Hemophilic Mice Research Articles

The Tissue Factor Pathway Inhibitor Gamma Knockout Mouse Is a Better Model Than the Wild Type Mouse for Studying Tissue Factor-Related Human Diseases

BACKGROUND. Tissue factor (TF) is critical for hemostasis. However, TF also plays a pathogenic role in a multitude of diseases, including: thrombosis, coagulation-associated inflammation, multi-organ failure associated with infections, tumor growth, angiogenesis and metastasis. Tissue Factor Pathway Inhibitor (TFPI), the regulator of TF actions, produces FXa-dependent feedback inhibition of the FVIIa/TF catalytic complex, directly inhibits FXa, and suppresses cellular signaling by FVIIa/TF/FXa complexes. In man, TFPI is expressed as isoforms TFPIα and TFPIβ. The pathophysiologic impacts of the individual α and β isoforms have yet to be elucidated. Mouse models have been used extensively to provide insight into human physiology and disease, including abnormalities concerning hemostasis and TF-associated diseases. Like man, mice express TFPI α and β, but unlike man, mice express a third transcript encoding TFPIγ. In fact, we recently reported that the vast majority (98%) of TFPI in mouse plasma is derived from TFPIγ. We also reported that anti-TFPI antibodies enhance in vitro thrombin generation in hemophilia mouse plasma that contains only the TFPIγ isoform, which demonstrated that TFPIγ possesses anticoagulant activity. RESULTS. We report herein that, while complete TFPI gene deletion is 100% embryonic lethal, TFPIγ only mice [TFPIαβ(-/-)] are viable and reproduce. Offspring genotypes from parents with intact TFPIγ(+/+) exons and TFPIαβ(+/-) were 33 TFPIαβ(+/+), 57 TFPIαβ(+/-) and 34 TFPIαβ(-/-) at weaning. Also, TFPIγ only mice produced similar numbers of pups per litter as wild type mice. The viability of TFPIγ only mice proves endogenous mouse TFPIγ possesses substantial functional activity. TFPIα is considered to be a much more potent anticoagulant than TFPIγ. Our preliminary thrombin generation assay data suggests that the sum total anticoagulant activity of the endogenous low level (0.07 nM) of TFPIα is similar to the sum total anticoagulant activity of the endogenous high level (3.5 nM) of TFPIγ. The presence of active TFPIγ in mice, not found in man, confounds attempts to translate results from wild type mouse studies to the human condition. The extents to which TFPIγ influences the various wild type mouse models of human diseases are unknown. The presence of mouse TFPIγ also precludes our ability to properly distinguish the roles of TFPI α and β isoforms independently. Deletion of the TFPIγ exon eliminates this confounder. Fortunately, in TFPIαβ mice the levels, properties and location of TFPIα and TFPIβ in mice reflect those in man. Importantly, we can now report that our CRISPR-Cas9-generated TFPIγ KO strains - TFPIαβ mice, TFPIα only mice, and TFPIβ only mice are viable and reproduce. To illustrate the potential influence of TFPIγ in a mouse model, we used a tail vein-rebleeding assay to assess bleeding in TFPI isoform-specific mice in a FVIII KO background. Reported as mean number of clots per 15 minutes, results were: 4.8 for hemophilia controls; 25.4 for controls with FVIII replacement; 4.2 for TFPIα KOs; and 13.5 for TFPIβγ KOs. (The S.E.M. of each group was <1.0 ). We find that deletion of TFPIα (including in platelets!) does not affect bleeding (P>0.5 vs controls), whereas deletion of TFPIβ and γ improves hemostasis ( P<0.001 vs controls). In hemophilia mice, suppressing TFPIα was without effect. We don't expect this observation to translate to man. Rather, suppressing TFPIα in man is anticipated to enhance coagulation in hemophilia patients. (Note, multiple trials show that TFPI antibodies that inhibit all TFPI forms enhance coagulation). Moreover, in the absence of the confounding influence of TFPIγ, we anticipate suppression of TFPIα will improve coagulation and reduce bleeding in TFPIγ deleted hemophilia mice. Further studies in our TFPIγ deleted strains are necessary to determine if this prediction is correct and could influence future anti-TFPI approaches for man. CONCLUSIONS. 1.) Endogenous mouse TFPIγ possesses significant functional activity. 2.) TFPIγ confounds the extrapolation of results in wild type mouse models to human diseases. 3.) The TFPIγ KO mouse possesses a TFPIαβ profile that is similar to man. 4.) For essentially all studies of human TF-associated diseases in mouse models, it would be prudent to delete TFPIγ. Disclosures No relevant conflicts of interest to declare.

