Factor VIII Knockout Mice Research Articles

Persistent Splenic-Derived IgM Preferentially Recognize Factor VIIIA2 and C2 Domain Epitopes but Do Not Alter Antibody Production

The most significant treatment complication for patients with hemophilia A is the development of neutralizing immunoglobin G (IgG), termed inhibitors, against factor VIII (FVIII) which prevent FVIII replacement therapy. Low titers of FVIII-specific immunoglobin M (IgM) have been identified in hemophilia A patients with and without inhibitors, as well as healthy individuals. However, the duration and influence of IgM on the immune response to FVIII remains unclear. To characterize the binding interactions of persistently secreted FVIII-specific IgM in hemophilia A mice and assess their effect on IgG antibody development. Splenic-derived monoclonal antibodies (MAbs) from immunized FVIII knockout mice were isolated and purified using hybridoma technology. Binding interactions were assessed utilizing a novel fluid-phase ELISA and computational modeling with HADDOCK to account for weak IgM binding. Sixteen porcine cross-reactive and non-inhibitory FVIII-specific IgM MAbs were identified. RNA sequencing of FVIII-specific IgM revealed 13 unique VDJ/VJ sequences indicating derivation from 13 unique B cell clones. IgM demonstrated polyclonal and polyreactive binding to FVIII in vitro and in silico. Molecular docking studies with reconstructed IgM VDJ/VJ regions identified frequent IgM interactions with amino acid residues K376, T381, K437, R2215 or K2249 within the FVIII A2 and C2 domains. Injections of individual IgM prior to FVIII exposure and co-injection of FVIII/IgM immune complexes did not affect de novo FVIII antibody production. Persistent FVIII-specific IgM are polyclonal but preferentially bind the A2 and C2 domains and FVIII/IgM immune complex formation do not significantly alter inhibitor development.

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice

AbstractHemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

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

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. FVIII-KO mice received prophylaxis with emicizumab or emicizumab+TxAc before trauma. FVIII-KO mice were given emicizumab 1.5mg/kg via IV injection. A second retro-orbital IV injection containing human FIX and FX (both 100U/kg) was given 24h later and 5min 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. 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. Our results suggest a potential benefit of TxAc when used in combination with emicizumab in prophylactic settings, especially in patients presenting breakthrough bleeds.

Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles.

Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat FVIII knockout (FVIII KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-Fe3O4) to carry the B-domain-deleted (BDD)-FVIII plasmid, which was delivered into the FVIII KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD-FVIII plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-Fe3O4 nanoparticles. The FVIII KO mice that received the DPPC-Fe3O4 plasmid complex were assessed by staining the ferric ion of DPPC-Fe3O4 nanoparticles with Prussian blue in liver tissue. The bleeding of the FVIII KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in FVIII KO mice and might be a promising approach to treating hemophilia A patients in clinical settings.

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.

Thrombin: A Driver or Attenuator of Bone Disease on Hemophilia?

