The dose can make the poison: lessons learned from adverse in vivo toxicities caused by RNAi overexpression

Since the seminal 1998 report of RNA interference (RNAi) in nematodes [1], the ascent of RNAi technologies from a curious phenomenon in worms to a widely and routinely used surrogate genetic tool in higher eukaryotes, as well as one of our most promising therapeutic modalities, has been nothing short of meteoric. Ironically, though, in the same year, 2006, that the rise of RNAi temporarily culminated in the Nobel Prize for its pioneers Andrew Fire and Craig Mello, Mark Kay's group published a startling study reporting fatal side effects from abundant RNAi expression in the livers of adult mice [2]. Since then a series of further studies in various species and tissues have solidified the original idea that one crucial mechanism underlying the observed in vivo toxicities or fatalities is adverse saturation of the endogenous miRNA machinery by ectopic RNAi triggers. Herein I briefly review these papers and findings before highlighting key lessons that we can learn and new avenues that we can now take.The 2006 Grimm et al. study [2] came as a surprise to the field, as the wealth of previous reports had proved RNAi's superior efficacy and thus fostered a rapid translation of RNAi technologies from bench to bedside. What was so different in this particular work was the unique combination of (1) an utmost potent viral RNAi delivery vector (self-complementary adeno-associated virus serotype 8 (scAAV8)), (2) a powerful promoter (U6, one of the strongest known RNA polymerase III promoters) driving small hairpin RNA (shRNA) expression and (3) delivery of high vector doses (directly into the hepatic circulation in some animals) [2]. This experimental setup not only ensured complete liver transduction in the injected mice but also introduced, on average, thousand RNAi expression templates into each hepatocyte, likely resulting in the transcription of hundreds of thousands of shRNA molecules per cell.Unsurprisingly, at least in retrospect, such massive overloading of the cells wi