Global Gene Functional Analysis with siRNA Libraries

描述(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。新的高通量功能基因组学工具的开发对于全基因组范围内发现和验证药物靶点至关重要，并为复杂人类疾病的药物治疗和诊断开辟了新的途径。最近引入的基于小干扰RNA(SiRNA)的基因敲除技术有望给基因功能分析带来革命性的变化。该计划的目标是在慢病毒载体中开发一套全面的全基因组siRNA文库，并使其在商业上可用，这些文库比目前可用的合成siRNA集合更有效、更经济。在第一阶段资助下，开发了Biosafe猫免疫缺陷病毒(FIV)慢病毒siRNA克隆载体和构建高复杂性siRNA文库的技术。构建了人类1.5K siRNA文库，该文库由1500个基因的7500个siRNA组成，并在抗辐射HT1080细胞模型中进行了功能测试。基于siRNA文库和Affymerix基因芯片(R)微阵列的组合，开发了高通量的抗辐射基因遗传筛选技术。慢病毒siRNA克隆载体已作为商业化产品发布，并可随时使用，预先包装在病毒颗粒中的人1.5K和8.5K siRNA文库目前正处于产品推出周期。在第二阶段的资助下，我们建议将该计划扩展到在慢病毒载体中开发和商业化一套全面的全基因组50K人、40K小鼠和30K大鼠的siRNA文库，并选择不同的报告器和tet调控的siRNAs表达。SiRNA文库将允许在全球范围内搜索与各种疾病有关的关键基因，并通过在活细胞环境中任意选择使疾病相关基因失活来验证新的药物靶点。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载体和siRNA文库，定制全基因组功能筛选服务，以及用于前列腺癌治疗的经验证的抗癌药物靶标。

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.