Introduction: Hemophilic bone disease is a pathology inherent to factor VIII (FVIII) and factor IX (FIX) deficiency present in both adult and pediatric persons with hemophilia (PWH). This pathology is also present in mouse models. We have previously demonstrated that FVIII replacement restores lost mineralization activity in osteoblast-like cells from FVIII-deficient mice. We hypothesized that bone disease in PWH is secondary to decreased thrombin generation in PWH. Methods: Murine bone marrow stromal cells isolated from FVIII knockout (FVIII-/-) and wild-type (FVIII+/+) C57BL mice and human osteosarcoma (HOS) TE85 cells were cultured in α-MEM with 0-10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL Amphotericin B. Ten millimolar ß-glycerophosphate and 50 µg/mL ascorbic acid were added to induce differentiation and mineralization. Mineralized deposits were stained with alizarin red. Alkaline phosphatase (ALP) activity and culture protein content were measured using colorimetric assays. Results: In , the addition of thrombin in serum-free conditions increases cell proliferation ~25% (p=5x10-4, ANCOVA) and decreases mineralization up to 84% (p=1x10-6) (Figure 1). Thrombin's effect on ALP activity in HOS TE85 cells is negative at low cell numbers and positive at high cell numbers (Figure 1). Osteoblast-like cells from FVIII-/- mice mineralize 70% less than those from FVIII+/+ cells (p=3x10-6). Administering FVIII to cells from FVIII-/- mice ameliorates the mineralization deficiency (Figure 1), while mineralization in cells from FVIII+/+ mice does not change (p=0.66). FVIII-/- cells express 30% lower levels of ALP activity than FVIII+/+ (p=3x10-4). FVIII+/+ cells proliferate 26% more rapidly than FVIII-/- cells (p=4x10-7, ANCOVA), but this is not affected by the addition of FVIII in vitro (p=0.45). The addition of FVIII causes a ~33% decrease in ALP activity in osteoblast-like cells from both FVIII-/- (p=2x10-4) and FVIII+/+ (p=5x10-5) mice. Although thrombin impacts osteoblast differentiation and function, it has paradoxical effects on their mineralization, suggesting thrombin deficiency is not the sole driver of reduced bone health in PWH. Deficient mineralization in cells from FVIII-/- mice compared to those from FVIII+/+ mice is corrected with FVIII replacement in vivo or in vitro, suggesting that circulating FVIII has a positive role in these cells' mineralization. More rapid proliferation in cells from FVIII+/+ mice likely contributes to the increase in mineralization, yet, as proliferation is unchanged by FVIII replacement, it appears unlikely that this effect occurs via the same mechanism. Thrombin causes increased proliferation of HOS TE85 cells, yet also causes a significant decline in mineralization activity (Figure 1). Whether this is a paradoxical effect present only in this transformed cell line is yet undetermined. ALP activity increases when HOS TE85 cells are given thrombin in vivo during the log phase of growth (Figure 1), indicating a positive effect on the osteoblast-like function of the cells. However, this notion is undermined by the decrease in mineralization. Identifying the role of FVIII, FIX, thrombin, and active protein C in bone mineralization in PWH is proving to be a challenging undertaking, yet it remains critical, especially as new drugs with the potential to supplant FVIII prophylaxis hit the market. Future experiments will include thrombin and a thrombin inhibitor, a PAR1 agonist and antagonist, a PAR2 antagonist, and an APC neutralizing antibody. Upcoming findings will be discussed. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership.

Elimination of factor VIII‐specific B cells by immunotoxins composed of a single factor VIII domain fused to Pseudomonas exotoxin A

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA exvivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested exvivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors.

Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8‐GP) and development of a human pharmacokinetic prediction model

Background N8-GP is an extended half-life recombinant factorVIII (FVIII) for the treatment of hemophiliaA. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals. Objective To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8-GP in preclinical models and predict the human pharmacokinetic (PK) profile. Methods The pharmacokinetics of subcutaneously administered N8-GP were evaluated in FVIII knockout (F8-KO) mice and cynomolgus monkeys; a human PK prediction model in hemophiliaA patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8-KO mice. The injection-site distribution and absorption of subcutaneously administered N8-GP were assessed in F8-KO mice by the use of temporal fluorescence imaging and immunohistochemistry. Results Subcutaneously administered N8-GP had a bioavailability, a first-order absorption rate and a half-life, respectively, of 24%, 0.094h-1 and 14h in F8-KO mice, and 26%, 0.33h-1 and 15h in cynomolgus monkeys. A dose-dependent effect of subcutaneously administered N8-GP on blood loss was observed in mice. A minimal amount of N8-GP was detected at the injection site 48-72h after single or multiple dose(s) in F8-KO mice. Subcutaneously administered N8-GP was localized to the skin around the injection site, with time-dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8-GP at a daily dose of 12.5IUkg-1 will provide FVIII trough levels of 2.5-10% in 95% of patients with severe hemophiliaA. Conclusions Subcutaneously administered N8-GP may provide effective hemophiliaA prophylaxis. A phaseI clinical trial is underway to investigate this possibility.

