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RNAi targeting heparin cofactor II promotes hemostasis in a canine model of acquired hemophilia A

Gene Therapy volume 32, pages 398–409 (2025)Cite this article

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Abstract

Heparin cofactor II (HCII) is a critical anticoagulant protein that inactivates thrombin. In our previous mouse studies, we demonstrated that GalNAc-HCII, a small interfering RNA (siRNA) targeting HCII conjugated with N-acetylgalactosamine (GalNAc), exhibited promising therapeutic effects in hemophilia A mouse models. Further evaluation in large animal models, especially with FVIII inhibitors, is essential before GalNAc-HCII can proceed to clinical trials. In this study, we successfully established, for the first time, an acquired hemophilia A canine model by multiple intravenous injections of a rabbit-dog chimeric neutralizing anti-canine FVIII antibody. In the control group, the Beagle dogs exhibited spontaneous bleeding symptoms accompanied by prolonged activated partial thromboplastin time (APTT). After administration, GalNAc-HCII (0.8 and 1.6 mg/kg) demonstrated potent, dose-dependent, and durable HCII inhibitory effects. After 5 days, in normal dogs, GalNAc-HCII reduced HCII levels to 32.67% ± 3.07% and 10.62% ± 1.74% with 0.8 and 1.6 mg/kg GalNAc-HCII, respectively. In hemophilic dogs, GalNAc-HCII treatment significantly improved hemostatic function. Specifically, in the carotid artery thrombosis model, the thrombus formation time was shortened [29.7 ± 2.08 min (0.8 mg/kg) and 18.0 ± 1.0 min (1.6 mg/kg) vs. 40 min (control), P < 0.01]; in the knee joint puncture-induced bleeding model, joint bleeding and synovitis were alleviated; and in the saphenous vein bleeding model, the number of hemostatic events increased. Furthermore, repeated administration of GalNAc-HCII effectively reduced the prolonged APTT. This study demonstrates the efficacy of GalNAc-HCII in hemophilic dogs, suggesting it as a promising novel therapeutic option for patients with hemophilia, including those with FVIII inhibitors.

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Fig. 1: Thrombin generation curves in HA plasma depleted of HCII activity to the indicated levels in the background of 25 μg/ml DS.
Fig. 2: Efficacy of siRNA-HCII in cellular systems and its GalNAc-conjugated chemical modifications.
Fig. 3: Pharmacokinetic profiling and hepatic targeting of GalNAc-HCII in canine models.
Fig. 4: Pharmacodynamic profiling of GalNAc-HCII in canine models.
Fig. 5: Functional in vitro assay of the rabbit-dog chimeric anti-FVIII neutralizing antibody chVIII-6E3.
Fig. 6: Hemostatic effects of GalNAc-HCII in a dog model of acquired hemophilia A via measurements of APTT.
Fig. 7: Hemostatic effects of GalNAc-HCII in HA dogs via carotid artery thrombosis models, knee joint needle-puncture-induced bleeding models, and saphenous vein bleeding models.
Fig. 8: Potential hepatotoxicity and thrombosis risk for GalNAc-HCII in HA dogs.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This work was supported by the National Natural Science Foundation of China (Nos. 82170131 and 82470134), the Chang Jiang Scholars Program (No. 2022161), the Young Top-notch Talent Cultivation Program of Hubei Province 202117, and the National Key R&D Program of China (Nos. 2022YFC2304600 and 2023YFC2509500).

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  1. These authors contributed equally: Yuyang Zhang, Tingting Liu.

Authors and Affiliations

  1. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Yuyang Zhang, Tingting Liu, Haiming Kou, Huafang Wang, Yu Hu & Liang V. Tang

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Contributions

LT and YH conceived and designed the study. YZ, HK, and TL collected and assembled the data. YZ, HW, HK, and LT analyzed and verified the data. YZ, LT, HK, and YH verified and interpreted the data. All authors wrote and approved of the article and are accountable for publication.

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Correspondence to Liang V. Tang.

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The authors declare no competing interests.

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We have received informed consent from all subjects. This study was reviewed and approved by the Medical Ethical Committee and the Animal Ethics Committee of Union Hospital, Huazhong University of Science and Technology. [2024] IACUC Number: 4357. All methods were performed in accordance with the relevant guidelines and regulations.

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Zhang, Y., Liu, T., Kou, H. et al. RNAi targeting heparin cofactor II promotes hemostasis in a canine model of acquired hemophilia A. Gene Ther 32, 398–409 (2025). https://doi.org/10.1038/s41434-025-00541-w

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