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

到目前为止，我们已经完全测定了由258个siRNA介导的敲低，对应于129个siRNA。 人类基因这些siRNA中约70%显示稳态水平降低50%， 所研究的基因的表达。这一数据发表在2007年的《核酸》杂志上 Research.我们已经对一些siRNA不能沉默的原因进行了广泛的研究， 已经确定，单核苷酸多态性，错误和变化的共识， 用于设计siRNA和差异表达的转录物和基因组序列 转录物变体都可能导致siRNA介导RNAi的失败。的 以下描述了该验证过程的影响的一个示例。使用合成 siRNAS对应的酶天冬酰胺合成酶，我们以前验证约翰博士 Weinsteins组（LMP，CCR）能够快速评估功能关系 ASNS表达和酶药物L-天冬酰胺酶（L-ASP）的活性之间的关系。这项工作 已经导致了专利申请的提交和详细描述这项工作的手稿 在Press。这项研究表明，用靶向ASNS的siRNA处理细胞， 降低ASNS表达并增强L-天冬酰胺酶的生长抑制活性 （L-ASP），一种FDA批准的用于治疗慢性淋巴细胞白血病的药物。该数据 表明L-ASP治疗可以应用于其他癌症，并表明ASNS可以 用作L-ASP临床有效性的生物标志物。这项研究提出了一个范例， 用于RNAi分析以进一步药物基因组学研究，最近已被 发表在《分子癌症治疗学》上。我们还研究了siRNA的能力， 同时降低多个基因的表达。我们的概念是确定 siRNA介导的RNAi可以比目前报道的更广泛地用于研究 多亚基蛋白质复合物，表现出功能冗余的蛋白质， 研究生物途径和网络内的相互作用。这种类型的分析 可能对抗癌药物的开发特别有价值，因为它可以帮助 鉴定产生特别致命表型的分子靶向组合。我们 确定至少六种不同的基因靶可以同时沉默， 充分表征的合成siRNA。这项工作已于最近出版。正在进行的研究 正在进一步研究可能影响合成siRNA介导的疗效的因素， RNA干扰

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.