Recombinant Human FVIII 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.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

Co-administration of FVIII with IVIG reduces immune response to FVIII in hemophilia A mice

Hemophilia A is an X-linked recessive congenital bleeding disorder. Exogenous infusion of FVIII is the treatment of choice, and the development of immunoglobulins against FVIII (inhibitors) remains the major challenge in clinical management of the disease. Here, we investigated the effect of co-administration of FVIII with intravenous immunoglobulin (IVIG) on the development of inhibitors in previously untreated hemophilia A mice. A group of hemophilia A mice (C57BL/6FVIII−/−) received weekly injections of recombinant human FVIII (rFVIII) for twelve consecutive weeks while a second group received co-injections of rFVIII + IVIG. An in-house enzyme-linked immunosorbent assay (ELISA) was designed to detect antibodies to rFVIII. Every mouse in the first group developed antibodies to rFVIII. In contrast, mice treated with rFVIII + IVIG showed significantly lower antibody titers. Interestingly, when co-administration of IVIG was discontinued after 12 weeks in some mice (rFVIII continued), these mice experienced an increase in antibody titer. In contrast, mice that continued to receive rFVIII + IVIG retained significantly lower titers. In conclusion, prophylactic rFVIII co-administration with IVIG modulated the immune response to FVIII and resulted in decreased anti-FVIII antibody titer. These findings suggest that co-injection therapy with IVIG could potentially be effective in the management of hemophilia A patients at risk of inhibitor development.

Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model.FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period.We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway.In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression after Hemarthrosis Differs between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein

IntroductionLocal and systemic molecular responses to hemarthroses in hemophilia are not well understood. Emerging clinical evidence suggests that treatment with FVIII-Fc Fusion protein (FcFVIII), using the Fc-portion of immunoglobulin for half-life extension, may mitigate joint inflammation and modulate immune stimulation. We analyzed gene expression profiles in synovium and spleen (a major immune regulatory organ) in FVIII-deficient mice at baseline and after hemarthrosis treated with recombinant human FVIII (rhFVIII) or murine (m)FcFVIII to characterize the respective differences and molecular pathways for each FVIII preparation.MethodsHemarthrosis was induced by sub-patellar needle puncture in FVIII-deficient mice treated with saline (vehicle), 100 IU/kg mFcFVIII (Fc murine due to species specificity) or 120 IU/kg rhFVIII (to account for differences in t 1/2) administered intravenously 2 hours before and 6 hours after injury. Spleen and synovium were harvested on day 3 or day 14 post-injury (n=3-5 per group). Spleen and synovium from uninjured mice 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 (SPIA). Molecular pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway databases.ResultsKnee injury in FVIII-deficient mice resulted in hemarthrosis and a drop in mean hematocrit from 44.7% to 27.5% (saline), which was prevented by rhFVIII and mFcFVIII prophylaxis (mean day 2 hematocrit ~45%).In the spleen, differential gene expression (DGE) in saline treated mice was pronounced but fleeting with 5295 and 9 differentially expressed genes (DEG) on day 3 and 14. DGE was abrogated by either rhFVIII or mFcFVIII, without significant expression differences between rhFVIII and mFcFVIII at the single gene level. However, differences were noted between rhFVIII and mFcFVIII during KEGG pathway analyses, where rhFVIII predominantly perturbed pathways related to inflammation, T-cell signaling and innate immune regulation, while mFcFVIII perturbed pathways related to B-cell signaling and cellular homeostasis.In the synovium, DGE in saline treated mice was also pronounced on day 3 (3388 DEG) but, in contrast to the spleen, continued on day 14 (1030 DEG). Unlike in the spleen, DGE was not fully corrected with rhFVIII or mFcFVIII; moreover, gene expression differed between rhFVIII and mFcFVIII. This was particularly evident on day 14, with a partial reduction to 603 and 294 differentially expressed genes with rhFVIII and mFcFVIII, respectively. Only 163 genes overlapped, whereas 440 were unique to rhFVIII and 131 to mFcFVIII. A comparison between gene expression with rhFVIII and mFcFVIII yielded that ≥ 2 log-fold expression difference was present for 74 genes, with 72 genes upregulated with rhFVIII relative to mFcFVIII. Many of these genes are involved in pathways related to innate and adaptive immune responses, cell cycle regulation and extracellular matrix organization in the Reactome database. This response was dampened by mFcFVIII.ConclusionsJoint bleeding in hemophilic mice induced pronounced systemic and synovial gene expression, which was diminished by rhFVIII or mFcFVIII. However, while there were many similarities with regard to regulation of gene expression and/or molecular pathways, there were also notable differences, especially involving immune regulatory, inflammatory and tissue repair functions. These observations provide new molecular insights regarding Fc-driven effects of FVIII after hemarthrosis, suggesting that the type of FVIII preparation used for bleed control may have implications beyond hemostasis. Disclosuresvon Drygalski: Hematherix, Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Super FVa; uniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Biomarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