Next-Generation FVIII Mimetic Shows Superior Effect in a FIX- and Fx-Humanized Mouse Model In Vivo

Next-Generation FVIII Mimetic Shows Superior Effect in a FIX- and Fx-Humanized Mouse Model In Vivo

Adeno-Associated Virus Mediated Delivery of a TFPI Antibody Restores Hemostasis in Mouse Model of Hemophilia

Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment of an active bleed. Some patients develop inhibitory antibodies making them refractory to replacement therapies. Although hemophilic patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. Augmenting the extrinsic tissue factor pathway is an attractive alternate approach to maintain hemostasis in hemophilic patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating protease functions of Factor Xa and Factor VIIa/Tissue Factor complex and as such, TFPI inhibition has been explored as an innovative option to restore thrombin generation. Previously, we demonstrated that antibody mediated TFPI inhibition restores hemostasis in mouse models of hemophilia (Jasuja et al, 2016). Here we investigated the impact of an adeno-associated virus (AAV) vector expressed anti-TFPI antibody in correcting bleeding disorders in hemophilic mice as a novel approach to restore hemostasis. An AAV vector expressing heavy and light chains of a neutralizing anti-TFPI monoclonal antibody was designed and constructed. In vitro evaluations confirmed that vector expressed antibody was functional using a dilute Prothrombin Time (dPT) assay. Next, male hemophilic A mice were systemically administered (intravenous) 1x1011 AAV vector genomes (vg) encoding anti-TFPI or control and had hemostasis evaluated 16 days post-treatment. Following recalcification of whole blood, thromboelastography (TEG) was used to measure clot formation times (R value), speed of clot formation (K value), as well as maximum amplitude. In hemophilia A mice treated with AAV-anti-TFPI, clot formation time (R value) was normalized to wild type levels, with a 10-fold reduction compared to hemophilia A mice administered a control Green fluorescent protein (GFP) encoding AAV vector. Mice administered the control AAV-GFP vector failed to correct additional parameters, including speed of clot formation (K value), angle and maximum amplitude, all of which remained similar to untreated hemophilia A mice. In contrast, for AAV-anti-TFPI dosed Hem A mice, all of these parameters were in the normal range similar to wild type mice. Our data demonstrate AAV mediated delivery of anti-TFPI antibody corrects coagulation abnormalities observed in a mouse model of Hemophilia A. Disclosures Rakhe: Pfizer Inc.: Employment. Jasuja:Pfizer Inc.: Employment. Shelke:Pfizer Inc.: Employment. Sawant:Pfizer Inc.: Employment. Somanathan:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment.

Prevention of Bleeding-Induced Vascular Abnormalities in the Hemophilic Mouse Joint By Increasing TAFI Levels or Inhibiting uPA Activity

Hemophilic arthropathy is characterized by vascular abnormalities in the joint soft tissue that may cause rebleeding and progressive joint destruction. Defective thrombin generation in hemophilia leads to impaired activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI). Activated TAFI (TAFIa) is a carboxypeptidase that cleaves C-terminal Lysine from fibrin or cell plasminogen receptors, thereby diminishing fibrinolysis or pericellular plasmin generation. In addition, TAFIa inactivates pro-inflammatory (i.e. C3a, C5a, bradykinin) and anti-angiogenic (i.e. VEGF-A, SDF-1α) substrates. We have recently shown that TAFI-gene deficiency causes vascular abnormalities in the mouse joints that phenocopy those in hemophilic arthropathy, either after joint bleeding or spontaneously with aging. Based on these mechanistic insights, we evaluated treatment with TAFI for its ability to mitigate bleeding-induced vascular abnormalities and associated bleeding susceptibility in the FVIII KO mouse joint. Knee joint bleeding in FVIII KO mice was induced by a subpatellar puncture injury that was standardized to cause severe bleeding as defined by a hematocrit (Hct) drop from 47 ± 2 % at baseline (BL) to 29 ± 11 % two days after injury. The vascular abnormalities formed in the hemophilic mouse joint 2 weeks after injury capture those in patients with hemophilic arthropathy: i.e. increased vascular permeability and abnormal vessel enlargement. Hydrodynamic gene delivery was used in mice for continuous treatment with human wild type TAFI (hTAFI-WT). To compensate for lower TAFI activation in hemophilia, two TAFI mutants were tested with extended enzymatic stability: hTAFI-CYQ (8-fold increased) and hTAFI-CIIYQ (180-fold increased). The TAFI constructs had comparable antigen expression profiles (reaching a similar level as in human plasma at day 2 and gradually returning to BL by week 2). Treatment efficacy to reduce vascular permeability, however, differed according to TAFIa's enzymatic stability in FVIII KO mice 2 weeks after injury. Without treatment, Evans blue-BSA extravasation in the joints was increased (1.8-fold compared to the contralateral joint; p= 0.0001 vs BL). hTAFI-WT overexpression initiated 2 days after injury had no effect, whereas hTAFI-CYQ and hTAFI-CIIYQ overexpression reduced the vascular permeability to 1.5-fold (n= 10; p= 0.10 vs untreated) and 1.3-fold (n= 8; p= 0.0032 vs untreated), respectively, indicating that hTAFI-CIIYQ was most effective. Histology at week 2 confirmed that hTAFI-CIIYQ reduced the number of excessively enlarged CD31+ vessels (with ≥ 20 µm diameter) by 3-fold (n= 6; p= 0.0011 vs untreated). To determine whether the protective effect of TAFI overexpression on bleeding-induced vascular abnormalities was associated with inhibition of the plasminogen activation system, continuous administration of tranexamic acid (TXA; 50 mg/ml in drinking water) or antibody-mediated blockage of uPA's proteolytic activity (mU1; 60 mg/kg; 2x weekly) was initiated in FVIII KO mice 2 days after injury. Histology at week 2 showed mU1, but not TXA, reduced vessel enlargement to the same extent as hTAFI-CIIYQ. Finally, the joint bleeding model was adopted to appreciate functional consequences of hypervascularity to changes in bleeding susceptibility. Aged TAFI KO mice (8 months) were used for their spontaneously (3-fold) increased number of enlarged vessels (≥ 40 µm diameter; p= 0.0076) in the joint that correlates with Power Doppler (PD) signal. A mild injury was optimized to cause minimal bleeding and showed that PD signals in joints of aged TAFI KO mice correlated negatively with the Hct 2 days after mild injury with injection of a FVIII inhibitor (r= -0.53, p= 0.024; n= 18), supporting hypervascularity as a major contributor to hemophilic joint bleeding. Furthermore, the same mild injury caused minimal bleeding in naive FVIII KO mice with low BL vascularity (median Hct 46 % vs 48 %; p= 0.81 vs BL; n= 7), but excessive bleeding in FVIII KO mice injured 2 weeks previously with vascular abnormalities (median Hct 38 %; p= 0.021 vs BL; n= 7). hTAFI-CIIYQ effectively reduced bleeding severity upon re-injury (median Hct 47 %; p> 0.99 vs BL; n=5). Our data provide an experimental link between vascular abnormalities and bleeding susceptibility in hemophilic joints and conclude that the vascular abnormalities were alleviated by increasing TAFI levels or inhibiting uPA activity. Disclosures von Drygalski: University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Mosnier:The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The Factor VIII Variant X5 Enhances Hemophilia a Gene Therapy Efficiency By Its Improved Secretion

