Over the course of the last four years, the Thermo Scientific R&D team in Lafayette, CO has collaborated with several academic and industrial groups to identify new and interesting applications of RNA interference (RNAi). During that time we have interacted with a talented group of researchers and successfully demonstrated applications for RNAi in gene function analysis¹, drug target validation², and gene mapping³. Our recent collaboration with Dr. Robert Davey at the University of Texas, Medical Branch (UTMB) has been particularly gratifying in that the results from his studies on Respiratory Syncytial Virus (RSV) have direct application to human disease.⁴

Dr. Robert Davey	Respiratory Syncytial Virus (RSV)

Dr. Davey is a member of Department of Microbiology and Immunology at UTMB with a special interest in host-pathogen interactions that affect viral entry. As most readers are aware, viruses take advantage of a number of host-encoded functions, including receptors, vesicular compartments, cytoskeletal matrices, and replication machinery, to enter and proliferate within the host cell. Understanding how the host contributes to pathogen reproduction is particularly important in that such knowledge can provide new and useful strategies for future drug development efforts.

Viral entry generally occurs by one of two mechanisms: fusion with the cell membrane or appropriation of one or more endocytic pathways. Historically, drugs that disrupt endocytosis by neutralizing the pH gradients of endosomal compartments (e.g. chloroquine and bafilomycin) have been used to characterize these two entry strategies. Specifically, pH-sensitive viruses such as influenza and rabies are believed to enter the cell by endocytosis, while pH-insensitive viruses (e.g. RSV, Human Immunodeficiency Virus, HIV, and Murine Leukemia Virus, MLV) are assumed to enter by cell fusion.

Dr. Davey has challenged the conventional interpretation of pH-sensitivity by combining focused collections of the Thermo Fisher Scientific SMARTpool siRNA reagents with a novel viral entry assay. Using the highly virulent RSV as a model pH-insensitive system, the Davey lab transfected cells with pools of siRNAs targeting 84 genes associated with endocytosis, cytoskeletal rearrangement, and membrane trafficking, and subsequently infected with RSV pseudoviral particles containing a GFP reporter. Surprisingly, even though RSV is classified as pH-insensitive, the virus exhibited a robust dependence on host genes associated with early events in clathrin-dependent endocytosis. These findings were supported by complementary genetic studies that employed dominant negative alleles of genes involved in clathrin-dependent endocytosis, thus lead the authors to speculate that RSV (and possibly other pH independent viruses) take advantage of endocytic pathways but exit the endosome prior to vesicle acidification.

While the results from the Davey Lab offer new insights into RSV entry, they also address a critical issue that has lately arisen during RNAi-based phenotypic screens, that is, specificity. Several recent studies in both mammalian and drosophila systems have acknowledged the problem of siRNA off-target effects, with some screens exhibiting a false positive rate of 90% or more.⁵ In the screen performed by Dr. Davey and his colleagues, 14 out of the 15 positives identified in the primary screen were subsequently shown (through deconvolution of the siRNA pools) to be true positives. This low level of false positives (6.6%) is likely to be attributed to the high degree of specificity of the their assay and the fact that pools induce fewer numbers of off-targets (and off-target phenotypes) than individual siRNA.

Overall, the findings by the Davey Lab provide an important entry point for future detailed studies of RSV pathogenesis and are likely to make a significant contribution to RSV drug development. As additional collaborative programs with scientists investigating rabies, small pox and other pathogens are currently ongoing, we expect the contributions of RNAi technologies to the understanding infectious disease to continue.

References