A Novel Von Willebrand Factor Fragment Acts As a Chaperone to Prolong the Half-Life of Recombinant Human FVIII (Simoctocog Alfa) in Hemophilia a Dogs and FVIII/VWF Double Knockout Mice

Introduction/Objective: Subcutaneous (SC) drug delivery represents a convenient method of administration compared to intravenous (IV) delivery. However, due to very low bioavailability this route is not yet applicable for the administration of FVIII as a therapy for hemophilia A (HA). von Willebrand factor (VWF), in particular the D'D3 domains, is known to protect FVIII from proteolytic degradation, rapid clearance and binding to membranes. Therefore VWF represents a promising candidate for a strategy to increase FVIII bioavailability after SC treatment. Different VWF fragments containing at least the D'D3 domains were engineered in order to achieve high FVIII binding affinity, prolonged half-life of the VWF fragment, and consequently high bioavailability of FVIII.Materials/Methods: A modified VWF fragment (OCTA12) or an unmodified D'D3 domain, both with a C-terminal Strep-Tag, was transiently expressed in HEK293 cells and purified by Strep-Tactin® affinity chromatography. Recombinant human FVIII (rhFVIII, simoctocog alfa) complexed with OCTA12, D'D3 or full-length plasma-derived VWF (flVWF, Wilate®) was administered via IV route to FVIII/VWF double knockout (FVIII/VWF DKO) mice at 240 IU FVIII/kg in order to test the ability of the fragments to stabilize FVIII in circulation. Blood samples were taken at 5 minutes and 1, 4, 8, 12, 24, 36 and 48 hours after treatment and analyzed for FVIII activity. Subsequently, IV and SC administration of the complex of rhFVIII and OCTA12 or rhFVIII alone was evaluated in a HA dog model. For this purpose, HA dogs were treated according to the scheme presented in Table 1. Blood samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 32, 48, 72 and 94 hours after treatment and analyzed for FVIII activity.Results: Five minutes after IV administration of rhFVIII complexed with different VWF variants to FVIII/VWF DKO mice, peak activity levels were determined (expressed in % mean ± SEM, n = 15): rhFVIII alone 78.9 ± 6.1; rhFVIII/D'D3 351.2 ± 22.3; rhFVIII/OCTA12 295.9 ± 17.4; rhFVIII/flVWF 404.4 ± 20.5. The FVIII activity levels were significantly higher (statistically evaluated by Student's t-test) when rhFVIII was administered with any VWF variant in comparison to rhFVIII administered alone. The three co-administered VWF species D'D3, OCTA12 or flVWF did not differ significantly in terms of measured FVIII levels. rhFVIII administered alone was rapidly cleared from the circulation (T1/2 = 0.07 h). Co-administration with flVWF or D'D3 resulted in a 4.5- or 6.4-fold prolongation of the half-life respectively (T1/2 = 0.32 h and T1/2 = 0.45 h respectively). The highest half-life prolonging effect (14.7-fold) was measured when rhFVIII was co-administered with OCTA12 (T1/2 = 1.03 h). The IV administration of rhFVIII alone (200 IU/kg) or complexed with OCTA12 (100 IU/kg) in the HA dog resulted in peak FVIII activity level of 215% or 134% respectively at 30 minutes post-infusion (mean of two injections). The SC administration of rhFVIII complexed with OCTA12 resulted in a 10.8% activity level at 30 minutes post-infusion and reached a peak level of 24.6% at 8 h post-infusion (mean of three injections). For calculation of bioavailability, the area under the curve (AUC) of FVIII activity after IV administration of rhFVIII alone was defined as 100%. The AUC of IV-administered rhFVIII/OCTA12 complex at 100 IU/kg (half the dose) was 90%. Bioavailability of FVIII after SC administration of the rhFVIII/OCTA12 complex was 37.7%.Conclusions: The co-administration of rhFVIII with the novel engineered VWF fragment OCTA12 results in high FVIII bioavailability after SC administration in HA dogs. The bioavailability of FVIII was also increased after IV administration of the FVIII/OCTA12 complex in FVIII/VWF DKO mice and HA dogs. Moreover, when complexed with OCTA12, FVIII exhibits prolonged half-life in FVIII/VWF DKO mice. [Display omitted] DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

