Murine Hemophilia Research Articles

Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors

BackgroundPlatelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic. ObjectivesTo evaluate the efficacy of Bu–Flu and Mel–Flu preconditioning in platelet gene therapy of HA with inhibitors. MethodsBu–Flu and Mel–Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet–FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests. ResultsBu–Flu, but not Mel–Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu–Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet–FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu–Flu conditioning. ConclusionBu–Flu preconditioning allows for successfully introducing platelet–FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors.

Von Willebrand factor modulates immune complexes and the recall response against factor VIII in a murine hemophilia A model

AbstractAchieving tolerance toward factor VIII (FVIII) remains an important goal of hemophilia treatment. Up to 40% of patients with severe hemophilia A (HA) develop neutralizing antibodies against FVIII, and the only proven treatment to achieve tolerance is infusion of FVIII over prolonged periods in the context of immune tolerance induction. Here, we addressed the role of von Willebrand factor (VWF) as a modulator of anti-FVIII antibody effector functions and the FVIII-specific recall response in an HA mouse model. Analytical ultracentrifugation was used to demonstrate formation of FVIII-containing immune complexes (FVIII-ICs). VWF did not fully prevent FVIII-IC formation but was rather incorporated into larger macromolecular complexes. VWF prevented binding of FVIII-ICs to complement C1q, most efficiently when it was preincubated with FVIII before the addition of antibodies. It also prevented binding to immobilized Fc-γ receptor and to bone marrow–derived dendritic cells. An in vitro model of the anti-FVIII recall response demonstrated that addition of VWF to FVIII abolished the proliferation of FVIII-specific antibody-secreting cells. After adoptive transfer of sensitized splenocytes into immunocompetent HA mice, the FVIII recall response was diminished by VWF. In summary, these data indicate that VWF modulates the formation and effector functions of FVIII-ICs and attenuates the secondary immune response to FVIII in HA mice.

HTRS2023.P2.13 Off-target toxicity of CART cell therapy in murine hemophilia A with inhibitors

HTRS2023.P2.13 Off-target toxicity of CART cell therapy in murine hemophilia A with inhibitors

Gut dysbiosis modulates the immune response to factor VIII in murine hemophilia A

AbstractThe development of neutralizing FVIII antibodies is the most serious complication of hemophilia A treatment. The currently known patient- and treatment-related risk factors for inhibitor development do not accurately predict this adverse event in all patients. The composition of the gut microbiota has been shown to influence immune-mediated diseases at distant anatomical sites (eg, lungs, brain, and joints). We demonstrate that a disrupted gut microbiota can be created in a mouse model of hemophilia A using a broad-spectrum antibiotic. Under controlled conditions, this sustained dysbiosis was associated with an increase in splenic B cells and the development of higher titer, FVIII-specific immunoglobulin G antibodies after FVIII challenge. Splenic and mesenteric lymph node cytokines, T cells, and dendritic cells were unaffected before administration of FVIII. However, the immune transcriptome of both aforementioned secondary lymphoid organs was significantly modified. Short-chain fatty acids (SCFAs), which are immunomodulatory microbial metabolites, were depleted in cecal contents of the dysbiotic mice. Furthermore, supplementation of the drinking water with butyrate, the most immunologically active SCFA, successfully achieved attenuation of the FVIII immune response. Collectively, data from this exploratory study suggest that the composition of the gut microbiota alters the FVIII immune response via the action of specific microbial metabolites on the immune cell transcriptome and that oral supplementation with butyrate effectively reduces the FVIII immune response.

In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy.

