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

Abstract

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.