Evaluation of Plasma-Derived FVII Efficacy for the Treatment of Bleeding in a Murine Model of Hemophilia a

BackgroundRecombinant human activated factor VII (rhFVIIa) has been used successfully to treat hemophilic patients with neutralizing antibodies to FVIII or FIX. High doses of FVIIa are known to be effective in opposing bleeding in hemophilia B mice and in a mouse model of antibody-induced hemophilia A. The therapeutic indications for human plasma-derived coagulation factor VII (pdFVII) include the prophylaxis and treatment of bleeding disorders in isolated congenital or acquired factor VII deficiency. These preparations are considered to be ineffective in hemophilia A in the presence of inhibitors as they do not contain activated factor VII and therefore cannot be used in haemophiliac patients with inhibitors.AimHere, we investigated the potential use of pdFVII to induce hemostasis in a mouse model of congenital hemophilia A with high-titer inhibitors and antibody-induced hemophilia A in wild-type mice.MethodsF8 KO mice were given 2 IU recombinant human FVIII intravenously weekly for 4 weeks. All mice that developed high-titer inhibitors (>5 BU/ml) were selected to be used in tail transection experiments. Additionally, a model of antibody-induced hemophilia was used by injecting C57BL/6 wild-type mice with 2 doses of murine plasma containing high-titer inhibitors. Mice were anesthetized and tails transected at a cross-sectional diameter of 1.5 mm; the tail was immediately placed in thermostated saline, and the duration of active bleeding was recorded. Bleeding was monitored for 10 minutes. Blood loss was determined by measuring the amount of released haemoglobin in the saline. Mice were pre-treated 15 minutes before haemostatic challenge with a single i.v. injection of saline, rhFVIIa (4 mg/kg) or pdFVII (500 IU/kg).ResultsAdministration of both rhFVIIa and pdFVII corrected the bleeding tendency in both animal models. In particular, pdFVII and rFVIIa treatments caused a marked reduction in the mean bleeding index score of hemophilia A mice with inhibitors in comparison to mice treated with vehicle only (vehicle, mean=5750, s.e.m.= 438; pdFVII, mean=1301, s.e.m.= 819; rFVIIa, mean=1101, s.e.m.=565; P < 0.001 for pdFVII vs vechicle and rFVIIa vs vehicle comparisons, NS for rFVIIa vs pdFVII; ANOVA followed by Bonferroni's multiple comparison test). Comparable results were obtained in the antibody-induced hemophilia model. No statistically significant differences were observed between groups treated with rFVIIa or pdFVII in both models.ConclusionsOur results provide evidence that pharmacological doses of both rFVIIa and pdFVII are effective in promoting hemostasis in animal models of congenital hemophilia A with inhibitors and antibody-induced hemophilia A in wild-type mice. DisclosuresNo relevant conflicts of interest to declare.

Pharmacology and Pharmacokinetics of NXT007; Emicizumab-Based Engineered Fixa/Fx Bispecific Antibody with Improved Properties

Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.