Our previous studies have demonstrated that platelet-targeted factor IX (FIX) gene therapy can introduce sustained platelet-FIX expression in hemophilia B (FIXnull ) mice. In this study, we aimed to enhance platelet-FIX expression in FIXnull mice with O6 -methylguanine-DNA-methyltransferase (MGMT)-mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter. Platelet-FIX expression in FIXnull mice was introduced by 2bF9/MGMT-mediated hematopoietic stem cell transduction and transplantation. The 2bF9/MGMT-transduced cells were effectively enriched after drug selection by O6 -benzylguanine/1,3-bis-2-chloroethyl-1-nitrosourea. There were a 2.9-fold higher FIX antigen and a 3.7-fold higher FIX activity in platelets, respectively, posttreatment compared with pretreatment. When a 6-hr tail bleeding test was used to grade the bleeding phenotype, the clotting time in treated animals was 2.6 ± 0.5 hr. In contrast, none of the FIXnull control mice were able to clot within 6 hr. Notably, none of the recipients developed anti-FIX antibodies after gene therapy. One of four recipients developed a low titer of inhibitors when challenged with rhF9 together with adjuvant. In contrast, all FIXnull controls developed inhibitors after the same challenge. Anti-FIX immunoglobulin G were barely detectable in recipients (1.08 ± 0.54 µg/ml), an 875-fold lower level than in the FIXnull controls. Our data demonstrate that using the MGMT-mediated drug selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune tolerance in FIXnull mice.

Fludarabine in Combination with Busulfan As Pretransplant Conditioning for Platelet Gene Therapy in Murine Hemophilia a with Inhibitors

Treatment of hemophilia A (HA) with inhibitors is costly, inconvenient, and incompletely effective. We have demonstrated that syngeneic transplantation of hematopoietic stem cells (HSCs) that are genetically modified to express FVIII in platelets restores hemostasis in HA mice with pre-existing anti-FVIII immunity under total body irradiation (TBI) preconditioning, a proof-of principle that platelet gene therapy could be a promising strategy for HA patients. Currently, Fludarabine along with busulfan (FB) has been shown to be highly effective as a preconditioning regimen for allogeneic HSC transplantation (HSCT) in the clinic. Thus, in this study, we aimed to evaluate if FB works as a clinically translatable preconditioning approach for platelet gene therapy of HA with pre-existing inhibitors. Fludarabine was intraperitoneally administered at 100mg/kg/day on Days -7 through -4, and busulfan was given at 25mg/kg/day on Days -2 and -1 into rhFVIII-primed FVIIInull mice. A TBI regimen of 6.6Gy was used as a parallel control. Platelet FVIII expression was introduced by transplantation of 2bF8 lentivirus (2bF8LV)-transduced HSCs. The peripheral blood recovery was monitored by blood count, and cell subset reconstitution was analyzed by flow cytometry. Animals were further analyzed by quantitative PCR (qPCR), FVIII assays, inhibitor assays, and tail bleeding test. Blood counts demonstrated that platelet numbers and hemoglobin levels in the FB group were similar to those in the TBI group after HSCT. The recovery of leukocytes in the recipients with FB conditioning was greater than that in the animals conditioned with TBI at the early stage (4-12 weeks after HSCT). The engraftments in the two groups were comparable in terms of donor- derived leukocyte chimerism during the study course. The percentage of leukocyte chimerism at each time point in the FB group (ranging from 68.6±12.3% to 83.5±8.5%, n=7) was not statistically significantly different than that in the TBI group (ranging from 66.5±15.8% to 74.5±14.8%, n=5-6). All recipients achieved more than 55% donor-derived leukocytes at 20 weeks after HSCT. When we looked into the cell subsets in the recipients, we found that FB preconditioning is favorable for donor-derived myeloid cell reconstitution (the average engraftment ranging from 81.3-85.4% in the FB group, 52.0-62.3% in the TBI group). qPCR results confirmed that HSCs were genetically modified by 2bF8LV in the recipients with either FB or TBI conditioning. The average copy number of the 2bF8 cassette per cell was 0.93±0.17 (n=7) in the FB group, which was not significantly different compared to the TBI group (0.79±0.11, n=6). The average platelet-FVIII levels at each time point ranged from 3.87-5.98 mU/108 platelets in the FB group and 2.21-10.47 mU/108 platelets in the TBI group, respectively. There was no significant difference between the two groups. To assess whether the bleeding phenotype was rescued in HA mice after receiving 2bF8 LV-transduced HSCs, we used a 6-hour (hr) tail bleeding test. All 2bF8 LV-transduced recipients stopped bleeding within 6 hrs with a remaining hemoglobin level of 62.7±20.6%, which was significantly higher than the FVIIInull control group (37.6±4.5%). Of note, none of the HA control mice stopped bleeding within 6 hrs. Notably, we found that both anti-FVIII inhibitor and total IgG titers declined with time in recipients under either FB or TBI preconditioning. When reduction of the inhibitor titer over time was calculated as a half-life (t1/2), there was no significant difference between the FB group and the TBI group. When the inhibitor titer dropped to undetectable, we further challenged recipients with 50 U/kg rhF8 weekly for 4 weeks. There were no detectable anti-FVIII inhibitors in the 2bF8LV-transduced recipients from either the FB or TBI group. In contrast, all the untransduced transplanted HA control mice produced various levels of inhibitors under the same challenge. These data indicate that 2bF8LV-transduced primed-HA mice under either FB or TBI conditioning may induce immune tolerance. In conclusion, fludarabine along with busulfan preconditioning successfully introduced therapeutic levels of platelet FVIII expression without evoking an anti-FVIII memory response in the transduced recipients with pre-existing immunity. Our data suggest that this approach may be a promising and clinically translatable strategy for gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

