Global Gene Functional Analysis with siRNA Libraries

描述（由申请人提供）：后基因组时代的最大挑战是揭示每个人类基因的功能。新型高通量功能基因组学工具的开发对于全基因组范围的药物靶点发现和验证至关重要，并且为复杂人类疾病的药物治疗和诊断的新概念开辟了途径。最近引入的基于小干扰RNA（siRNA）的基因敲除技术有望彻底改变基因功能分析。该计划的目标是开发并商业化一套全面的慢病毒载体全基因组siRNA文库，比目前可用的合成siRNA集合更有效和更具成本效益。在I期资助下，开发了生物安全的猫免疫缺陷病毒（FIV）慢病毒siRNA克隆载体和用于构建高复杂性siRNA文库的技术。构建了人1.5K siRNA文库，包含1500个基因的7500个siRNA的冗余组，并在辐射抗性HT 1080细胞模型中进行了功能测试。基于siRNA文库和Affymerix GeneChip（r）微阵列的组合，开发了用于辐射抗性基因遗传筛选的高通量技术。慢病毒siRNA克隆载体作为商业产品和即用型产品发布，预包装在病毒颗粒中，人1.5K和8.5K siRNA文库目前处于产品发布周期。在第二阶段的资助下，我们建议将该计划扩展到慢病毒载体中的一套全基因组50K人类，40K小鼠和30K大鼠siRNA文库的开发和商业化，并选择不同的报告基因和tet调控的siRNA表达。siRNA文库将允许对涉及各种疾病的关键基因进行全局搜索，并通过在活细胞中任意选择性灭活疾病相关基因来验证新的药物靶点。该技术将与克利夫兰临床基金会合作，用于确定针对前列腺癌的药物开发目标。拟议的研究和产品开发计划的预期成果将是一套商业化的试剂盒，包括慢病毒载体和siRNA文库，用于高通量基因功能分析，定制全基因组功能筛选服务和经验证的抗癌药物靶点，用于治疗前列腺癌。

DESCRIPTION (provided by applicant): The greatest challenge of the post genomic era is to uncover functions of every individual human gene. Development of novel high throughput functional genomics tools are critical for genome-wide discovery and validation of drug targets, and to open avenues for newer concepts in drug therapy and diagnostics of complex human diseases. The recent introduction of gene knockdown technology based on small interfering RNAs (siRNA) holds a promise to revolutionize gene functional analysis. The goal of the proposed program is to develop and make commercially available a comprehensive set of genome-wide siRNA libraries in lentiviral vectors that are more effective and cost-efficient than the currently available collection of synthetic siRNAs. Under Phase I funding, the biosafe feline immunodeficiency virus (FIV) lentiviral siRNA cloning vectors and technology for construction of high complexity siRNA libraries were developed. The Human 1.5K siRNA library, comprising of a redundant set of 7500 siRNAs for 1500 genes, was constructed and functionally tested in the radiation resistance HT1080 cell model. High-throughput technology for genetic screens of radiation-resistance genes, based on a combination of siRNA libraries and Affymerix GeneChip(r) microarrays, was developed. Lentiviral siRNA cloning vectors were released as commercial products and ready-to-use, prepackaged in viral particles, human 1.5K and 8.5K siRNA libraries are currently in the product launch cycle. Under Phase II funding, we propose to extend the program towards development and commercialization of a comprehensive set of genome-wide 50K human, 40K mouse and 30K rat siRNA libraries in lentiviral vectors with a selection of different reporters and tet-regulated expression of siRNAs. siRNA libraries will allow the performance of global searches for key genes involved in a variety of diseases and to validate new drug targets by arbitrary selective inactivation of diseaseassociated genes in the context of the living cells. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development, aiming at prostate cancer. The anticipated outcomes of the proposed research and product development program will be a commercially available set of kits comprising of lentiviral vectors and siRNA libraries for high-throughput gene functional analysis, custom genomewide functional screen service and validated anticancer drug targets for treatment of prostate cancer.