Platelet-Targeted Gene Therapy Induces Robust Immune Tolerance Even in a Primed Model Via Peripheral Clonal Deletion of CD4 and CD8 T Cells and Expansion of Treg Cells

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) results in effective immune tolerance induction to FVIII and FIX as well as the non-coagulant protein ovalbumin (OVA) in the unprimed model. Here we explored how the reactivity of immune responses affects the efficacy of transgene expression and the potential immune tolerance mechanisms in platelet-specific gene transfer in the primed model. We used OVA as a surrogate protein for this study. Recipient wild-type (WT) CD45.1 mice were primed with OVA protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either WT/CD45.2 or OTII/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. We found that pre-existing high-reactive immune responses devastate platelet-OVA expression in recipients in both the WT/WT and OTII/WT models. Using the WT/WT mouse model, 6 of 7 2bOVA-transduced recipients had sustained platelet-OVA expression with levels ranging from 4.53-8.84 ng/108 platelets. Anti-OVA total IgG titers declined with time in 2bOVA-transduced OVA-primed recipients even when rechallenged with OVA. In contrast, recall memory immune responses were elicited in 2bGFP and untransduced transplanted controls. Furthermore, full-thickness tail skin grafts from CAG-OVATg mice were successfully engrafted onto 2bOVA-transduced recipients and survived throughout the remaining lifetimes of the animals. In contrast, skin grafts were completely rejected in control 2bGFP-transduced and untransduced transplanted recipients. To ensure that the immune system was not inactive in 2bOVA-transduced recipients, animals were immunized with unrelated antigen recombinant human FVIII (rhF8) using a protocol known to induce anti-F8 immune responses even in WT mice. All 2bOVA-transduced recipients developed anti-F8 inhibitory antibodies after rhF8 immunization with no differences between the 2bOVA group and the 2bGFP and untransduced control groups. Using the OVA-specific CD4 TCR transgenic model (OTII/WT), we showed that sustained platelet-OVA expression was achieved in 90% of 2bOVA-transduced recipients. OVA-specific CD4 T cells were deleted and Treg cells were expanded in peripheral lymphoid organs in 2bOVA-transduced OVA-primed recipients. The levels of platelet-OVA expression negatively correlates with the percentages of OVA-specific CD4 T cells, but positively correlates with Treg cells. Immune tolerance was also achieved in the OTII/WT model even though all donor-derived CD4 T cells are OVA-specific. However, our studies showed that high-reactive immune responses could influence the neoprotein expression in platelet-targeted gene therapy. Using the OVA-specific CD8 TCR transgenic model (OTI/WT), in which all donor-derived CD8 T cells are OVA-specific, we found that low levels of platelet-OVA expression were obtained at week 5 after transplantation (0.72±0.65 ng/108 platelets) and then dropped to barely detectable at subsequent time points. Flow cytometry analysis showed that there were 33.9±12.9% platelets positive with GFP in 2bGFP LV-transduced recipients, demonstrating that high transduction efficiency and successful transplantation were achieved. Of note, OVA-specific CD8 T cells in the 2bOVA group were 48% lower than in the 2bGFP control group, demonstrating that antigen-specific CD8 T cells were partially deleted in the OTI/WT model after platelet-targeted OVA gene transfer. Interestingly, we found that the percentage of the antigen-specific CD8+Foxp3+ cells in the 2bOVA group was significantly higher than in the 2bGFP group (0.19±0.14% vs. 0.05±0.01%, respectively). These results suggest that there is a counterpace process between immune reaction and immune tolerance after platelet-targeted gene therapy. In conclusion, our studies demonstrate that platelet-targeted gene therapy can induce antigen-specific immune tolerance even when the immune response has been mounted via peripheral clonal deletion of antigen-specific CD4 and CD8 T cells and expansion of Tregs, suggesting that platelet-targeted gene therapy is a promising approach to induce immune tolerance for treatment of diseases with undesired immune responses or even with pre-existing immune responses, such as hemophilia A with inhibitors or autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

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.