In utero therapy for hemophilia A using EGFP amniotic fluid stem cells

Hemophilia A is an X chromosome recessive genetic disease, that causes clotting factor VIII (FVIII) deficiency leading to blood coagulation dysfunction. The activity of coagulation factor in serious hemophilia patients is less than one percent. The current therapeutic methods of hemophilia A are mainly for giving either the coagulation factor supplementary injection or gene therapy. However, both methods have some shortcomings. Therefore, an alternative potential treatment might be desired. The amniotic fluid stem cells (AFSCs) are derived from amniotic fluid. The amniotic fluid is the medical waste and it don't involve in ethical issues. Moreover, AFSCs can differentiate to diverse cells of ectoderm, mesoderm, and endoderm lineages. In utero transplantation (IUT) of AFSCs is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant. In human, certain disease, such as osteogenesis imperfect, was successfully treated by using this method. By transplanting AFSCs into fetus, the AFSCs can directly involve in the development of the fetus and achieve therapeutic effect. The aim of this research is to assess therapeutic effect of hemophilia A after in utero transplantation by amniotic fluid‐derived EGFP‐stem cells (EGFP‐AFSCs) into the liver of FVIII knockout mice. In this study, the B6;129S‐F8tm1Kaz/J knockout mice was used as a hemophilia A animal model. The C57BL/6 mice was used as a normal control and untreated hemophilia A mice was used as a negative control. To establish IUT animal model, the pregnant CD‐1 mice (14.5 days) underwent abdominal surgery and transplanted EGFP‐AFSCs (5 × 104 cells per fetus) into the liver of fetus. Three weeks after birth, the mice were sacrificed and the EGFP signal in organs were analyzed by RT‐PCR, histological examination and flow cytometry. Results showed that EGFP signal can be detected in the liver and intestine. Furthermore, we used several methods including activated partial thromboplastin time, FVIII activity assay, tail clip survival rate, and hemoglobin concentration to analyze whether the FVIII activity in the hemophilia A mice can be restored or not after IUT operation by EGFP‐AFSCs. By conducting this research, we expect to solve the hemophilia patient's therapeutic problems and increase better quality of life.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abnormal von Willebrand factor secretion, factor VIII stabilization and thrombus dynamics in type 2N von Willebrand disease mice

Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex, with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII. Objectives In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization and thrombus formation in a murine model. Methods Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles. Results Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII knockout (KO) mice demonstrated impaired thrombus formation associated with decreased thrombus stability. Conclusions The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis.

FVIII-specific human chimeric antigen receptor T-regulatory cells suppress T- and B-cell responses to FVIII

AbstractReplacement therapy with factor VIII (FVIII) is used in patients with hemophilia A for treatment of bleeding episodes or for prophylaxis. A common and serious problem with this therapy is the patient's immune response to FVIII, because of a lack of tolerance, leading to the formation of inhibitory antibodies. Development of tolerogenic therapies, other than standard immune tolerance induction (ITI), is an unmet goal. We previously generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T-cell clone. The resulting engineered T cells suppressed both T- and B-cell effector responses to FVIII. In this study, we have engineered an FVIII-specific chimeric antigen receptor (ANS8 CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Transduced ANS8 CAR T cells specific for the A2 domain proliferated in response to FVIII and ANS8 CAR Tregs were able to suppress the proliferation of FVIII-specific T-effector cells with specificity for a different FVIII domain in vitro. These data suggest that engineered cells are able to promote bystander suppression. Importantly, ANS8 CAR-transduced Tregs also were able to suppress the recall antibody response of murine splenocytes from FVIII knockout mice to FVIII in vitro and in vivo. In conclusion, CAR-transduced Tregs are a promising approach for future tolerogenic treatment of hemophilia A patients with inhibitors.

Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice.As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein.To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response.In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. DisclosuresNo relevant conflicts of interest to declare.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure

AbstractAnalysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34low cells within Lin−Sca+Kit+ progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte–colony-stimulating factor (G-CSF)–induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF–induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro–computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

The bone disease associated with factor VIII deficiency in mice is secondary to increased bone resorption