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector efficiency could be improved by employing more potent liver-specific promoters, but this might come at the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause oxidative stress, and if not resolved may even induce cell death. Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5 variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene therapy. Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells and purified to apparent homogeneity using standard procedures. The preparations were assayed for total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice (E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic, humanized HLA-DRB1*1501). Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5 variant revealed similarity of the two proteins and their specific activities in particular, indicating that introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5 transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any increased immunogenic risk compared to BDD-FVIII. Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy. Disclosures Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler:Baxalta Innovations GmbH, a Takeda company: Employment. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. De La Rosa:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Hoellriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Reipert:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

Targeting Translation Termination Machinery with Antisense Oligonucleotides for Diseases Caused by Nonsense Mutations.

Efforts to develop treatments for diseases caused by nonsense mutations have focused on identification of small molecules that promote translational read-through of messenger RNAs (mRNAs) harboring nonsense stop codons to produce full-length proteins. However, to date, no small molecule read-through drug has received FDA approval, probably because of a lack of balance between efficacy and safety. Depletion of translation termination factors eukaryotic release factor (eRF) 1 and eRF3a in cells was shown to promote translational read-through of a luciferase reporter gene harboring a nonsense mutation. In this study, we identified antisense oligonucleotides (ASOs) targeting translation termination factors and determined if ASO-mediated depletion of these factors could be a potentially effective and safe therapeutic approach for diseases caused by nonsense mutations. We found that ASO-mediated reduction of either eRF1 or eRF3a to 30%–40% of normal levels in the mouse liver is well tolerated. Hemophilia mice that express a mutant allele of human coagulation factor IX (FIX) containing nonsense mutation R338X were treated with eRF1- or eRF3a-ASO. We found that although eRF1- or eRF3a-ASO alone only elicited a moderate read-through effect on hFIX-R338X mRNA, both worked in synergy with geneticin, a small molecule read-through drug, demonstrating significantly increased production of functional full-length hFIX protein to levels that would rescue disease phenotypes in these mice. Overall our results indicate that modulating the translation termination pathway in the liver by ASOs may provide a novel approach to improving the efficacy of small molecule read-through drugs to treat human genetic diseases caused by nonsense mutations.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.

TNF-α/TNF-R System May Represent a Crucial Mediator of Proliferative Synovitis in Hemophilia A.