Effects of Hemostasis Correction on Vascular Remodeling and Synovial Gene Expression Changes in Mice after Induced Hemarthrosis

IntroductionRepeated joint bleeding in patients with hemophilia leads to hemophilic arthropathy (HA), which cannot be entirely prevented by clotting factor replacement. Vascular remodeling and permeability are associated with hemarthrosis and may contribute to HA progression; however, the mechanisms and effects of hemostasis correction are poorly understood. Here, we explored synovial vascular and gene expression changes in FVIII-deficient mice after induced hemarthrosis +/- FVIII replacement, and in wild-type mice with reversible hemostasis suppression.MethodsHemarthrosis was induced in FVIII-deficient mice by sub-patellar needle puncture +/- 100-200 IU/kg recombinant human FVIII (rhFVIII) 2 hours before and 6 hours after injury. Wild-type mice were treated with 10 µg/ml warfarin for 7 days and 0.25 mg/kg anti-FVIII 2 hours before injury (hypoBALB/c). After injury, warfarin was continued for 2 weeks or reversed on day 2 with 100 IU/kg 4-factor prothrombinase complex concentrate (4F-PCC). Vascularity and gene expression were analyzed at baseline and 2 weeks post-injury. Vessel number and remodeling were assessed by histology with Safranin-O-Fast Green and α-smooth muscle actin (αSMA) staining, respectively, and microvascular flow was detected by musculoskeletal ultrasound with Power Doppler. The permeability of synovial vessels was determined by quantification of extravasated albumin-bound Evans blue dye in knee joints. For synovial gene expression studies, libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit and sequenced on an Illumina NextSeq500 (single-end; 75bp reads). The limma-voom method (R BioConductor) was used for differential expression analyses and functional enrichment was performed using Signaling Pathway Impact Analysis.ResultsIn FVIII-deficient mice, knee injury caused profound hemarthrosis that was largely prevented with rhFVIII prophylaxis (day 2 hematocrit: 26.4% and 46.3%). Soft tissue proliferation increased to a similar extent, as did various vascular parameters: microvascular flow (vehicle: 1.8-fold; rhFVIII: 1.5-fold), vessel number (vehicle: 2.3-fold, p=0.0005; rhFVIII: 2.0-fold, p=0.004), vessels with diameter ≥ 20 µm (vehicle: 2.9-fold, p=0.02; rhFVIII: 2.7-fold, p=0.02), and αSMA area per vessel (vehicle: 2.3-fold, p>0.05; rhFVIII: 3.6-fold, p=0.0006). Vascular permeability also increased significantly (1.7-fold, p=0.0007) and was only partially rescued by rhFVIII prophylaxis (1.3-fold, p>0.05). In hypoBALB/c mice (day 2 hematocrit: 29.6%), significant but less pronounced vascular changes occurred regardless of hemostasis correction, and without associated permeability, suggesting this is uniquely associated with FVIII-deficiency. RNA sequencing in FVIII-deficient mice revealed a strong transcriptional response to hemarthrosis (1527 differentially expressed genes (DEG), 13 perturbed pathways) that was only partially dampened with rhFVIII treatment (891 DEG, 20 pathways). Perturbation of extracellular matrix (ECM)-receptor interactions was highly significant in both groups (vehicle: pNDE=7.7x10-10; rhFVIII: pNDE=6.8x10-9). Similarly, numerous genes relating to angiogenesis and ECM remodeling, including collagens and MMPs, were up-regulated and minimally affected by rhFVIII treatment. These transcriptional changes may facilitate the observed vascular remodeling after hemarthrosis.ConclusionsHemarthrosis triggers profound changes in synovial gene expression, notably ECM components, that may drive the associated soft tissue and vascular changes, including vessel remodeling and leakiness. These processes are incompletely mitigated by hemostasis correction and may exacerbate (re-) bleeding tendencies. Therefore, further exploration is needed to identify key molecular pathways that can be targeted to intercept the progression of HA. Disclosuresvon Drygalski:UniQure BV, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy.

