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 (si F12 ) 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 si F12 (here, si F12 -A) on plasma coagulation through thrombin generation (TG). Besides a routine negative control siRNA (siNEG), we included extra siRNA controls: one siRNA similar to si F12-A except for positions 9-11 of the siRNA that are replaced with its complementary base pairs (si F12 -A ^C9/11 ), and a second siRNA against F12 (si F12 -B). Three days after injection, a significant increase in TG peak height was observed solely for animals injected with si F12 -A and si F12 -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.