Osteopenia and osteoporosis have increasingly become a recognized morbidity of factor VIII (FVIII) deficiency. Recently, we demonstrated that FVIII knockout (KO) mice had significantly decreased bone mass and bone strength despite the fact that they did not have haemarthroses. The aim of this study was to explore the mechanism of bone disease associated with FVIII deficiency. We compared biochemical markers of bone formation and osteoclastogenesis, inflammatory cytokines, as well as static and dynamic histomorphometry of genetically engineered FVIII KO male mice to those of wild-type (WT) controls. At 20 weeks of age, FVIII KO mice, as well as WT controls, were sacrificed. Serum and bones were obtained at the time of sacrifice to study biochemical markers of bone formation (osteocalcin) and osteoclastogenesis (receptor activator of nuclear factor kappa-β and osteoprotegerin), levels of inflammatory cytokines (interleukin-1α and interferon-β) and to perform static and dynamic histomorphometry of tibia cancellous bone. There was no difference in the biochemical markers of bone formation or osteoclastogenesis. However, there were differences in the two bone-associated cytokines studied. In addition, histomorphometric examination revealed cancellous osteopenia in FVIII KO mice as evidenced by decreased bone area and trabecular number and increased trabecular separation. Bone formation parameters were normal in FVIII KO mice. In contrast, osteoclast-lined bone perimeter was increased. These data demonstrate that bone disease in FVIII KO mice is due to an increased rate of bone resorption.

IL-6 receptor antagonist as adjunctive therapy with clotting factor replacement to protect against bleeding-induced arthropathy in hemophilia

The most common morbidity that results from hemophilia is bleeding-induced hemophilic arthropathy (HA), which once established may not be interrupted completely even by prophylactic clotting factor replacement. Specific therapies to oppose inflammatory cytokines, including Interleukin 6 (IL-6) receptor antagonists, have become important in the management of inflammatory arthritides. We investigated combining therapy using MR16-1, a rat IgG antibody directed against mouse IL-6 receptor (anti-IL-6R), with factor VIII (FVIII) replacement to protect against bleeding-induced arthropathy in hemophilia A mice. Three recurrent hemarthroses were induced in the knee joint capsule of FVIII knockout mice. Treatment at the time of each hemorrhage included either: no treatment; FVIII replacement given at the time of hemorrhage; FVIII replacement at hemorrhage plus anti-IL-6R as 4-weekly injections; FVIII replacement with non-specific control antibody (rat IgG); and anti-IL-6R alone without FVIII replacement. Six weeks following the first hemarthosis, joints were harvested and histopathology was scored for synovitis, for cartilage integrity and for macrophage infiltration. Animals that received anti-IL-6R as an adjunct to FVIII replacement demonstrated the best survival and the least acute joint swelling and pathology on histologic examination of the synovium and cartilage (P<0.05 for each parameter). All histopathologic parameters in the mice receiving FVIII+anti-IL-6R were limited and were comparable to findings in injured hemostatically normal mice. The major benefits of adjunctive anti-IL-6R were decreasing synovial hyperplasia, hemosiderin deposition and macrophage infiltration. Short-course specific inhibition of inflammatory cytokines as an adjunct to replacement hemostasis may be an approach to minimize hemophilic joint degeneration.

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Reduced Bone Mineral Density in Factor VIII Deficient Mice and the Role of Inflammatory Cytokines