The Tissue Factor Pathway Inhibitor Antibody, PF-06741086, Increases Thrombin Generation in Rare Bleeding Disorder and Von Willebrand Factor Deficient Plasmas

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration.The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. DisclosuresRakhe:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Anti‐C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model

Background The development of neutralizing anti-factorVIII (FVIII) antibodies remains a challenging complication of modern hemophiliaA care. Invitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance invivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophiliaA and low-titer inhibitors.

Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies.

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Concurrent influenza vaccination reduces anti-FVIII antibody responses in murine hemophilia A

AbstractInflammatory signals such as pathogen- and danger-associated molecular patterns have been hypothesized as risk factors for the initiation of the anti–factor VIII (FVIII) immune response seen in 25% to 30% of patients with severe hemophilia A (HA). In these young patients, vaccines may be coincidentally administered in close proximity with initial exposure to FVIII, thereby providing a source of such stimuli. Here, we investigated the effects of 3 vaccines commonly used in pediatric patients on FVIII immunogenicity in a humanized HA murine model with variable tolerance to recombinant human FVIII (rhFVIII). Mice vaccinated intramuscularly against the influenza vaccine prior to multiple infusions of rhFVIII exhibited a decreased incidence of rhFVIII-specific neutralizing and nonneutralizing antibodies. Similar findings were observed with the addition of an adjuvant. Upon exposure to media from influenza- or FVIII-stimulated lymph node or splenic lymphocytes, naïve CD4+ lymphocytes preferentially migrated toward media from influenza-stimulated cells, indicating that antigen competition, by means of lymphocyte recruitment to the immunization site, is a potential mechanism for the observed decrease in FVIII immunogenicity. We also observed no differences in incidence or titer of rhFVIII-specific antibodies and inhibitors in mice exposed to the live-attenuated measles-mumps-rubella vaccine regardless of route of administration. Together, our results suggest that concomitant FVIII exposure and vaccination against influenza does not increase the risk of inhibitor formation and may in fact decrease anti-FVIII immune responses.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

Effective Therapy for Murine Hemophilia A by Enhancing Factor VIII Expression in Platelets

