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Will off-target silencing prove too great a challenge to the future success of RNAi?


A.We have devised strategies to prioritize the potential genes of interest and, upon matching these criteria, we can examine the specificity of the siRNAs in our assays by re-ordering new sequences against additional regions of the same gene. When we have observed off-target effects, we can use bioinformatics approaches to determine the possible off-target gene that the siRNA species might be hitting and remake siRNAs specifically against this gene. If the new siRNAs are also effective then we can conclude that the effects we are seeing are due to the gene that was initially identified as an off-target effect.

David Evans
Head, Drug Discovery
Translational Genomics Research Institute


A.It’s the scope of what you’re doing. For us, if we’re just looking in vitro at a proof of concept experiment — is gene X involved in this pathway? — it’s probably not a huge issue. If you start having off-target effects on housekeeping or metabolic genes then therapeutically that could clearly be problematic. As with antisense and the other technologies that came before it, the proof is going to be in the pudding. You’re going to have to do the clinical trials.

David Uhlinger
Principal scientist, Bioinformatics
Johnson & Johnson

A.There will be some potential applications for which off-target silencing will not be unacceptable. For example, in therapeutic applications where unpredictable side effects might not be too severe to make the therapy unacceptable — if it comes to a choice between silencing the putative defect that is creating tumor cells, and some unintended silencing with perhaps innocuous effects — then off-target silencing is not too serious a problem. However, if one is looking specifically to identify downstream effects resulting from silencing of a specific gene, the change in the profile of expression cannot be relied upon to be coming from the putative target. That’s a serious problem.

Rudy Spangler
Rockefeller University

A.Dealing with the so-called off-target issues is probably the largest hurdle that I see. These nonspecific side effects lead me to believe that the long-term future of siRNA as a tool is limited (although it is clear that chemical modification is still not explored thoroughly). The bottleneck is still dealing with off-target effects, as the information generated is only going to be useful if the tool is specific. At this stage I think many applications for [siRNAs] are being explored but for me the selectivity issue is the Achilles’ heel of all of them.

Jamie Timmons
Department of Physiology and Pharmacology
Karolinska Institute


A.There’s a big debate, and it’s a legitimate debate, that we still don’t know how important they are, or how much they affect RNAi in vivo. What we’re looking at are transcriptional effects — those are small compared to the on-target effects. What we don’t know are what the effects are on the proteome. One of the things we found about the off-target effects is that they actually resemble microRNAs. If the off-target effects are reminiscent of microRNAs, microRNAs exert far greater effect on the proteome. That’s really the question that needs to be answered now.

Peter Linsley
Executive director, Cancer Biology
Rosetta Inpharmatics


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