RNAi targeting heparin cofactor II promotes hemostasis in hemophilia A

Abstract

Hemophilia A is a hemorrhagic disease due to congenital deficiencies of coagulation factor VIII (FVIII). Studies show that hemophilia patients with anticoagulant deficiency present less severe hemorrhagic phenotypes. We aimed to find a new therapeutic option for hemophilia patients by RNA interference (RNAi) targeting heparin cofactor II (HCII), a critical anticoagulant protein inactivating the thrombin. The optimal small interfering RNA (siRNA) was conjugated to an asialoglycoprotein receptor ligand (N-acetylgalactosamine [GalNAc]-HCII), promoting targeted delivery to the liver. After administration, GalNAc-HCII demonstrated effective, dose-dependent, and persistent HCII inhibition. After 7 days, in normal mice, GalNAc-HCII reduced HCII levels to 25.04% ± 2.56%, 11.65% ± 2.41%, and 6.50% ± 1.73% with 2, 5, and 10 mg/kg GalNAc-HCII, respectively. The hemostatic ability of hemophilia mice in the GalNAc-HCII-treated group significantly improved, with low thrombus formation time in the carotid artery thrombosis models and short bleeding time in the tail-clipping assays. After repeated administration, the prolonged activated partial thromboplastin time (APTT) was reduced. A 30 mg/kg dose did not cause pathological thrombosis. Our study confirmed that GalNAc-HCII therapy is effective for treating hemophilia mice and can be considered a new option for treating hemophilia patients.

Keywords: RNA interference; hemophilia; hemostasis; heparin cofactor II; thrombin.

Graphical abstract

Figure 1

A simple description of the coagulation pathway, expounding the inactivation effect of HCII for thrombin with the help of DS An anti-coagulation pathway was also depicted with anticoagulants, including antithrombin (AT), protein C (PC), and protein S (PS).

Figure 2

Thrombin generation curves in HA plasma depleted of HCII activity to 9.7% in the background of DS with various levels (A–C) Thrombin generation conducted in the absence of DS (A), with DS at a concentration of 250 (B) or 25 μg/mL (C). Healthy control plasma was generated from the mixture plasma of 15 volunteers. Further control plasma was produced by adding back rhFVIII (1 IU/mL) to recover the thrombin generation to an average level. All measurements were run with 1 pM tissue factor and 4 mM phospholipids. The ordinate represented the alterations of thrombin levels over time.

Figure 3

The reduction effects of siRNA-HCII in cellular assays and the synthesis of GalNAc-modified siRNA-HCII (A and B) Analysis of mRNA transcript levels (A) and HCII protein levels (B) of HCII gene in Hepa 1–6 and Hepg2 cell lines transfected with siRNA-HCII or a control siRNA (siRNA-control) by Lipofectamine 3000 reagent. The housekeeping gene GAPDH was used to normalize the abundance of HCII mRNA in quantitative real-time PCR or as an internal control to normalize the gray values of HCII protein in western blot. Untreated hepatocytes were evaluated as control (mock). The graph bars represent the mean value ± SD in three independent experiments. (C) Structure diagram of GalNAc-HCII showing the siRNA-HCII conjugated with a GalNAc ligand. Also presented are the modifications of the nucleotides performed in this study, including 2′-O-methyl (2′-OMe) and 2′-deoxy-2′-fluoro (2′-F). Phosphorothioate (PS) linkages are used for the modifications of the backbone.

Figure 4

Pharmacokinetics and liver targeting of GalNAc-HCII in WT mice (A and B) Drug levels of GalNAc-HCII in plasma (A) and liver (B) after a single administration in WT mice at different dosages. Two mice were sacrificed in each dose group at each time point. All detection values were plotted, and those lower than the minimum quantitation were considered 0 μg/mL in plasma or 0 μg/g in the liver. (C) Fluorescence intensity of Cy3-labeled GalNAc-HCII (excited, 550 nm; visualized, 570 nm) in liver at 1, 2, 4, 12, and 24 h after a single administration with a dose of 1 mg/kg. The nucleus of tissue sections is incubated with DAPI (excited, 358 nm; visualized, 461 nm). WT mice treated with PBS were used as control. Scale bar, 50 μm.

