Pascal, how can you make sure the behavioral affects you observed are not off-target effects and are truly specific?
First, a scrambled DsiRNA negative control should be used and should not induce the effect by itself. Second, at least two different DsiRNAs targeting different regions of the same gene of interest should provide similar behavioral results. The final validation is that pharmacological effect observed after agonist injection was selectively reversed by the two DsiRNA.
Pascal, was there any inflammatory response following the two in vivo injections of DsiRNA? Is it safe to inject more than two times to maintain a continuous therapeutic effect?
In terms of live animal inflammation, very little was observed. After dissection, a small red spot was observed at the injection site under the skin, but no apparent sign of discomfort or edema. In terms of molecular inflammation, we tested multiple cytokines, including IL6 and TNF-a, and no significant differences were observed compared to control or scramble DsiRNA injected animals. Based on these observations, more than two injections could be feasible in order to maintain continuous effect.
Pascal, you have been using very low doses of DsiRNA. If you used higher doses, would the behavioral effect (silencing) be maintained longer, or would there be a risk of priming an inflammatory or off-target effect?
There is no clear answer to that question, the in vivo stability of the transfection reagent/DsiRNA complex might vary depending on the transfection reagent used and site of injection. Increasing the dose might increase silencing effect without inducing off-target effect since the dose we use is very low (from 1 to 5µg). However, increasing doses to a much higher level (in the range of 50 to 100 µg, comparable to study with siRNA) will increase chances to induce off-target effect.
Once I have selected my DsiRNA candidate, can IDT help me with the modification process?
Absolutely. Our research group is continually working towards identifying the pattern of chemical modifications in DsiRNAs that will result in optimal potency and stability while minimizing off-target effects.
Pascal, in your study, did you compare the DsiRNAs with traditional 19nt siRNA?
No we did not compare with traditional 19 or 21nt siRNA.
I am currently validating dsiRNAs for my target. Can IDT help me with this process?
Yes. In fact, there is a recent webinar that may be helpful in these initial stages of the experimental process–Planning and Executing siRNA Experiments: Good Practices for Optimal Results.
Pascal, do you use stablization modified dsiRNA for the in vivo study?
Not in those specific experiments, we used unmodified DsiRNAs. However, it is possible that we would have observed better silencing with methyl-modified DsiRNAs.