Hemophilic arthropathy (HA) typically begins with proliferative synovitis that shares some similarities with inflammatory arthritides, in which the proinflammatory cytokine tumor necrosis factor (TNF)-α has a crucial pathogenetic role. Inappropriate release of TNF-α was shown to contribute to arthropathy development following intra-articular bleeding in hemophilic mice. Here, we were interested in determining whether systemic levels of TNF-α and synovial tissue expression of the TNF-α/TNF receptor (TNF-R) system could be increased and related to joint damage in hemophilia A patients with severe HA. Serum levels of TNF-α measured by quantitative enzyme-linked immunosorbent assay (ELISA) were significantly increased in HA patients (n = 67) compared to healthy controls (n = 20). In HA patients, elevated TNF-α levels were significantly associated with the number of hemarthroses, the grade of synovial hypertrophy, and both the clinical World Federation of Hemophilia score and ultrasound score. The expression of TNF-α, TNF-R1, and TNF-R2 was strongly increased in HA synovium (n = 10) compared to the non-inflamed osteoarthritis control synovium (n = 8), as assessed by both immunohistochemistry and Western blotting. Increased protein levels of TNF-α, TNF-R1, and TNF-R2 were retained in vitro by HA fibroblast-like synoviocytes (n = 6) with respect to osteoarthritis control fibroblast-like synoviocytes (n = 6). Stimulation with TNF-α resulted in a significant increase in HA fibroblast-like synoviocyte proliferation quantified by the water-soluble tetrazolium (WST)-1 assay, while it had no relevant effect on osteoarthritis fibroblast-like synoviocytes. Quantification of active/cleaved caspase-3 by ELISA demonstrated that TNF-α did not induce apoptosis either in HA or in osteoarthritis fibroblast-like synoviocytes. The TNF-α/TNF-R system may represent a crucial mediator of proliferative synovitis and, therefore, a new attractive target for the prevention and treatment of joint damage in HA patients. Our findings provide the groundwork for further clinical investigation of anti-TNF-α therapeutic feasibility in hemophiliacs.

Novel Piperazino-Enaminones Decrease Pro-inflammatory Cytokines Following Hemarthrosis in a Hemophilia Mouse Model.

Hemarthrosis is the primary cause of hemophiliac arthropathy (HA). Pro-inflammatory cytokines are thought to play an important role in the pathogenesis of HA, and thus, anti-cytokine approaches may be used as an adjuvant therapy. A novel series of enaminone compounds (JODI), that contain the N-aryl piperazino motif, have been shown in vitro to reduce pro-inflammatory cytokines and thus may be efficacious in vivo. In this report, we will assess whether JODI can suppress multiple cytokines which might be potentially responsible for joint inflammation in a mouse model of hemarthrosis. The results showed that JODI significantly improved the survival after LPS treatment, and most pro-inflammatory cytokines/chemokines were decreased significantly after JODI administration. In the hemophilia mouse model, hemarthrosis resulted in local cytokine/chemokine changes, represented by elevated pro-inflammatory (IL-6, MCP-1, MIP-1α, MIP-1β) and pro-angiogenic (VEGF and IL-33) cytokines, and decreased anti-pro-inflammatory cytokines IL-4 and IL-10. The changes were reversed by administration of JODI, which can be used as a novel approach to manage hemophilia arthropathy.

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice

AbstractHere we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)–secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

Suppressing protein Z‐dependent inhibition of factor Xa improves coagulation in hemophilia A

Essentials Protein Z (PZ) catalyzes PZ-dependent proteinase inhibitor (ZPI) inactivation of factor (F)Xa. Gene-deletion of PZ or ZPI improves coagulation in hemophilia (FVIII knockout) mice. A PZ blocking antibody enhances thrombin generation in human hemophilia plasma. Suppression of the PZ/ZPI pathway may ameliorate the phenotype of severe hemophilia. SUMMARY: Background Hemostasis requires a balance between procoagulant and anticoagulant factors. Hemophiliacs bleed because of a procoagulant deficiency. Targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a cofactor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of activated factor X at phospholipid surfaces. Objectives To evaluate the effects of PZ or ZPI gene deletion in hemophilic mice, and of blocking PZ in human hemophilic plasma. Methods A tail vein rebleeding assay (TVRB) was developed on the basis of the serial disruption of clots forming over a period of 15 min following tail vein laceration in an anesthetized mouse. Wild-type (WT)/FVIII knockout FVIIIKO, PZ knockout PZKO/FVIIIKO and ZPI knockout ZPIKO/FVIIIKO mice were evaluated in this model, and their plasmas were tested in thrombin generation assays. A mAb against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results The numbers of clot disruptions (mean ± standard error of the mean) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice supplemented with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to that in FVIIIKO plasma supplemented with ~ 15% recombinant FVIII. A mAb against PZ added to human hemophilic plasma enhanced thrombin generation to an extent similar to the addition of ~ 15% FVIII. Conclusions Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia.