Introduction: Platelets may comprise an ideal vehicle for delivering factor VIII (FVIII) in hemophilia A (HemA) as FVIII stored in platelet α-granules is protected from neutralization by inhibitory antibodies and, during bleeding, activated platelets locally excrete their contents to promote clot formation. In our previous study, it was demonstrated that intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a transgene encoding human factor VIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) successfully transduced hematopoietic stem cells (HSCs) in HemA mice without the requirement of preconditioning as in ex vivo gene therapy. FVIII expressed and then stored in platelets can partially correct the HemA phenotype over 5 months in mice with or without pre-existing inhibitors.Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to improve the efficiency of human FVIII cDNA by testing a new human FVIII variant (a kind gift from Dr. Weidong Xiao), F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain). The other was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents.Results:We first tested the FVIII expression levels from two human FVIII variants by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 μg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Based on this result, two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+ 293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses (Figure 1). These results indicated that F8X10K12 may further enhance FVIII gene expression for more effective therapy. Two LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated. Secondly, the immune competent C57BL6 mice were pretreated with both dexamethasone (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21). IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0 (Figure 2a). On day 7, drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (11.8±2.1% vs 6.9±3.1%, P =0.005) and GFP+ Lineage- Sca1+ cKit+ HSCs (48.3±6.1% vs 44.4±17.2%, P =0.31) compared with LV-only treated mice (n=3) (Figure 2b). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P <0.001) in the total bone marrow and GFP+ HSCs (10.7±3.3% vs 2.6±0.6%, P <0.001) were observed in drugs + LVs treated mice (n=3) compared with LV-only treated mice on day 160 after LV infusion (n=3) (Figure 2c).Conclusion: We found that a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. In addition, administration of dexamethasone that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+ HSCs in treated mice up to 160 days. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with dexamethasone and antiCD8α mAb can be used to further enhance and prolong transgene expression levels in platelets for effective correction of hemophilia A. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The important role of von Willebrand factor in platelet‐derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies

Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. 2bF8 transgenic mice in the FVIII(-/-) background (2bF8(tg+/-) F8(-/-) ) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. Only 18% of 2bF8(tg+/-) F8(-/-) VWF(-/-) animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000 BU mL(-1) . In contrast, 82% of 2bF8(tg+/-) F8(-/-) VWF(+/+) mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50,000 BU mL(-1) . All 2bF8(tg+/-) F8(-/-) VWF(-/-) mice without inhibitors survived tail clipping and no VWF(-/-) F8(-/-) mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors.

Expanding the ortholog approach for hemophilia treatment complicated by factor VIII inhibitors

The formation of neutralizing antibodies (inhibitors) directed against human coagulation factor VIII (hFVIII) is a life-threatening pathogenic response that occurs in 20-30% of severe congenital hemophilia A patients and 0.00015% of the remaining population (i.e. acquired hemophilia A). Interspecies amino acid sequence disparity among FVIII orthologs represents a promising strategy to mask FVIII from existing inhibitors while retaining procoagulant function. Evidence for the effectiveness of this approach exists in clinical data obtained for porcine FVIII (pFVIII) products, which have demonstrated efficacy in the setting of congenital and acquired hemophilia. In the current study, recombinant (r) ovine FVIII (oFVIII) was evaluated for antigenicity and procoagulant activity in the context of human patient-derived and murine model-generated FVIII inhibitors. The antigenicity of roFVIII was assessed using (i) inhibitor patient plasma samples, (ii) murine anti-FVIII MAbs, (iii) immunized murine hemophilia A plasmas and (iv) an in vivo model of acquired hemophilia A. Overall, roFVIII demonstrated reduced reactivity to, and inhibition by, anti-hFVIII immunoglobulin in patient plasmas. Additionally, several hFVIII epitopes were predicted and empirically shown not to exist within roFVIII. In a murine hemophilia A model designed to mimic clinical inhibitor formation, it was demonstrated that inhibitor titers to roFVIII were significantly reduced when compared with the orthologous immunogens, rhFVIII or rpFVIII. Furthermore, in a murine model of acquired hemophilia A, roFVIII administration conferred protection from bleeding following tail transection. These data support the investigation of FVIII orthologs as treatment modalities in both the congenital and acquired FVIII inhibitor settings.

High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

AbstractNeutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed “inhibitors,” bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified “functional” B-cell epitopes, defined as those residues contributing appreciable antigen–antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigen–antibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients.

