RNAi Targeting Heparin Cofactor ? Promotes Hemostasis In Hemophilia

Hemophilia is a hemorrhagic disease due to congenital deficiencies of coagulation factors VIII(FVIII)or IX(FIX)[1].Studies show that hemophilia patients with anticoagulant deficiency present less severe hemorrhagic phenotypes[41,42].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 siRNA was conjugated to an N-acetylgalactosamine(Gal NAc-HCII),promoting targeted delivery to the liver.After a single administration,Gal NAc-HCII demonstrated effective,dose-dependent,and persistent HCII inhibition in wild-type(WT)mice.After 7 days of dosing,Gal NAc-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 Gal NAc-HCII,respectively.The hemostatic ability of hemophilia mice in the Gal NAc-HCII treated group significantly improved and was dose-dependent,with low thrombus formation time in the carotid artery thrombosis models and short bleeding time in the tail-clipping assays.Gal NAc-HCII recovered the hemostatic ability of hemophilia mice to that of normal mice at a dose of 10 mg/kg.Although still different from WT mice,the activated partial thromboplastin time(APTT)of hemophilia mice was significantly reduced in Gal NAc-HCII treated group,which lasted longer than the recombinant human FVIII(rh FVIII)replacement treated group.In addition,30 mg/kg dose did not increase the potential risk of thrombosis and no pathological thrombosis was found in hemophilia mice,even combined with rh FVIII therapy.Our study confirmed that Gal NAc-HCII therapy is effective and safe for treating hemophilia mice and can be considered a new option for treating hemophilia patients.Part I The effect of HCII neutralization on thrombin generation in vitroObjective: To observe the effect of HCII inhibition on plasma thrombin generation in vitro.Methods: The depletion of HCII in vitro was performed by adding HCII neutralizing antibody to the FVIII-deficient plasma;the HCII activity level was detected by using chromogenic substrate method;thrombin generation assay was used to evaluate thrombin levels in HCII inhibited plasma.Results: The activity of HCII was significantly reduced to 9.7±0.8% in a ratio of 1:9(neutralizing antibody vs plasma);in the absence of DS,HCII inhibition had little effect on the thrombin generation in the FVIII deficient plasma,while the thrombin level was improved in the HCII-reduced group with DS;it demonstrated an additive thrombin generation response to 1 IU/ml rh FVIII in the background of reduced HCII but not surpassed that of healthy control.Conclusion: In the presence of DS,HCII inhibition could significantly promote the thrombin generation levels in hemophilia plasma;the potential risk of thrombosis,caused by thrombin overproduction,might be low in the HCII-reduced plasma even with the rh FVIII.Part II The selection of siRNA targeting liver HCII geneObjective: To screen out the optimal siRNA to specifically knock down HCII protein in human and mouse hepatocytes.Methods: siRNA,targeting the common sequence of human and mouse HCII m RNA,was designed and transfected into hepatocytes cell lines(mouse: Hepa1-6;human: Hepg2)by lipofectamine3000;total RNA was extracted using the Trizol reagent and the relative abundance of HCII m RNA normalized to the housekeeping gene GAPDH was detected by real-time quantitative PCR;total protein was extracted by RIPA lysis buffer and protein levels of HCII relative to the internal control(GAPDH)was determined by Western blot.Results: The optimal siRNA(siRNA-HCII,5?