Generation of a Unique Cohort of Hemophilia A Dogs Tolerant to Human FVIII for Evaluating the Safety and Efficacy of AAV Delivery of Wild Type and Variant Human FVIII

The hemophilia A (HA) dogs have been a valuable model for evaluating the efficacy of novel hemophilia therapeutics. These dogs have a mutation that is analogous to the most common mutation in humans with severe disease, the intron 22 inversion. The advantages of the hemophilia dog model include: (1) it was predictive of the therapeutic adeno-associated viral (AAV) vector dose in human factor IX (hFIX) clinical trials, (2) it is an outbred immunocompetent species, (3) it demonstrates a clinical bleeding phenotype consistent with patients, including hemarthrosis, and (4) it permits long-term investigation of both efficacy and safety. Our previous studies delivering AAV-canine factor VIII (cFVIII) in the HA dogs demonstrated long-term (up to 10 years of follow-up) dose-dependent cFVIII expression without evidence of an immune response to the cFVIII protein. In hemophilia B preclinical studies, the comparable biological characteristics between canine FIX and hFIX allows preclinical results using cFIX to be translated to clinical studies. In contrast, for hemophilia A, our studies of recombinant cFVIII and human FVIII (hFVIII) proteins demonstrate that cFVIII is a more stable protein that has higher biological activity and is secreted better than hFVIII (Sabatino et al. 2009). Thus, expression of cFVIII following AAV delivery does not accurately predict the therapeutic dose of AAV-hFVIII which is relevant for translation to clinical trials. The challenge of administering AAV-hFVIII to the HA dogs is that this expressed xenoprotein (hFVIII) results in inhibitor formation that precludes the ability to measure transgene (hFVIII) expression. We hypothesized that tolerizing HA dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response of AAV8-hFVIII versus a novel hFVIII variant that has increased activity and secretion (Nguyen et al. 2017). In this study we used a neonatal retroviral (RV) delivery approach to tolerize the HA dogs (n=5) to B-domain deleted hFVIII (hFVIII-BDD). HA neonatal male dogs (S28, S29, V06, V26, V27) were treated with the retrovirus (RV-hAAT-hFVIII-BDD-WPRE) (3x10e9 TU/kg) on day 2 of life. The levels of hFVIII expression after RV delivery were 0.3-6% 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. At 5-6 months of life the dogs (S28, S29, V06) were challenged with hFVIII-BDD (Xyntha; 25IU/kg; I.V.) weekly for 6 consecutive weeks. Samples were collected before and 15 minutes after each protein challenge to demonstrate the successful infusion of the protein. At 4 weeks after the final challenge, no anti-hFVIII IgG1 or IgG2 antibodies were detected consistent with no evidence of an inhibitor. At 4.5 years of age, S28 and S29 had stable expression of 0.5-1% of hFVIII and were rechallenged with hFVIII-BDD (Xyntha; 25IU/kg per week x 6 wks). No anti-hFVIII IgG1 or IgG2 antibodies were detected 4 weeks after the final protein challenge. Thus, these data demonstrate that all of the RV-hAAT-hFVIII-BDD-WPRE treated dogs that have been challenged (n=3) have been tolerant to hFVIII-BDD. Since the goal of this study is to generate a cohort of HA dogs tolerant to hFVIII-BDD to address the efficacy and safety of AAV8-hFVIII-BDD versus a hFVIII-BDD variant, we treated the first dog (S29) 12 weeks after the second series of protein challenges with an optimized AAV vector cassette containing a codon-optimized hFVIII sequence with a modified transthyretin (TTRm) promoter, AAV8-TTRm-hFVIII-BDD (2x10e12 vg/kg). Prior to AAV administration the hFVIII activity was 0.2%. At 8 weeks post AAV administration, the hFVIII activity increased to 3.5%. No anti-hFVIII-BDD IgG1 or IgG2 was detected after AAV-hFVIII administration. These data demonstrate that low levels of sustained hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII. Overall, these studies demonstrate that the neonatal HA dogs treated with a retrovirus targeting hFVIII expression to the liver are tolerant to hFVIII and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of our novel FVIII variants. DisclosuresSabatino:Spark Therapeutics: Patents & Royalties.