Figure 5

Pharmacodynamics of GalNAc-HCII in WT mice (A) Dose-response and maintenance of HCII reduction after a single injection of GalNAc-HCII at different dosages. Plasma HCII levels were detected using an ELISA kit. Relative HCII protein levels at each time point for each mouse were determined by normalizing to HCII levels seen in PBS-treated WT mice. The graph bars represent the group mean (n = 4) ± SD. The black arrow below the vertical axis represented the time of dosing (0 days). (B) Stable maintenance of HCII reduction after repeated dosing of GalNAc-HCII at different dosages. Relative HCII protein levels at each time point for each mouse were determined by normalizing to HCII levels detected in PBS-treated WT mice. The graph bars represent the group mean (n = 4) ± SD. Black arrows below the vertical axis represented the time of dosing (0, 7, and 14 days).

Figure 6

Hemostatic effects of GalNAc-HCII in HA mice via carotid artery thrombosis models, tail-clipping assays, and measurements of APTT (A and B) Thrombus formation time of carotid artery in FeCl₃-induced thrombosis models (A) and bleeding time of tail artery in tail-clipping assays (B) on HA mice administered with GalNAc-HCII at different dosages (n = 5 for each dosage) or rhFVIII at a concentration of 100 IU/kg (n = 4). WT (n = 4) and HA mice (n = 4) are injected PBS as controls. Bars represent the group mean ± SD. A one-way ANOVA test followed by a multiple comparison test was used. ^ap < 0.05 versus WT mice with PBS; ^bp < 0.05 versus HA mice with PBS; ^cp < 0.05 versus HA mice with rhFVIII. (C) Alterations of APTT over time in HA mice after a single dosing of GalNAc-HCII with different doses or rhFVIII at 100 IU/kg. WT and HA mice with PBS were used as controls. Plasma from two mice was mixed to generate a plasma sample. Six mice were used for each group. Bars represent the group mean ± SD. (D) APTT correction in HA mice after three weekly injections of 2 or 5 mg/kg GalNAc-HCII for 2 weeks (n = 3 for each group). WT (n = 4) and HA mice (n = 4) with PBS were used as controls (35.7 ± 5.3 s for WT mice and 75.8 ± 4.9 s for HA mice). Bars represent the group mean ± SD. A one-way ANOVA test followed by a multiple comparison test was used. ^ap < 0.05 versus WT mice with PBS; ^bp < 0.05 versus HA mice with PBS.

Figure 7

Potential hepatotoxicity and thrombosis risk for GalNAc-HCII in HA mice (A) The toxicity effect on liver functions after repeated dosing of GalNAc-HCII or GalNAc-control at a concentration of 30 mg/kg in WT and HA mice (n = 6 for each group), including ALT, AST, T-BIL, D-BIL, and ALB. Bars represent the group mean ± SD. A one-way ANOVA test followed by a multiple comparison test was used. ^ap < 0.05 versus WT mice with PBS; ^bp < 0.05 versus HA mice with PBS. (B) H&E staining of liver sections prepared to explore the microscopic appearance of hepatotoxicity in WT and HA mice (n = 6 for each group) dosed with GalNAc-HCII or GalNAc-control at 30 mg/kg for six times. WT (n = 6) and HA mice (n = 6) with PBS were used as controls. The black arrow represented punctate hepatocyte necrosis with leukocyte infiltration, and the black triangle represented hepatocyte degeneration. (C) Thrombin generation curves in HA mice dosed with GalNAc-HCII at 30 mg/kg every 2 days for six times or 100 IU/kg rhFVIII once a day for 3 days after the last dosing of GalNAc-HCII. WT and HA mice with PBS were used as controls. Four mice were used for each group. (D) APTT detections in HA mice after frequent dosing of GalNAc-HCII at 30 mg/kg or rhFVIII at 100 IU/kg. WT and HA mice with PBS were used as controls. Four mice were used for each group. Bars represent the group mean ± SD. A one-way ANOVA test followed by a multiple comparison test was used. ^ap < 0.05 versus WT mice with PBS; ^bp < 0.05 versus HA mice with PBS.