RNA-Seq Dose Response Experiments for Quantification of Off-Target Effects with RNAi Therapeutics

RNAi therapeutics can be designed to silence almost any gene of interest and have demonstrated high levels of efficacy and acceptable safety profiles in pre-clinical and clinical development for cardio-metabolic, hepatic infectious, central nervous system, and rare diseases. Minimizing microRNA-like off-target activity while maintaining on-target silencing is a means to maximize the safety profile. One strategy to mitigate off-target activity is to incorporate thermally destabilizing residues such as glycol nucleic acid in the seed region of the antisense strand of a double-stranded RNA. Here we demonstrate the benefit of this strategy using Alnylam's ESC+ conjugate platform by performing RNA-Seq in dose response to measure both on-target and off-target effects. Diverse measures and visualizations of transcriptomic noise will be presented, as well as estimates of relative on-target to off-target effects as a function of dose. These results show that ESC+ conjugates are capable of simultaneously achieving high levels of on-target silencing while maintaining low levels of transcriptomic noise.