的RNAi筛选技术，以及用于数据验证并将这些信息集成到基于操作细胞模型中的实验和生物信息学工具。为了解决这些问题，在第一阶段，我们开发了第二代经过功能验证的（FV）人类毒素基因组慢病毒15K shRNA文库，我们已经证明了它们用于解密细胞信号传导途径的实用性。第二阶段研究的最终目标是开发和建立一个具有成本效益的新型功能基因组学平台，以促进发现治疗性分子靶标。具体而言，我们建议扩大并商业化一组人和小鼠基因组宽的FV SHRNA库。这些库将具有改进的性能，并设计用于通过高通量（HT）测序对效应子进行成本效益的合并格式筛选。作为支持工具，我们将开发用于体外和体内筛查的协议，试剂和软件工具命中验证和治疗目标优先级。为了测试我们功能基因组学平台的性能，我们建议使用新型的RNAi资源来描述乳腺上皮细胞中肿瘤发生的过程。我们将在独特的同基因人乳腺上皮细胞（HMEC）系中执行合成的致死性筛选，该细胞（HMEC）构成了最相关的乳腺癌遗传改变。此外，我们将使用完全转化的HMEC和常见的乳腺癌细胞系来验证异种移植模型中体外筛查的结果。然后，我们的发现将与从科学出版物中收集的数据相结合，并在公共知识库中介绍，其最终目标是开发信号通路模型，这些模型专门控制乳腺癌细胞的增殖和存活。这些开发的RNAI筛选，验证和软件工具将被商业化为产品和自定义服务，以为研究社区提供高度模块化的，具有成本效益的方法，以理解和整合信号转导网络中动态变化并最终描述疾病特异性表型的动态变化。结果，我们预见到这些工具集将显着提高效率，经济性和易于阐明和建模疾病特异性信号转导网络，并为基础研究人员提供对现有市售试剂和软件的优先，具有成本效益的替代方案。拟议的RNAi筛查和生物信息学策略具有系统地识别治疗干预的新抗癌目标的巨大潜力，并促进了高度特定的药物，生物标志物和新型治疗概念的开发。公共卫生相关性：第二阶段项目的最终目标是开发并使商业上可用的新型正交功能基因组学平台，以促进对治疗分子靶标的发现和验证。作为第一步，我们建议开发并使商业上提供一组第二代全功能验证的全基因组和小鼠65K合并的shRNA慢病毒库，具有改进的性能和优化的设计设计，用于具有成本效益的基因筛选。作为确认工具，我们将开发用于使用合并的SHRNA subliblaries在屏幕上鉴定出的药物目标候选物的高通量和活体验证的方案。从生物信息学的角度来看，我们将制造软件工具，以将RNAi筛选数据与转录组分析和从科学文献开采的分子网络信息集成。拟议的功能基因组学平台将被应用和验证，以在独特的同源性人类乳腺上皮细胞（HMEC）系列中发现新的癌症治疗靶标，这是最常见的乳腺癌基因改变。这些研究的结果是，我们将重建合成的致死途径，并使公开可用的乳腺癌知识数据库。这些开发的RNAI筛选，验证和软件工具将被商业化为产品和自定义服务，以为研究社区提供高度模块化的，具有成本效益的方法，以理解和整合信号转导网络中动态变化并最终描述疾病特异性表型的动态变化。结果，我们预见到这些工具集将显着提高效率，经济性和易于阐明和建模疾病特异性信号转导网络，并为基础研究人员提供对现有市售试剂和软件的优先，具有成本效益的替代方案。拟议的RNAi筛查和生物信息学策略具有系统地识别治疗干预的新抗癌目标的巨大潜力，并促进了高度特定的药物，生物标志物和新型治疗概念的开发。