Short hairpin RNAs Dave Lyons May 2009.

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Presentation transcript:

Short hairpin RNAs Dave Lyons May 2009

Short hairpin RNAs Discovery of interfering RNAs shRNAs in biology shRNAs in medicine

Short hairpin RNAs Discovery of interfering RNAs shRNAs in biology shRNAs in medicine

Discovery of RNAi . . . 1990 2000 2010

“Invention” of shRNA 2000 2010 Paddison et al. 2002

“Invention” of shRNA Paddison et al. 2002; Rao et al. 2009

Short hairpin RNAs Discovery of interfering RNAs shRNAs in biology shRNAs in medicine

shRNA in biological research Large-scale screens In vivo studies Validation

shRNAs for large-scale screens Dykxhoorn and Lieberman 2005

shRNAs for in vivo studies 1.) Mouse KO / KI / BAC 2.) Transplantation of infected SCs 3.) Infect mouse with vector Dykxhoorn and Lieberman 2005

shRNAs for in vivo studies 4.) Tail vein injection Pol II promoter Pol III promoter Dykxhoorn and Lieberman 2005; Giering et al. 2008

shRNA - validation 4.) Tail vein injection Off-target effects? Pol III promoter Pol III promoter Pol II promoter Pol II promoter Dykxhoorn and Lieberman 2005; Giering et al. 2008

shRNA - validation 1.) need a phenotypic assay to accurately confirm on-target effect. 2.) need to control for specific and non-specific off-target effects. - expression profiling of transfected controls - general immune response? - bottleneck effects (e.g. nuclear export)

Short hairpin RNAs Discovery of interfering RNAs shRNAs in biology shRNAs in medicine

shRNA in medicine shRNA is difficult to deliver without viruses but in vivo experiments in mouse suggest better overall for specificity than siRNA. Successful viral targeting is central to future of shRNA therapeutics. Clinical trials with siRNA outnumber shRNA currently.