The Severe Spontaneous Bleeding Phenotype in a Novel Hemophilia a Rat Model with an Inversion Mutation Is Rescued By Platelet-Targeted FVIII Expression

Previous studies by our group and others have demonstrated that platelet targeted FVIII expression can rescue the bleeding phenotype in hemophilia A (HA) mice in both the inhibitor and non-inhibitor models. The clinical efficacy has been further proven in a dog HA non-inhibitor model. While these are informative, neither the canine nor mouse model entirely mimic human disease because 1) neither HA mice nor dogs exhibit the frequent spontaneous bleeding in severe HA patients, and 2) canine platelets do not contain FVIII carrier protein VWF and therefore store and release FVIII differently, thus precluding examining the efficacy of platelet gene therapy in the presence of inhibitors.The efficacy of platelet-FVIII gene therapy, therefore, needs to be examined in an animal model more representative of human disease. Since the inversion of FVIII gene is the most common mutation that causes severe HA in patients, using a CRISPR/Case9 strategy we generated a novel HA rat model in a Dahl/Salt Sensitive (Dahl) inbred congenic background (Dahl-F8KO) with the nearly entire murine FVIII gene inverted causing a translational stop 6 amino acids after the signal sequence of FVIII. There is no detectable FVIII in plasma in Dahl-F8KO rats (LOD is 1 U/dL). Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 56.5% of homozygous affected animals (n=46) by the age of 8-weeks and up to 82.6% by the age of 32-weeks. This was significantly higher (P<0.0001) than the occurrence of spontaneous bleeding in another colony in Sprague Dawley (SD) background developed by Nielsen et al. using zinc finger nuclear strategy (J Thromb Haemost 2014) with a 13-bp deletion in exon 16 of FVIII. In SD-F8KO rat model, spontaneous bleeds occur in 23.3% and 42.1% of homozygous affected animals (n=85) that were housed in our facility by the age of 8- and 32-weeks, respectively. Likewise, the percentages of homozygous affected animals with 2, 3, or 4 bleeding episodes in the Dahl-F8KO colony were significantly higher than those in the SD-F8KO colony. In contrast, none of the heterozygous (F8+/-) animals ever have a spontaneous bleed. Similar to SD-F8KO rats, Dahl-F8KO rats can develop anti-FVIII inhibitory antibodies after 2 doses of recombinant human FVIII (rhF8) treatment. Thus, our novel Dahl-F8KO rat model is a unique animal model with the severest spontaneous bleeding phenotype yet reported.To investigate if platelet-derived FVIII can rescue the spontaneous bleeding phenotype in HA rats, we generated transgenic rats (2bF8Tg) in both SD- and Dahl-background in which FVIII expression is under control of the platelet-specific αIIb promoter (2bF8) using lentivirus-mediated oocyte transduction transgenesis. Bone marrow (BM) mononuclear cells were isolated from femurs of 2bF8Tg rats and transplanted into F8-/- rats preconditioned with 950 R TBI. Animals were closely monitored after BM transplantation (BMT), and blood samples were collected for FVIII assays and whole blood thrombin generation assay (wbTGA). After BMT from SD-2bF8Tg rats, no spontaneous bleeding was observed in SD-F8KO recipients within the entire study course (1 year at the time of this report) with a sustained platelet-FVIII expression of 20.9±8.1 mU/108 platelets (n=3). Similarly, we performed BMT from Dahl-2bF8Tg rats into Dahs-F8KO and found that 1 of 3 recipients had a bruise at the early stage of BM reconstitution. However, no any other spontaneous bleeding has yet been observed (5 months). To confirm that the bleeding diathesis in F8-/- rats was ameliorated after 2bF8 gene expression, wbTGA was performed. All parameters, including Lag Time, Peak Time, Peak Thrombin, Endogenous Thrombin Potential in 2bF8Tg-BMT recipients were significantly different compared to F8-/- control rats, but were similar to those obtained in WT control animals. Of note, neither detectable levels of plasma FVIII nor anti-F8 antibodies were detected in F8-/- recipients after receiving BMT from 2bF8Tg rats.In summary, we have developed a novel HA rat model with both the pathophysiology and clinical phenotype found in severe HA patients, which could be an ideal model for evaluating the clinical efficacy of new therapeutics. Our studies demonstrated that transplantation of BM cells that are genetically modified to express FVIII only in platelets can efficiently prevent the severe spontaneous bleeding in HA rats with no anti-F8 antibody development. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

Clinical analysis on one patient with hemophilia A complicated with primary immune thrombocytopenia