-UGGUGGAGAGAUGGCAAAAAA-3?)was screened out.HCII m RNA levels were significantly reduced in the siRNA-HCII treated group,about 6.65±1.61% and 6.17±4.50% relative to the siRNA-control treated group in Hepa1-6 or Hepg2 cells;HCII protein levels were reduced to 10.06±6.89% and 13.38±9.04% with siRNA-HCII in Hepa1-6 and Hepg2 cell lines.Conclusion: siRNA-HCII effectively reduced the expressions of HCII in mouse and human hepatocytes.Part III Pharmacokinetics and pharmacodynamics of siRNA targeting HCII in miceObjective: To observe the pharmacokinetics and pharmacodynamics of siRNA-HCII in mice.Methods: siRNA-HCII was covalently conjugated to N-acetylgalactosamine(Gal NAc)to generate Gal NAc-HCII;C57BL / 6 mice were subcutaneously dosed Gal NAc-HCII labeled with Cy3 dye,and the fluorescence intensity of frozen sections of tissue samples was observed by a fluorescence microscope;C57BL / 6 mice were subcutaneously administered Gal NAc-HCII at 2,5,or 10 mg/kg,and drug levels in plasma and liver at different time point were detected via stem-loop q RT-PCR assays;C57BL / 6 mice were subcutaneously administered Gal NAc-HCII at different doses for once or repeatedly,and the HCII antigen levels of plasma at different time point were detected by enzyme-linked immunosorbent assay(ELISA).Results: The drug specifically targeted the liver with utmost fluorescence intensity emerging at 4 h after dosing,and no drug accumulation was observed in other organs;drug levels of Gal NAc-HCII were predominantly dependent on the administered dosage and time point,and reached the peak concentrations in plasma and liver at 30 min or 4 h after administration respectively;dose-dependent inhibition of HCII protein levels was observed in Gal NAc-HCII treated mice with the lowest HCII level appearing at 7 d after a single dosing,and when dosed repeatedly,HCII protein decreased to stable levels(~ 20%)after 2 weeks of administration.Conclusion: Gal NAc-HCII had a significant liver targeting effect and could be maintained for a long time in mice to effectively inhibit the HCII levels of plasma and liver.Part IV The effect of HCII inhibition on the hemostatic capacity of miceObjective: To observe the effect of Gal NAc-HCII on the hemostatic capacity of hemophilia mice.Methods: Hemophilia mice were subcutaneously dosed Gal NAc-HCII at 2,5 or 10 mg/kg,and the thrombus formation time was evaluated using an acute injury model of carotid artery induced by Fe Cl3;tail-clipping assay was used to evaluate the bleeding time of mice;blood samples were collected to detect activated partial thromboplastin(APTT)at different time points after a single injection;blood samples were collected to detect APTT to evaluate the effect of multiple administration after repeated administration subcutaneously with Gal NAc-HCII at different doses.Results: Gal NAc-HCII significantly shortened the thrombus formation time of carotid artery and bleeding time of tail artery in mice in a dose-dependent manner,and the hemostatic ability of mice was restored to the normal level at the dose of 10mg/kg;Gal NAc-HCII significantly reduced APTT,which was still different from the normal level even after repeated dosing;compared with rh FVIII treated group,the maintenance of APTT was more durable in Gal NAc-HCII dosed mice.Conclusion: Gal NAc-HCII significantly improved the hemostatic ability of hemophilia mice,especially for traumatic bleeding.Part V Toxicity studies of siRNA targeting HCIIObjective: To study the toxicity of Gal NAc-HCII in hemophilia mice.Methods: Hemophilia mice were subcutaneously administered Gal NAc-HCII every 2 days at 30 mg/kg for 6 times;the clinical manifestations,such as weight,diet,activity,limbs and survival,were observed in administration duration;plasma of mice was collected to detect APTT,complete blood count(CBC),liver and kidney function,myocardial enzyme and thrombin generation;the tissues and organs of mice were collected to evaluate the size,shape,and color;pathological sections of mice were obtained to evaluate the cytotoxicity of Gal NAc-HCII.Results: Mice well tolerated Gal NAc-HCII with no obvious abnormality in diet and activity;the slight elevation of liver function indexes was observed and histopathological sections indicated punctate hepatocyte necrosis in the focal perivascular;no abnormalities was observed in the size and shape of tissues and no pathological thrombosis was found;Gal NAc-HCII promoted the generation of thrombin and significantly reduced APTT,both did not surpassed levels of normal mice,even combined with rh FVIII.Conclusion: Gal NAc-HCII showed hepatotoxicity,but did not increase the potential risk of thrombosis in hemophilia mice.