Extravascular factor IX pool fed by prophylaxis is a true hemostatic barrier against bleeding.

BACKGROUND: Factor (F)IX can bind to type IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. OBJECTIVES: The aim of the study was to evaluate the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds. METHODS: The capacity of 4 different FIX molecules (plasma-derived and recombinant) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knockout mice. RESULTS: A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50 to 100 IU/kg, while plasma-derived FIX generated a FIX signal at all doses, including 50 IU/kg. Such a signal was also detected after five 100 IU/kg daily infusions of recombinant FIX, suggesting that FIX can accumulate in the extravascular space during prophylaxis. The extravascular procoagulant activity of FIX, assessed in saphenous vein bleeding assays, was significantly higher in hemophilia B mice after these 5 days of prophylaxis compared to a single infusion of 100 IU/kg of FIX and assessment of FIX activity 7 days later. CONCLUSION: Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection toward bleeding.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

INTRODUCTION AND AIM: Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5 IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. MATERIALS AND METHODS: Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. RESULTS AND CONCLUSION: We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

In vivo intranasal delivery of coagulation factor IX: a proof-of-concept study.

BACKGROUND: Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB is prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) with standard half-life or extended half-life. Unmet needs remain regarding the development of non-invasive administration routes for coagulation factors. The aim of this study was to evaluate the effectiveness of intranasal delivery (IND) of rFIX and rFIX fused to Fc fragment (rFIX-Fc) in mice. METHODS: Drops of rFIX and rFIX-Fc were deposited in the nostrils of wild-type, FcRn knock-out, FcRn humanized, and FIX knock-out mice. rFIX mucosal uptake was evaluated by measuring plasma FIX antigen and FIX activity (FIX:C) levels, and by performing histologic analysis of the nasal mucosa following IND. RESULTS: After IND, both rFIX and rFIX-Fc were equally delivered to the blood compartment, irrespective of the mouse strain studied, mostly through a passive mechanism of transportation across the mucosal barrier, independent of FcRn receptor. Both plasma FIX antigen and FIX:C activity levels increased following IND in FIX knock-out mice. CONCLUSION: This proof-of-concept study describes evidence supporting the nasal route as an alternative to FIX i.v. infusion for the treatment of HB.

Haemostatic effect of adding tranexamic acid to emicizumab prophylaxis in severe haemophilia A: A preclinical study.

BACKGROUND: Patients with severe haemophilia have impaired haemostatic response, delayed clot formation and fibrin clots that are vulnerable to fibrinolysis. Emicizumab is a bispecific antibody that mimics activity of activated factor VIII (FVIII) and increases haemostatic capacity to the level of moderate-to-mild haemophilia, thereby used for prophylaxis. Regardless of the impressive clinical performance of emicizumab, breakthrough bleeds may still occur. We aimed to study, in FVIII knockout mice (FVIII-KO), whether haemostasis is improved with the addition of tranexamic acid (TxAc) to emicizumab. METHODS: FVIII-KO mice received prophylaxis with emicizumab or emicizumab+TxAc before trauma. FVIII-KO mice were given emicizumab 1.5 mg/kg via IV injection. A second retro-orbital IV injection containing human FIX and FX (both 100U/kg) was given 24 h later and 5 min before the tail amputation or knee trauma. After trauma-induced knee joint bleeding, magnetic resonance imaging (MRI), and histological analysis were used to compare haemostatic efficacy of the two prophylactic strategies. Thrombin generation (TG) was measured and clots obtained with TG experiment were analysed by scanning electron microscopy. RESULTS: In FVIII-KO mice, blood loss after tail clip was lower after prophylaxis with emicizumab+TxAc compared to emicizumab. MRI results and histological analysis of knee joints showed that the addition of TxAc significantly decreased joint bleeding. Fibrin fibre diameters of mice treated with emicizumab only was thicker than those who received combined prophylaxis with emicizumab+TxAc. CONCLUSION: Our results suggest a potential benefit of TxAc when used in combination with emicizumab in prophylactic settings, especially in patients presenting breakthrough bleeds.

Hemostatic effect of tranexamic acid combined with factor VIII concentrate in prophylactic setting in severe hemophilia A: A preclinical study.

