Prevailing drug discovery approaches focus on compounds with molecular selectivity, inhibiting disease-relevant targets over others in vitro. However in vivo, many such agents are not therapeutically selective, either because of undesirable activity at effective doses or because the biological system responds to compensate. In theory, drug combinations should permit increased control of such complex biology, but there is a common concern that therapeutic synergy will generally be mirrored by synergistic side-effects. Here we provide evidence, from 94,110 multi-dose combination experiments representing diverse disease areas and large scale flux balance simulations of inhibited bacterial metabolism, that multi-target synergies are more specific than single agent activities to particular cellular contexts. Using an anti-inflammatory combination, we show how multi-target synergy can achieve therapeutic selectivity in animals through differential target expression. Synergistic combinations can increase the number of selective therapies using the current pharmacopeia, and offer opportunities for more precise control of biological systems.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that downregulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA expression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.genetic screen ͉ lentivirus ͉ MAGIC