Abstract 21 Background:Low bone mineral density (BMD) and increased risk of fracture are increasingly being identified in patients with hemophilia. Multiple clinical studies have shown that both children and adults with hemophilia have significantly decreased BMD, with up to 70% of adult patients affected. Decreased BMD in this population has previously been attributed to inactivity due to hemophilic arthropathy and comorbid conditions including infection with HIV and hepatitis C.The factor VIII (FVIII) knockout (KO) mouse model has been extensively characterized with respect to bleeding and the development of inhibitors. However, the skeletal status of these animals has not been described. Therefore, the purpose of this study is to examine the skeletal phenotype of FVIII deficient mice and measure serum markers of bone turnover and regulation. Methods:We compared the skeletal phenotype of 11 male FVIII KO mice and 8 matched wild-type (WT) controls at 20 weeks of age. BMD was measured using dual energy x-ray absorptiometry (DXA), bone geometry was examined by μCT and, after sacrifice, femoral breaking strength was measured by three-point bending until failure (ultimate force).In a second group of 24 mice (12 KO and 12 WT), serum was obtained under uniform conditions at 20 weeks of age via cardiac puncture. Serum levels of receptor activator of nuclear factor kappa-B ligand (RANK-L), osteoprotegerin (OPG), interleukin 1α (IL-1α) and osteocalcin were measured using commercially available ELISAs. Alkaline phosphatase (alk phos) activity was measured using a colorimetric assay. The RANK-L/OPG system regulates bone turnover, osteocalcin and alk phos are markers of bone turnover and IL-1α is an inflammatory cytokine. Results:No differences in body weight, length, percent fat, or femoral length were observed between FVIII KO and WT mice (data not shown). No spontaneous bleeding was observed in any animal. As shown in Table I, KO mice had significantly decreased BMD, cortical thickness and stiffness compared to WT mice. This resulted in decreased resistance to fracture as measured via ultimate force. The data presented here demonstrate that adult FVIII KO male mice exhibit lower cortical bone mass resulting in femora less resistant to fracture compared with WT controls. Because FVIII KO mice do not have spontaneous joint hemorrhages and exhibit normal behavior and activity levels, these data suggest that there is an underlying connection between the coagulation system and bone metabolism that results in lower BMD and bone strength.Table I- Femoral bone measurements of mice. Standard deviations are in parentheses.BMD (mg/cm2)Cortical Thickness (mm)Cross-sectional Area (mm2)Stiffness (N/m)Ultimate Force (N)KO55.327 (2.522)0.159 (0.006)1.171 (0.102)97.143 (7.192)20.163 (1.263)WT58.375 (3.110)0.165 (0.004)1.196 (0.126)106.921 (9.750)22.007 (2.061)P value0.0300.0200.6320.0330.031To understand why KO mice have decreased BMD, we measured serum levels of regulators of bone metabolism, markers of bone turnover and inflammatory cytokines. There was no difference in RANK-L, OPG or the RANK-L/OPG ratio between the KO and WT mice. In addition, there was no observed difference in osteocalcin and alk phos. However, WT animals had significantly higher levels of IL-1α expression (P = 0.008). All of the KO animals had undetectable IL-1α levels under physiologic conditions whereas the mean IL-1α level in WT mice was 32.2 pg/ml. Thrombin production is reduced in hemophilia and thrombin has previously been shown to stimulate inflammatory cytokine production. Therefore, these data suggest that thrombin may be necessary for IL-1α production under non-stress conditions and that lower levels of IL-1α may be detrimental to bone health. Although increased inflammatory cytokines are typically associated with decreased BMD, prior research has shown that physiologic levels of cytokines are necessary to maintain bone health. Conclusions:These data provide compelling evidence that the link between FVIII deficiency and impaired skeletal health is real and independent of differences in physical activity and other medical co-morbidities. In addition, the decreased levels of IL-1α observed in KO mice suggest that inflammatory cytokines are involved in this pathophysiology. Ongoing work in our laboratory is aimed at further exploring these connections. Disclosures:No relevant conflicts of interest to declare.

Deletion or Inhibition of Fc Gamma Receptor IIb (CD32) Prevents the Memory B Cell Response to Factor VIII in a Hemophilia A Mouse Model

Abstract▪204▪This icon denotes a clinically relevant abstractThe formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI. Disclosures:No relevant conflicts of interest to declare.

Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice

Blood collection in mice can be a challenge, in particular for samples used for coagulation analysis as initiation of coagulation during the procedures can influence the results. Blood collection from the retrobulbar venous plexus is commonly used but the method remains controversial. Several alternatives exist but not all are applicable to mice with a compromised coagulation system because of subsequently excessive bleeding. We therefore wanted to explore whether blood collection by puncture of the submandibular vein could replace blood collected from the retrobulbar venous plexus during pharmacokinetic and pharmacodynamic studies in mice lacking coagulation factor VIII (FVIII). The plasma concentrations of recombinant activated factor VII were independent of the blood collection method in a pharmacokinetic study. The same applied to the thromboelastographic profile of mice with normal coagulation in a pharmacodynamic study. However, excessive haemorrhages were observed in all FVIII knockout mice after a single puncture of the submandibular vein and 60% of the mice were euthanized 2-4 h after the blood collection. In contrast, no or only slight haemorrhage was observed in animals subjected to blood collection from the retrobulbar venous plexus. No signs of distress determined by blood glucose level or clinical abnormalities of the eye were observed after puncture of the retrobulbar venous plexus. In conclusion, blood collected by puncture of the submandibular vein and retrobulbar venous plexus has a quality which allows it to be used in coagulation assays. However, because of excessive bleedings, puncture of the submandibular vein is not recommended in mice lacking FVIII.