Characterization of the Interaction of Activated FVII with the Endothelial Protein C Receptor (EPCR) in the Rat

Activated FVII (FVIIa) has been shown to interact with EPCR resulting in long-term extravascular tissue presence in mice, well beyond its short circulating half-life. In addition, data in hemophilia patients on short term FVIIa prophylaxis indicate that clinical improvements can persist even in the post-prophylaxis period. Taken together, these data suggest that EPCR may sequester administered FVIIa in the extravascular space that retains activity, potentially explaining the hemostatic improvements that persist long after its infusion has ceased. As such, the EPCR-FVIIa interaction may have mechanistic and translational ramifications in the treatment of bleeds in hemophilia which needs to be further investigated. Unfortunately, hemophilic mice do not model the human disorder in terms of bleeding diathesis. In contrast, the novel rat model of hemophilia A (Nielsen LN et al, 2014) exhibits spontaneous bleeds that, if untreated, can be fatal. Therefore, this model can be utilized to better understand the FVIIa-EPCR interaction in the on-demand and prophylactic action of FVIIa administration in hemophilia. As an initial step towards that goal, here we characterize for the first time the rat FVIIa-EPCR interaction in vitro. We generated rat FVIIa by introduction of a PACE/Furin intracellular cleavage site (RKRRKR) between the light and heavy chains of rat FVII, an approach we have previously shown to result in secretion of two-chain activated FVII protease of various species (human, mouse and canine) in vitro. Recombinant rat FVIIa was purified in a two-chain form using conditioned medium from a HEK-293 stable cell line. The affinity of rat FVIIa to rat EPCR was assessed by incubation of the protease on engineered CHO-K1 cells that stably expressed full length rat EPCR. We found that rat FVIIa exhibited minimal binding to rat EPCR; in contrast human FVIIa bound rat EPCR with a Kd of ~400 nM. The lack of EPCR binding of rat FVIIa, similar to what we previously observed with mouse FVIIa (Pavani G et al., 2014 and 2016), complicates studies aimed at deciphering the hemostatic effects of the FVIIa-EPCR interaction in hemophilia using the rat. Therefore, we decided to engineer a rat FVIIa molecule with EPCR binding capacity so that it can be compared to rat FVIIa (non-EPCR binder) in subsequent in vivo experiments. For this, a rat FVIIa chimeric protease was generated using the first 11 amino acids of the Gla domain of rat protein C (rat FVIIa-FVRAG, with substitutions L4F, L8V, W9R, S10A, S11G). Using CHO-K1 expressing rat EPCR, we found that purified recombinant rat FVIIa-FVRAG bound rat EPCR with a Kd of ~500 nM, comparable to human FVIIa. Considering that the Gla domain, where the modifications are located, is essential for protease function, we also determined the procoagulant activity of rat FVIIa-FVRAG vs. FVIIa. We performed a thrombin generation assay in which each protease was added at increasing concentration (0-500 nM) and we measured the lag time, peak thrombin, endogenous thrombin potential. We found that rat FVIIa, rat FVIIa-FVRAG and human FVIIa (control) behaved similarly in all measured parameters for each concentration tested. In conclusion, we have defined the lack of EPCR binding of rat FVIIa and characterized a recombinant rat FVIIa molecule that can bind rat EPCR in vitro as a gain of function, but is otherwise similar to rat FVIIa. This molecule can now be used to probe the FVIIa-EPCR interaction in the novel and relevant hemophilia A rat model. DisclosuresMargaritis:Bayer Hemophilia Awards: Research Funding; Bristol Myers Squibb: Other: Salary (spouse); Novo Nordisk A/S: Research Funding.

Systemic Transcriptional Responses to Hemarthrosis and FVIII Replacement in FVIII-Deficient Mice

IntroductionHemarthrosis in patients with hemophilia (PWH) leads to local inflammation and vascular changes in the joint, but little is known about the extent and nature of systemic responses to joint bleeding. Since the spleen is a major systemic immune-modulatory organ, we quantified changes in splenic gene expression profiles in FVIII-deficient mice at baseline and after induced hemarthrosis, and in the presence and absence of FVIII replacement therapy.MethodsHemarthrosis was induced in FVIII-deficient mice by sub-patellar needle puncture +/- 100 IU/kg recombinant human FVIII (rhFVIII) intravenously 2 hours before and 6 hours after injury. Spleens were harvested on day 3 or 2 weeks post-injury (n=3-5). Spleens from uninjured mice +/- rhFVIII treatment served as controls. RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq500 platform (single-end; 75bp reads). The limma-voom method (R Bioconductor) was used for differential expression analyses. The criteria for differential expression were: i) a log fold-change (logFC) >1 or <-1, and ii) an adjusted p value <0.05. Functional enrichment was performed using Signaling Pathway Impact Analysis and the STRING database of protein-protein interactions.ResultsKnee injury in FVIII-deficient mice caused gross hemarthrosis that was largely prevented with rhFVIII prophylaxis (day 2 hematocrit: 26.4% and 46.3%). Pronounced alterations in splenic gene expression profiles occurred in vehicle-treated mice on day 3 post-injury, with 4227 differentially expressed genes (DEG) and 41 perturbed pathways. This response was markedly improved with rhFVIII treatment (386 DEG; 5 pathways), and almost entirely corrected by 2 weeks. Multiple pathways relating to immune processes, inflammation, and cell survival were highly perturbed on day 3 post-injury, including cytokine-cytokine receptor interactions (pNDE=1.4x10-6) and cell cycle (pNDE=5.3x10-5). The cell cycle pathway remained significantly altered despite rhFVIII treatment (pNDE=3.5x10-6), while other pathways were comparable to uninjured mice. Analysis of the top 50 DEG that are mutual to both treatment groups revealed a striking difference in directionality, with up-regulation in the vehicle group, and down-regulation in the rhFVIII group. Together, these findings demonstrate a significant effect of rhFVIII treatment on systemic transcriptional responses to joint bleeding. Treatment with rhFVIII in the absence of hemarthrosis resulted in only 97 DEG on day 3 by the same criteria, and 233 DEG after lowering the logFC threshold from (-)1 to (-)0.5. Of these 233 genes, STRING analysis revealed perturbation of the platelet activation pathway (FDR: 7.7x10-10) for up-regulated genes (93 DEG) and the T cell receptor signaling pathway (FDR: 1.3x10-8) for down-regulated genes (140 DEG), which may corroborate a role of T cell responses to rhFVIII treatment in the development of inhibitors. These responses to rhFVIII were unique to uninjured mice and did not occur in mice with induced hemarthrosis and rhFVIII treatment.ConclusionsJoint bleeding in hemophilic mice leads to acute, profound changes in multiple systemic pathways, including immune and inflammatory processes, as shown by gene expression profiling. Prophylactic treatment with rhFVIII largely corrects this response to hemarthrosis. Interestingly, in the absence of hemarthrosis, rhFVIII treatment affects platelet activation and T cell receptor signaling, whereby further analyses are required to determine if these effects stimulate or dampen immune responses. This approach can be used to explore the systemic mechanisms contributing to progressive hemophilic arthropathy in PWH, elucidate immune responses that may facilitate inhibitor formation, and lead to development of novel therapeutic strategies. Disclosuresvon Drygalski:UniQure BV, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy.

