CCR RNAi Initiative: Validation of siRNAs Against Cancer

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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.