Another approach is the use of lentiviruses to deliver short hairpin RNAs (shRNAs) for screening. “Current-generation lentiviral vectors have been engineered to allow delivery into most cell types,” explains Michele A. Cleary, Ph.D., research fellow, Rosetta Inpharmatics (www.rii.com). “This allows for RNAi in cells that cannot be transfected with siRNAs.”
Lentiviral delivery of RNAi can provide sustained silencing, according to Dr. Cleary. When cells are transduced with shRNA-encoding lentiviral vectors, the shRNA expression cassette becomes stably integrated into the host genome. “This is a major advantage, particularly because siRNAs silence genes for only five to seven days. But some assays need a longer time, for example, 14 days, to achieve an effect.”
Lentivirus-mediated delivery also creates cell lines capable of expressing shRNAs that target genes whose silencing is lethal to the cell. This can be done by manipulating expression by using inducible promoters that can be turned on or off as desired.
“It can also help when looking for off-target effects. We simply turn on knockdown, assess the effects by gene-expression profiling, and compare those profiles to when the shRNA is silent. Using this approach, we have learned that, like siRNAs, shRNAs have off-target effects, but the extent and magnitude is less. This may be because shRNAs enter the RNAi pathway at a different point.”
But just as with siRNAs, shRNAs must be carefully designed. “It’s critical to choose an effective shRNA core sequence using validated algorithms.”
Rosetta is utilizing this system for cancer biology, but other applications can be much broader. “Because lentiviruses can be used on nondividing cells, such as neurons that are traditionally difficult to transfect, the system has a wide repertoire of target cells. However, delivery of RNAi to living tissues via lentiviruses still needs some optimization. The use of other viral vectors is being explored to help achieve that goal.”