A Novel Humanized Hemophilia-a Mouse Model to Facilitate Preclinical In Vivo Studies of Human Specific Fviiia-Mimetic Bispecific Antibodies

Introduction: To improve treatment options for Hemophilia A patients with and without Factor VIII inhibitors, we and others are developing bispecific antibodies that replace the function of FVIII by bringing together activated FIX and FX to activate FX and restore normal hemostasis. Thus far, preclinical in vivo evaluation of these novel reagents has been limited to non-human primates, due to their lack of species cross-reactivity.To accommodate pharmacokinetic, pharmacodynamic and in vivo efficacy and safety studies of novel FVIIIa-mimetic bispecific antibodies we set out to generate two humanized FIX;FX double knock-in (DKI) mouse models: one with normal murine FVIII activity (hFIX;hFX-DKI) and one with hemophilia A (hFIX;hFX-DKI;mFVIII-KO).Methods and Results: Human FIX-knock-in (KI) and human FX-KI mice were generated, using classical C57/Bl6 embryonic stem cell targeting. Both hFIX and hFX encoding sequences replaced their murine counterparts with expression of the human genes driven by the intact murine promotor. hFIX;hFX-DKI mice were generated by intercrossing both lines to homozygosity. hFIX;hFX-DKI;mFVIII-KO mice were generated by CRISPR/Cas9 mediated knockout of murine FVIII in hFIX-KI mice, prior to intercrossing with hFX-KI mice. Both hFIX;hFX-DKI and hFIX;hFX-DKI;mFVIII-KO mice are fertile and viable.Plasma protein expression levels of both hFIX and hFX in single-KI, DKI, and hFIX;FX-DKI;mFVIII-KO mice were determined by human specific ELISAs and were approximately 1 and 2 µg/mL, respectively. These protein concentrations correspond to 20 % FIX and 10 % FX normal human plasma levels. Chromogenic assays showed matching plasma clotting factor activities compared to normal human plasma, i.e. 10-20% activity of hFIX and 5-10% of hFX. Similar data were obtained by one stage assay. Finally, functional FVIII deficiency in the hFIX;hFX-DKI;mFVIII-KO mice was confirmed by both chromogenic and one stage clotting assays.Importantly, hFIX;hFX-DKI mice showed only slightly reduced whole blood clotting activity compared to wild-type mice by rotational thromboelastometry (ROTEM; mean clotting times: 910 sec and 516 sec, respectively) whereas hemophilic mice showed significant prolonged clotting times (2500 sec). These data suggest a mild hemophilic phenotype due to reduced human clotting factor expression. Nonetheless the hFIX;hFX-DKI mice showed 100% survival in the tail vein transection model compared to nearly 0% survival for HemA and HemB mice. These findings indicate that hFIX;hFX-DKI;mFVIII-KO should be suitable for the tail vein transection bleeding efficacy evaluation of FVIIIa-mimetic antibodies. In contrast, the more severe tail clip bleeding model using hFIX;hFX-DKI mice showed only slightly less bleeding compared to hemophilia mice (median blood loss, 581 µl versus 766 µl), showing that 10% human clotting factor plasma levels compared to wild-type mice (62 µl) is not sufficient to stop bleeding in this efficacy model. The latter finding is in line with reported data showing that near normal FVIII or FIX clotting factor levels are needed in the severe tail clip bleeding model to stop bleeding in HemA or HemB mouse models, respectively (Dumont, Blood, 2012, Toby et al PLOS One 2016).Conclusions: We generated and characterized novel mouse models enabling pharmacokinetic, pharmacodynamic and in vivo efficacy and safety studies for novel FVIII-mimetic bispecific antibodies. Ongoing and future studies using these novel mouse models aim to elucidate the in vivo mode of action of this new class of non-replacement clotting factor drug candidates as well as their in vivo interactions with other bypass reagents DisclosuresLi:Bioverativ a Sanofi company: Employment, Equity Ownership, Honoraria. Hu:Biogen: Employment, Equity Ownership, Honoraria. Leksa:Bioverativ a Sanofi company: Employment. Aleman:Bioverativ, a Sanofi company: Employment. Peters:Bioverativ a Sanofi company: Employment, Equity Ownership. Salas:Bioverativ a Sanofi company: Employment, Equity Ownership. Van Der Flier:Bioverativ a Sanofi company: Employment, Equity Ownership, Honoraria.

