CCR RNAi Initiative: Validation of siRNAs Against Cancer-Associated Genes

到目前为止，我们已经完全检测了对应于129个人类基因的258个sirna介导的敲低。大约70%的sirna在研究基因的稳态表达水平上下降了50%。这一数据发表在2007年的《核酸研究》杂志上。我们对一些siRNA无法沉默的原因进行了广泛的研究，并确定用于siRNA设计的共识转录物和基因组序列中的单核苷酸多态性、错误和变化以及转录物变体的差异表达都可能导致siRNA介导RNAi的失败。下面描述了此验证过程影响的一个示例。使用合成的与天冬酰胺合成酶相对应的sirna，我们之前已经验证了John weinstein博士小组（LMP， CCR）能够快速评估ASNS表达与酶药物l -天冬酰胺酶（L-ASP）活性之间的功能关系。这项工作已经导致了专利申请的归档和详细描述这项工作的手稿正在出版中。该研究表明，用靶向ASNS的sirna处理细胞可降低ASNS的表达并增强l -天冬酰胺酶（L-ASP）的生长抑制活性，L-ASP是FDA批准用于治疗慢性淋巴细胞白血病的药物。这一数据提示L-ASP治疗可应用于其他癌症，并提示ASNS可作为L-ASP临床疗效的生物标志物。这项研究为RNAi分析在进一步的药物基因组学研究中提供了一个范例，并于最近发表在《分子癌症治疗学》上。我们还研究了sirna同时降低多个基因表达的能力。我们的想法是确定sirna介导的RNAi是否可以比目前报道的更广泛地用于研究多亚基蛋白复合物、显示功能冗余的蛋白质以及研究生物途径和网络中的相互作用。这种类型的分析对于抗癌药物的开发尤其有价值，因为它可以帮助识别产生特别致命表型的分子靶标组合。我们确定至少六个不同的基因靶点可以同时沉默使用表征良好的合成sirna。这项工作最近刚发表。正在进行的研究正在进一步研究可能影响合成siRNA介导的RNAi疗效的因素。

To date we have fully assayed the knockdown mediated by 258 siRNAs corresponding to 129 human genes. Approximately 70% of these siRNAs show a 50% decrease in the steady state levels of the expression of the gene under study. This data was published in 2007 in Nucleic Acids Research. We have conducted extensive study of the reasons why some siRNAs fail to silence and have established that single nucleotide polymorphisms, errors and changes in the consensus transcript and genomic sequences used for the design of siRNAs and differential expression of transcript variants can all contribute to the seeming failure of an siRNA to mediate RNAi. The following describes one example of the impact of this validation process. Using synthetic siRNAS corresponding to the enzyme asparagine synthetase we had previously validated Dr. John Weinsteins group (LMP, CCR) were able to rapidly assess a functional relationship between ASNS expression and the activity of the enzyme drug L-asparaginase (L-ASP). This work has lead to the filing of a patent application and a manuscript describing this work in detail is In Press. This study showed that treatment of cells with siRNAs targeted against ASNS reduces ASNS expression and potentiates the growth inhibitory activity of L-asparaginase (L-ASP), a FDA approved drug used for the treatment of chronic lymphocytic leukemia. This data suggests that L-ASP treatment could be applied to other cancers and suggest that ASNS could be used as a biomarker for the clinical effectiveness of L-ASP. This study presents a paradigm for the use of RNAi analysis to further pharmocogenomic studies and has recently been published in Molecular Cancer Therapeutics. We also investigated the ability of siRNAs to simultaneously decrease the expression of multiple genes. Our concept was to determine if siRNA-mediated RNAi could be used more extensively, than currently reported, for the study of multi subunit protein complexes, proteins exhibiting functional redundancy and for investigation of interactions within biological pathways and networks. This type of analysis could be especially valuable for the development of anti-cancer drugs, as it could assist in identifying molecular target combinations that generate particularly lethal phenotypes. We determined that at least six different gene targets could be simultaneously silenced using well-characterized synthetic siRNAs. This work has been recently published. On-going studies are further investigating factors that may influence the efficacy of synthetic siRNA mediated RNAi.