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

Murine Hemophilia A With Or Without Pre-Existing Anti-Factor VIII Inhibitors Is Partially Corrected By Factor VIII Stored In Platelets After Intraosseous Infusion Of Lentiviral Vectors Into Bone Marrow Without Preconditioning

IntroductionPlatelets may comprise an ideal vehicle for delivering FVIII in hemophilia A (HemA) as FVIII stored in platelet α-granules is protected from neutralization by inhibitory antibodies and, during bleeding, activated platelets locally excrete their contents to promote clot formation. In order to avoid specific challenges posed by ex vivo gene delivery including, in particular, the requirement to pre-condition the subject, we evaluated intraosseous (IO) infusion of self-inactivating lentiviral vectors (LV) for in situ gene transfer into bone marrow cells. In previous studies, we confirmed that hematopoietic stem cells (HSCs) can be efficiently transduced to express GFP after IO administration of LV driven by a MND promoter (M-GFP-LV). MethodsIn the current study, we aimed at limiting transgene expression to the megakaryocyte lineages using IO delivery of 20 µL LV containing either GFP (G-GFP-LV) or a B-domain variant human FVIII (G-F8-LV) gene under the control of megakaryocyte strictly specific promoter glycoprotein 1bα (Gp1bα). ResultsIn M-GFP-LV treated control mice, GFP was detected in 6.4% of HSCs, 3.4% of B220+, and 9.0% of CD11c+ bone marrow cells on day 29. In contrast, in G-GFP-LV (6.0E+08 TU/mL) treated mice, GFP was undetectable in bone marrow HSCs, B220+, CD11c+ or CD11b+ cells. GFP expression level in platelets of G-GFP-LV treated mice was ten folds of that in M-GFP-LV treated mice (0.1% vs 0.01%). It indicated that in platelets, the activity of Gp1bα was stronger than that of MND. More importantly, GFP expression levels were stable over 100 days, suggesting that platelets containing the transgene products did not elicit transgene-specific immune responses. Next, we treated HemA mice with G-F8-LV (6.0 E+07 TU/mL). There was no detectable hFVIII expression in bone marrow HSCs on day 8 or in blood cells (CD3ε+, B220+, CD11c+ or CD11b+) on day 35. However, up to 3% platelets express hFVIII on day 91. These results suggested that HSCs in HemA mice were successfully transduced by G-F8-LV after IO infusion, and in the long term, FVIII was synthesized in megakaryocytes and stored in platelet α-granules. In treated mice, the average percentage of platelets expressing hFVIII was stable at 1-2% from day 27 to day 160. The average FVIII antigen level in platelets on day 112 was 1 mU per 1 × 108 platelets, which was comparable with platelet FVIII in transgenic and ex vivo gene therapy treated mice. We also evaluated LV-treated HemA animals for phenotypic correction of bleeding diathesis by tail clip assay. The blood loss was 41% (n=7), 48% (n=5) and 33% (n=5) compared with control HemA (normalized to 100%), mock treated HemA (∼100%), and wild-type (2.5%) mice on days 35, 118 and 160, respectively. Additionally, there was neither detectable FVIII activity nor anti-FVIII antibodies in blood on day 160, which indicated that there was insignificant leaky expression of FVIII in other cells. Finally, we also infused G-F8-LV into HemA inhibitor mice. Inhibitors were induced by repeated injection of 3U recombinant hFVIII. The average antibody level was 80 Bethesda Unit before IO infusion of the vectors. In G-F8-LV treated mice, the average hFVIII antigen level on day 27 was 0.74 mU per 1 x 108 platelets (n=5). Bleeding assay was performed on day 160. The blood loss of treated mice was significantly reduced compared with untreated HemA mice, indicating that IO infusion of G-F8-LV can overcome anti-FVIII antibodies and correct hemophilia phenotype. ConclusionWe have successfully transduced HSCs in situ by a single infusion of LVs into bone marrow to correct hemophilia A. Gp1bα promoter in lentiviral vectors can specifically direct the transgene expression in mouse platelets. Following IO infusion of G-F8-LV, FVIII stored in platelets can persistently and partially correct the HemA phenotype for at least five months (experimental duration) in mice with and without pre-existing inhibitors. Overall, direct transduction of bone marrow cells targeting platelet-specific FVIII expression may provide an effective therapy to treat severe hemophilia A patients with high-titer inhibitors. Disclosures:No relevant conflicts of interest to declare.

