Enhancing hemostasis potency in hemophilia with a small interfering RNA targeting protein S.

Abstract

Background: One hemophilia treatment concept focuses on rebalancing coagulation and anticoagulation to restore normal blood clotting. Targeting the coagulation regulator, protein S (PS), in hemophilia shows promise to increase the generation of thrombin-a critical enzyme in the clotting process.

Objectives: This study aimed to: (1) assess whether inhibiting PS increases thrombin generation (TG) in plasma from individuals with hemophilia A (HA) and B (HB); and (2) develop a hepatocyte-targeted PS-silencing RNA (siRNA) therapy using N-acetylgalactosamine (GalNAc) conjugation to restore hemostasis in hemophilia without increasing thromboembolic risks.

Methods: We assessed TG in plasma from patients with HA and HB. To target the liver specifically, we developed a PS-siRNA conjugated with GalNAc. This approach ensures that PS levels remain adequate in other cells, thereby minimizing the risk of thrombosis. Additionally, we evaluated the therapeutic potential of PS-siRNA in preclinical models.

Results: Inhibiting PS with a polyclonal antibody in plasma resulted in a 3-5 fold increase in TG in HA and a 4-9 fold increase in HB plasma, with a 70% reduction in plasma PS. In preclinical models, subcutaneous PS-siRNA therapy in HA mice and non-human primates successfully lowered PS levels and improved clot formation. It also prevented bleeding in both saphenous vein puncture and knee-injury models in HA mice. Notably, it enhanced clotting without triggering widespread clot formation.

Conclusions: Reducing PS levels enhances TG in hemophilia models, and PS-siRNA therapy shows promise in improving hemostasis. This approach warrants further clinical investigation as a potential treatment for hemophilia.