BACKGROUND: Hemophilia is characterized by a compromised hemostatic response with delayed development of a clot and the formation of clots that are vulnerable to fibrinolysis. We proposed to study, in vitro and in factor VIII knockout mice (FVIII-KO), whether hemostasis is improved with the addition of tranexamic acid (TXA) to low FVIII plasma concentrations. METHODS: In vitro, blood samples from adults with severe hemophilia-A, spiked to final concentrations of 0-3-10 and 30IU.dL-1 of FVIII, were studied with and without TXA 0.1 mg/mL using thromboelastography in the presence of tPA (ROTEM-tPA), thrombin generation (TG) assay, and scanning electron microscopy. FVIII-KO mice received prophylaxis before trauma, to obtain circulating plasma FVIII at 3 IU.dL-1 or FVIII 3IU.dL-1  + TXA 0.1 mg/mL. After trauma-induced knee joint bleeding, magnetic resonance imaging, histological analysis, and tail clip assay were used to compare hemostastic efficacy of the two prophylactic strategies. RESULTS: A dose-dependent improvement of TG was observed with recombinant FVIII (rFVIII) alone (P = .024). As expected, no effect of TXA on TG capacity was observed. Fibrin fiber diameters were significantly decreased with TXA + rFVIII compared to rFVIII, suggesting a stronger fibrin network. Surprisingly, ROTEM-tPA was normalized with TXA alone. In FVIII-KO mice, blood loss after tail clip was lower after prophylaxis with rFVIII + TXA compared to rFVIII, with no statistical significance (P = .15). However, MRI results and histological analysis of knee joints showed that the addition of TXA significantly decreased joint bleeding (P = .022). CONCLUSION: Our results suggest a potential benefit of TXA when used in combination with FVIII in prophylactic settings.

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice.

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins.

INTRODUCTION: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV-based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half-life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). AIM: Adeno-associated viral vectors (AAV) mediating expression of hFIX-Alb and hFIX-Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half-life translates to higher plasma levels of FIX. METHODS: Single-stranded cross-packaged AAV2/8 vectors expressing hFIX-Alb, hFIX-Fc and hFIX were evaluated in vitro, and in mice. RESULTS: Both hFIX-Alb and hFIX-Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV-mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX-fusion proteins were comparable to wild-type hFIX. However, their expression levels were threefold lower than wild-type hFIX in vivo most likely due to inefficient secretion. CONCLUSION: This, the first, evaluation of hFIX-fusion proteins in the context of AAV gene transfer suggests that the hFIX-fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

Fusion of Factor IX to Factor XIII-B Sub-Unit Improves the Pharmacokinetic Profile of Factor IX.

Prophylaxis is currently considered the optimal care for severe haemophilia. For patients and their families one of the major difficulties with prophylaxis is the need for frequent venipunctures. The half-life of standard factor IX (FIX) concentrates is approximately 18 hours, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention in patients with severe haemophilia B. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life recombinant FIX (rFIX) concentrates have been developed. We designed a new rFIX molecule fused to coagulation FXIII-B sub-unit. This sub-unit is responsible for the long half-life of the FXIII molecule (10-12 days). The rFIX-LXa-FXIIIB fusion protein contains a short linker sequence cleavable by activated FX (FXa), to separate rFIX from the carrier protein as soon as traces of FXa are generated, leaving rFIX free to perform its enzymatic role in the tenase complex. The rFIX-LXa-FXIIIB fusion protein was expressed in human hepatic Huh-7 cells and Chinese hamster ovary cells, and both wild-type rFIX (rFIX-WT) and rFIX-LXa-FXIIIB showed similar clotting activity and thrombin generation capacity in vivo after injection in haemophilia B mice compared with rFIX-WT. The half-life of the rFIX-LXa-FXIIIB molecule in WT mice and rats was 3.9- and 2.2-fold longer, respectively, compared with rFIX-WT. A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIII-B, which might accelerate fibrin clot formation and thus improve haemostatic capacity of the molecule.

Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

BACKGROUND: Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail. OBJECTIVE: We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation. METHODS: Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C

Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity.

INTRODUCTION: Hemophilia B is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). The main treatment for hemophilia B is replacement therapy using FIX concentrates. Prophylactic treatment in severe hemophilia B is very effective but is limited by cost issues. Production of a recombinant FIX (rFIX) with enhanced clotting activity, offering the possibility of fewer infusions and fewer costs with similar efficacy, is one of the current challenges for hemophilia B treatment. The present study focused on an important amino acid sequence known to be involved in the interaction of activated FIX (FIXa) with its cofactor, activated factor VIII (FVIIIa). MATERIALS AND METHODS: Using site-directed mutagenesis of glutamate E410 (c240, chymotrypsin numbering), four recombinant FIX-E410 (E410H, A, L and N) mutants were developed and produced by the human hepatoma cell line Huh-7. RESULTS: The in-vitro clotting activity of mutant FIX molecules was 3 to 5-fold higher than wild-type recombinant FIX (FIX-WT). FIX-E410H compound showed the highest in-vitro procoagulant activity. Enhanced specific activity was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In hemophilia B mice, we observed significantly higher in-vivo clotting activity and thrombin generating capacity with FIX-E410H compared to FIX-WT. We demonstrated that increased procoagulant activity of FIX-E410H was mainly explained by 2.5- fold enhanced affinity of the mutant for human FVIIIa. CONCLUSION: We have engineered and characterized four improved FIX proteins with enhanced in-vitro and in-vivo activity. Future studies are required to evaluate the immunogenicity of FIX-E410.

Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications.

Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.

B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways.

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization.

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.