The Potential to Enhance Hemostasis by Inhibiting Tissue Factor Pathway Inhibitor

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factors (TF) X and VIIa. As such, it is a major regulator of the extrinsic clotting pathway. Compounds that block TFPI activity, or “inhibit the inhibitor,” effectively restore hemostasis in animal models of hemophilia. These compounds are thought to act by allowing unfettered TF-mediated thrombin generation through the extrinsic pathway in a manner that bypasses the need for the intrinsic pathway proteins, factor VIII and factor IX. Improved understanding of TFPI tissue expression and function is needed to better recognize how pharmaceutical agents targeting TFPI alter biochemical and physiological mechanisms of blood coagulation. Several advances have been made recently. TFPI is an alternatively spliced protein with two primary isoforms, TFPIα and TFPIβ, which have distinct cellular expression patterns and anticoagulant functions. TFPIα is produced by endothelial cells, and appears to localize within an intracellular granule with release following stimulation with thrombin or heparin. TFPIα is also produced by megakaryocytes and is released from activated platelets. Platelet TFPIα limits clot growth following vascular injury and alters bleeding in a murine hemophilia model. Interestingly, the anticoagulant activity produced by platelet TFPIα appears to be isoform specific and not mediated through inhibition of TF. TFPIβ is made by endothelial cells, localizes to endothelium, and is not present in platelets. TFPIβ is an effective inhibitor of TF-mediated cellular migration and dampens the effects of TF-induced intravascular coagulation in mice. This new knowledge of TFPI isoform expression patterns and anticoagulant activities has important implications for the development of TFPI-blocking pharmaceuticals to treat hemophilia. The recent findings also provide new insights into the biochemical mechanisms that activate blood coagulation pathways and regulate TF-mediated thrombotic and inflammatory disease. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Hemarthrosis in hemophilic mice results in alterations in M1-M2 monocyte/macrophage polarization

Joint bleedings result in iron-mediated synovitis and cartilage destruction. Monocyte/macrophage polarization affects their role in iron homeostasis. This study evaluates the effects of hemarthrosis on monocyte/macrophage polarization. Using a murine hemophilia model of acute joint bleeding and flow cytometry, we evaluated monocyte/macrophage polarization in blood, spleen, synovium, and knee lavage at day 1, 2, and 7 following the induction of hemarthrosis. Induction of hemarthrosis resulted in a transient shift of blood monocytes towards a M1 type (control 13 vs. 1847 counted cells at day 1; p<0.01), a temporary decrease of spleen M1 monocytes (control 2841 vs. 1086 counted cells at day 1; p=0.02), and a sustained decrease of spleen M2 red pulp macrophages (control 1853 vs. 673 counted cells at day 7; p=0.01). In addition, an increase in M1 (control 119 vs. 592 counted cells at day 1; p=0.04) and M2 (control 247 vs. 650 counted cells at day 1; p=0.02) synovial macrophages was noted. In the joint lavage, a temporary increase in M1 monocytes (control 20 vs. 125 counted cells at day 1; p=0.04) and a more sustained increase in M2 monocytes (control 73 vs. 186 counted cells at day 2; p<0.01) was observed. This study demonstrates alterations in monocyte/macrophage polarization following hemarthrosis resulting in a blood monocyte M1 phenotype and a combined M1-M2 monocyte/macrophage phenotype in the joint. Based on the different capabilities of M1 and M2 cells, modulating polarization of distinct monocyte/macrophage populations might represent interesting prophylactic or therapeutic approaches for joint bleedings.