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

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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.