Systemic Vascular Basement Membrane Markers Linked to Synovial Vascular Remodeling Are Bio-Markers of Hemarthrosis in Patients with Hemophilia

Introduction: Acute painful joint episodes in hemophilia are not always associated with hemarthrosis. Point-of-Care Musculoskeletal Ultrasound with Power Doppler (POC MSKUS/PD) is a fast and sensitive imaging modality to determine the presence of hemarthrosis and direct management. Systemic collagen cleavage products reflecting extracellular matrix, bone and soft tissue turnover may serve as an alternative tool for the detection of hemarthrosis. Based on previous observations of pronounced vascular remodeling within the synovial tissue during hemarthrosis in patients and hemophilic mouse models, we hypothesized that cleavage products of collagen IV, found in the basement membrane of blood vessels may differentiate between bleeding and non-bleeding states.Methods: Joint bleeding was induced in FVIII-/- mice (+/- FVIII-treatment before and shortly after injury). Synovial vascular remodeling was assessed 14 days later by MSKUS/PD and histology including αSmooth Muscle Actin (αSMA)-staining. Murine plasma samples were analyzed for C4M2 and P4NP-7S, which are type IV collagen turnover products. To determine human relevance, 31 adult patients with hemophilia (PWH), ≥ age 21, were studied prospectively for 3 years with MSKUS/PD during pain-free intervals and painful events for bleed status, vascular flow and 11 plasma markers of collagen turnover(C2M, ARGS, C3M, Pro-C3, C5M, Pro C5, C4M2, P4NP-7S, Col18N, DCN- CS, ColNeo10, NIC).Results: Type IV collagen turn-over markers were significantly elevated in plasma of FVIII-/- deficient mice 14 days after joint bleeding when compared to baseline (C4M2: 2.575 ng/mL vs. 1.94 ng/mL, p=0.029 and P4NP-7S: 80.3 ng/mL vs. 67.81 ng/mL, p= 0.0152 respectively ). This increase coincided with a significant increase in vessel number (2.3 fold, p=0.0008), vascular flow by PD (2.3 fold, p= 0.014) and αSMA staining (5.1 fold, p= 0.0007). C4M2 and P4NP7S plasma levels correlated negatively with αSMA staining, suggesting a role for αSMA in vessel stabilization (C4M2 p=0.02, Pearson r= -0.5707; P4NP-7S p= 0.0130, Pearson r= -0.6238). FVIII-treatment reduced plasma C4M2 and P4NP-7S levels to baseline (C4M2 1.98 ng/mL vs. 1.94 ng/mL, P4NP-7S 59.98 ng/mL vs. 67.81 ng/mL), but had no effect on the other vascular parameters. In patients, results from 91 visits were compiled. Twenty five were due to acute painful episodes with 16 confirmed hemarthroses. Of the 11 biomarkers evaluated, only C4M2 and P4NP-7S were transiently but significantly elevated in plasma during joint bleeding compared to pain free intervals or to non-bleeding painful episodes. Plasma C4M2 levels during acute painful bleeding episodes when compared to non-bleeding states were 41.65 vs. 29.69 ng/mL (p=0.0003), and P4NP-7S levels were 418.9 vs 278.1 ng/mL (p=0.0007), respectively. Baseline levels of C4M2 and P4NP-7S in PWH were above the lab reported normal range, indicating ongoing high vascular basement membrane turnover. Additionally, PWH who developed joint bleeding episodes had a higher level of collagen IV biomarkers at baseline when compared to PWH who did not experience joint bleeding (C4M2 33.91 vs 29.07 ng/mL, P4NP7s 320.6 vs 292.8 ng/mL), although this was not statistically significant. Hemarthrosis was accompanied by pronounced abnormal synovial microvascular flow evidenced by PD, while plasma levels of both biomarkers correlated positively with joint PD signal (C4M2, p= 0.0400, Pearson r=0.4409; P4NP7S-p=0.0141, Pearson r=0.5154).Conclusions: Our findings suggest that systemic vascular collagen turn-over products may be novel biomarker candidates to identify patients at high risk for joint bleeding and to diagnose acute hemarthrosis. Hemarthrosis in hemophilia is associated with pronounced synovial vascular remodeling. Cleavage products of type IV collagen, but not of any other collagen, were systemically elevated in PWH and distinguished bleeding from non-bleeding painful episodes. Type IV collagen is found exclusively in basement membranes and in the case of joint bleeding, the presumed source of these turnover markers is newly formed synovial blood vessels. Therefore, our findings support the concept that vascular instability during neovascularization is involved in the dynamics of hemophilic joint bleeding. These findings broaden the scope of potential diagnostic tools substantially, opening new avenues for personalized treatment of PWH. Disclosuresvon Drygalski:Bioverativ/Sanofi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; HemaBiologics: Consultancy; Genentech: Consultancy; Biomarin: Consultancy, Honoraria; Uniqure: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Hematherix: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Coagulation phenotype of wild-type mice on different genetic backgrounds.

