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Use of "C9/11 Mismatch" Control siRNA Reveals Sequence-Related Off-Target Effect on Coagulation of an siRNA Targeting
Marco Heestermans1,2, Annika de Jong1,2, Sander van Tilburg1,2
11Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands.
Abstract:
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-AC9/11), 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-AC9/11, 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.
Insights
Small interfering RNA targeting coagulation factor XII (siF12) unexpectedly increased blood clot formation. This prothrombotic response was found to be an off-target effect, not related to F12 inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Small interfering RNA (siRNA) targeting coagulation factor XII (siF12) was previously reported to cause a prothrombotic response.
- The mechanism behind this unexpected response requires further investigation.
Purpose of the Study:
- To investigate the prothrombotic effect of siF12-A on plasma coagulation.
- To elucidate the off-target mechanisms of siRNA in coagulation studies.
Main Methods:
- Evaluated thrombin generation (TG) in mice injected with siF12-A, a mismatch control (siF12-A^C9/11), a second F12 siRNA (siF12-B), and a negative control (siNEG).
- Assessed coagulation parameters three days post-injection.
Main Results:
- A significant increase in TG peak height, indicative of a prothrombotic state, was observed in animals treated with siF12-A and siF12-A^C9/11.
- The observed prothrombotic effect was independent of coagulation factor XII (F12).
Conclusions:
- The prothrombotic response induced by siF12-A is an off-target effect.
- Mismatch controls, like the C9/11 variant, are crucial for accurate siRNA studies.
- Plasma coagulation assessment should be integrated into off-target analyses for siRNA research.