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

Dykxhoorn and Lieberman 2005

1.) Mouse KO / KI / BAC

2.) Transplantation of infected SCs

3.) Infect mouse with vector

shRNAs for in vivo studies

Dykxhoorn and Lieberman 2005; Giering et al. 2008

4.) Tail vein injection

Pol II promoter

Pol III promoter

shRNA - validation

Dykxhoorn and Lieberman 2005; Giering et al. 2008

4.) Tail vein injection

Pol II promoter

Pol III promoter Pol III promoter

Pol II promoter

Off-target effects?

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.