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The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection

Gene Therapy (2024)Cite this article

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Abstract

Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.

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Fig. 1: GAPDH mRNA expression in HuAoSMCs at 24 h in the setting of different anticoagulants.
Fig. 2: Heparin dose effect on GAPDH mRNA expression in HuAoSMCs at 24 h.
Fig. 3: Donor variation in GAPDH mRNA expression in HuAoSMCs at 24 h.
Fig. 4: B2M mRNA expression in HuAoSMCs at 24 h in the setting of different anticoagulants.
Fig. 5: SiGLO Transfection in HuAoSMCs at 24 h.
Fig. 6: B2M mRNA expression in HuAoSMCs at 48 h in the setting of different anticoagulants.
Fig. 7: GAPDH mRNA expression in HuCoECs at 24 h in the setting of different anticoagulants.
Fig. 8: GAPDH protein expresion in HuAoSMCs at 60 h.
Fig. 9: Heparin dissociation assay.

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Acknowledgements

We thank Dr. Christiane Ferran and Dr. Cleide Angolano at the Center for Vascular Biology Research at Beth Israel Deaconess Medical Center/Harvard Medical School for their assistance with the generation of the fluorescence images, technical support in qt-PCR, and assistance with developing the research design.

Funding

This work has been supported by grants from The National Heart, Lung, and Blood Institute of the National Institutes of Health under award number R01HL021796, R01HL086741, 5T32HL007734, and 5T35HL110843.

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  1. Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Lucas Mota, Max Zhu, John N. Tomeo, Melina Recarey, Nyah Patel, Leena Pradhan-Nabzdyk, Frank W. LoGerfo & Patric Liang

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Contributions

LM, MZ, JNT, MR, LPN, FWL and PL contributed to the conception of the study. LM, MZ, LPN, NP, FWL and PL contributed to the design of the experiments. LM, MZ, JNT, and MR contributed to maintaining SMC culture. LM, MZ, JNT and MR contributed to the siRNA transfection agent solution creation, in vitro transfection, sample analysis and data generation. LM, MR, and NP contributed to siGLO experiments and IF imaging. LM, JNT, and NP contributed to protein isolation and western blot experiment. LM, NP, LPN, FWL and PL contributed to the supervision of the studies and data review. All authors reviewed and edited the manuscript and provided approval on the final submitted version.

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Correspondence to Patric Liang.

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Ethical approval was not required as we used cell lines for the in vitro experiments and no primary tissues or primary cell cultures were used.

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Mota, L., Zhu, M., Tomeo, J.N. et al. The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection. Gene Ther (2024). https://doi.org/10.1038/s41434-024-00460-2

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