Genetically engineered mouse models are used to investigate beneficial treatment in haemophilia by comparison with wild-type mice. It has been recognized that wild-type and haemophilic mice of different genetic backgrounds show different bleeding phenotypes. We assessed ex-vivo coagulation parameters in nine wild-type substrains of 129S1/Sv, BALB/c and C57BL/6 mice applying thromboelastography (TEG), activated partial thromboplastin time (aPTT), prothrombin time (PT) and fibrinogen levels. The comprehensive ex-vivo data are discussed in view of results from a tail-tip bleeding assay. Time to first clot formation (R-time) showed higher within-substrain (CV range: 28–54%) and higher between-substrain (median range: 25.53–42.60 min) variation for BALB/c than for C57BL/6 mice (CV range: 14–31%; median range: 22.45–24.93 min). Median R-time for 129S1/Sv mice was 30.42 min (CV: 33%). No distinct strain differences were observed for maximum amplitude (MA), aPTT, or PT, but males generally showed higher MA and shorter aPTT than females. Males of all substrains had higher fibrinogen levels than females. The heightened in-vivo variability (CV range: 81–171%; median range: 36.00–469.50 mg) in the tail-tip bleeding assay and increased blood loss in wild-type C57BL/6 male mice was not reflected in ex-vivo coagulation parameters. In general, ex-vivo coagulation results appeared consistent within substrains, but showed substrain and sex differences of variable magnitudes. We conclude that alignment of the mouse substrain genetic background to the experimental model is critical to reduce data variability and animal numbers.

Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo

AbstractHemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to FVIII epitopes. To specifically target FVIII-specific B cells, we engineered immunodominant FVIII domains (A2 and C2) as a chimeric antigen receptor expressed by both human and murine cytotoxic T cells. This FVIII domain engineered B-cell antibody receptor (BAR) that expresses T cells was capable of killing FVIII-reactive B-cell hybridomas in vitro and in vivo. Moreover, FVIII BAR CD8 T cells blocked the development of specific antibody from unimmunized spleen cells stimulated polyclonally with lipopolysaccharide in vitro. In addition, adoptive transfer of FVIII A2- and C2-BAR CD8 T cells significantly reduced the anti-FVIII antibody formation in hemophilic mice. These data suggest that BAR-engineered T cells are a promising approach for future prophylactic treatment for patients with severe hemophilia A who are at high risk of developing inhibitors.

Blood-induced bone loss in murine hemophilic arthropathy is prevented by blocking the iRhom2/ADAM17/TNF-α pathway

AbstractHemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII-deficient mice (F8−/− mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture–induced bleeding leads to increased TNF-α levels in the affected joint of F8−/− mice. Moreover, inactivation of TNF-α or iRhom2 in F8−/− mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients.

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non‐mucosal venous injury model in haemophilic rodents

Background Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. Objective To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard exvivo clotting/fibrinolysis assay can be used as a predictive surrogate for invivo efficacy. Methods A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non-mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays. Results The beneficial effect of systemic TXA therapy observed exvivo could not be confirmed invivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice. Conclusions The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non-mucosal tissues.

Autologous and Heterologous Cell Therapy for Hemophilia B toward Functional Restoration of Factor IX

SUMMARYHemophilia B is an ideal target for gene- and cell-based therapies because of its monogenic nature and broad therapeutic index. Here, we demonstrate the use of cell therapy as a potential long-term cure for hemophilia B in our FIX-deficient mouse model. We show that transplanted, cryopreserved, cadaveric human hepatocytes remain functional for more than a year and secrete FIX at therapeutic levels. Hepatocytes from different sources (companies and donors) perform comparably in curing the bleeding defect. We also generated induced pluripotent stem cells (iPSCs) from two hemophilia B patients and corrected the disease-causing mutations in them by two different approaches (mutation specific and universal). These corrected iPSCs were differentiated into hepatocyte- like cells (HLCs) and transplanted into hemophilic mice. We demonstrate these iPSC-HLCs to be viable and functional in mouse models for 9–12 months. This study aims to establish the use of cells from autologous and heterologous sources to treat hemophilia B.