Use of “C9/11 Mismatch” Control siRNA Reveals Sequence-Related Off-Target Effect on Coagulation of an siRNA Targeting Mouse Coagulation Factor XII

Recently, our group reported that a small interfering RNA (siRNA) targeting coagulation factor XII (siF12) leads to an unexpected prothrombotic response in a mouse model where venous thrombosis follows inhibition of endogenous anticoagulants. In this study, we aimed to clarify this unexpected response by evaluating the effects of this siF12 (here, siF12-A) on plasma coagulation through thrombin generation (TG). Besides a routine negative control siRNA (siNEG), we included extra siRNA controls: one siRNA similar to siF12-A except for positions 9-11 of the siRNA that are replaced with its complementary base pairs (siF12-A(C9/11)), and a second siRNA against F12 (siF12-B). Three days after injection, a significant increase in TG peak height was observed solely for animals injected with siF12-A and siF12-A(C9/11), which is considered prothrombotic. As this change in coagulation was unrelated to FXII we conclude that it was off-target. For siRNA studies we now recommend to include mismatch siRNA controls, such as the C9/11 mismatch control used in this study, and to consider plasma coagulation in off-target analysis.