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

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.