CCR RNAi Initiative: Validation of siRNAs Against Cancer

为了获得大规模的合成siRNA资源，基因沉默组与Qiagen公司达成了一项研究合作协议。到目前为止，Qiagen已经设计和合成了大约400个基因对应的sirna。为了充分利用这些资源，我们开发了96孔板格式的高通量自动合成sirna -脂质“反向转染”方案（biorbot 8000, Qiagen Inc.）。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%。我们对一些siRNA无法沉默的原因进行了广泛的研究，并确定了用于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。我们已经使CCR研究人员能够访问100个人类基因的机密RNAi分析数据，这导致了30多个内部材料转移协议的执行，并将适当的信息转移给CCR研究人员。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博士的团队（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。本研究为RNAi分析在进一步药物基因组学研究中的应用提供了一个范例。为了进一步使用RNAi分析，我们需要建立大规模的RNAi筛选方法。我们早期考虑使用阵列siRNA介导的RNAi筛选表明，这种格式与许多实际限制有关。这些限制包括可能需要广泛的自动化液体处理和/或需要大量的转染和分析试剂，这可能是成本过高的。此外，由于每次可检测的样品数量有限，一些检测平台与大规模分析不兼容。其中许多检测也可能是劳动密集型的。此外，表现出给定分析表型的靶标数量(即：“命中”)可能只对应于在更大的无偏见屏幕中评估的总目标的一小部分，回顾性地使大多数此类屏幕成为多余的。虽然已经解决了shrna的一些问题，但还没有研究检查与合成sirna相关的问题。为了解决这个问题，我们开发了一种新的策略，使用与多个基因靶点对应的合成siRNA的多重组合，以简化siRNA RNAi筛选。我们已经确定，多重sirna可以使至少6个基因沉默，其程度与单独靶向基因时相同。然后，我们生成含有随机多路sirna的sirna文库，并将其用于细胞活力筛选。利用这种策略，确定了几个影响乳腺癌细胞系生存能力的基因靶点。包括已知或推测的抗肿瘤分子靶点。

To access large-scale synthetic siRNA resources Gene Silencing Section, Genetics Branch, has developed a research collaboration agreement with Qiagen Inc. To date Qiagen has designed and synthesized siRNAs corresponding to approximately 400 genes. To make full use of these resources we have developed a high throughput automated synthetic siRNA-lipid "reverse transfection" protocol in a 96 well plate format (BioRobot 8000, Qiagen Inc.). 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. We have conducted extensive study of the reasons why some siRNAs fail to silence and have established the 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. We have enabled CCR investigators to access the confidential RNAi analysis data for 100 human genes, and this has resulted in the execution of over 30 intramural material transfer agreements and the transfer of appropriate information to CCR investigators. 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 Weinstein's 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.To go further in our use of RNAi analysis we have needed to establish large-scale RNAi screening methods. Our early consideration of siRNA mediated RNAi screens using arrayed siRNAs suggested that this format is associated with a number of practical limitations. Such limitations include the potential need for extensive automated liquid handling and/or the requirement for large amounts of transfection and assay reagents, which can be cost-prohibitive. In addition, some assay platforms are incompatible with large-scale analysis because of the limited number of samples that can be assayed at any one time. Many of these assays can also be labor intensive. Furthermore, the number of targets exhibiting a given assayed phenotype (i.e. "hits") is likely to correspond to only a small fraction of the total targets evaluated in larger unbiased screens, retrospectively making the majority of such screens superfluous. While some of these issues have been addressed for shRNAs no studies have examined this in relation to synthetic siRNAs. To address this we developed a novel strategy using multiplexes of synthetic siRNAs corresponding to multiple gene targets in order to streamline siRNA RNAi screens. We have established that multiplexed siRNAs can silence at least six genes to the same degree as when the genes were targeted individually. We then generated libraries of siRNAs containing randomly multiplexed siRNAs and used them in a screen of cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. Included were known or putative anti-tumor molecular targets.