Objective To investigate the diagnosis, treatment and prognosis of hemophilia A combined with primary immune thrombocytopenia (ITP). Methods The hemophilia A patient with ITP (male, 30 years old)admitted to the Department of Hematology, Xuzhou Medical University Affiliated Hospital on February 21, 2017 was selected as the study subject. During hospitalization, he was repeatedly treated with coagulation factor replacement therapy strategy, as well as symptomatic treatment measures such as hemostasis, platelet transfusion, intravenous infusion of glucocorticoids, and intravenous immunoglobulin (IVIG). By retrospective analysis, his clinical data were collected, and the clinical features, diagnosis and treatment were summarized. This study met the requirements of the Helsinki Declaration of the World Medical Association revised in 2013, and the informed consent of the subjects was obtained, and informed consent was signed with him. Results ① This patient was diagnosed as hemophilia A more than 20 years ago. During that period, the patient achieved a obvious effect by cryoprecipitation and recombinant human FⅧ infusion. About a month ago, without obvious inducement, the patient developed hematuria, epistaxis and oral hemorrhage. His activity of FⅧ was only 21.3%, and the platelet count was just 3×109/L. The patient received phenethylamine, recombinant human FⅧ, methylprednisolone, and intravenous immunoglobulin (IVIG) , but which was not effective for him. ② After admission to our hospital on February 21, 2017, the results of laboratory tests showed that the patient′s platelet count was very low, only for 2×109/L. His activity of FⅧ was 14.3%, and activity of FⅨwas 49.8%. Activated partial thromboplastin time (APTT) prolonged to 50.2 s, APTT was 32.1 s after correction, APTT (incubated at 37 ℃ for 2 h) prolonged to 50.0 s, and APTT (incubated at 37 ℃ for 2 h) was 37.9 s after correction. Morphological examination of bone marrow cells showed that bone marrow cells proliferated actively, granulocytes and erythroid line cells were proliferated, and the morphology was normal, as well as megakaryocyte maturation was accompanied by thrombocytopenia. Abdominal ultrasound results showed that the liver area was dense, the gallbladder wall was rough, and the spleen was not large (about 9.7 cm × 7.0 cm). ③ After admission, combining with previous medical history, results of relevant laboratory examinations and auxiliary examinations, the patient was diagnosed as hemophilia A combined with ITP by excluding other secondary diseases which caused thrombocytopenia. ④ After admission, the patient was repeatedly treated with recombinant human FⅧ. But the patient′s highest activity of FⅧ was only 25.8%, and APTT prolonged to 45.0 s, which suggested that the patient was partially tolerant to treatment. After receiving multiple platelet transfusions, the patient′s platelet count was still low, and did not exceed to 20×109/L, which suggested that the treatment is invalid. On May 5, 2017, the patient started oral eltrombopag (75 mg/d). After 3 months of treatment, the patient′s platelet count was up to 55×109/L, and the evaluation of efficacy was effective. However, after one month of discontinuation of eltrombopag, the patient′s platelet count fell to 10×109/L, which suggested that his disease relapsed. Conclusions Patients with hemophilia A combined with ITP, were clotting factor deficiency and thrombocytopenia, the risk of bleeding of them were high, and the treatment is difficult. In addition to infusion of recombinant human FⅧ, glucocorticoid and other drugs should be given to elevate platelet count, but the prognosis of this patient is not good. Patients with hemophilia A combined with ITP are rare in clinic, and its standard and effective treatment strategy requires a large sample size and multi-center study to further explore. Key words: Hemophilia A; Purpura, thrombocytopenic, idiopathic; Hemorrhage; Drug therapy; Prognosis; Primary immune thrombocytopenia

Co-Administration of Factor XIII with Hemostatic Agents in Hemophilia Promotes Clot Stability and Composition

Hemophilia A results from the absence, deficiency, or inhibition of factor VIII. Bleeding is treated with use of two broad classes of hemostatic agents; replacement factors (recombinant or plasma-derived human FVIII [FVIII] or factor IX) or bypassing agents (recombinant activated factor VIIa [rFVIIa], plasma-derived anti-inhibitor coagulant complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment with hemostatic agents, some hemophilia patients experience refractory bleeding. Factor XIII-A2B2 (FXIII) is a thrombin-activated protransglutaminase. Activated FXIII (FXIIIa) crosslinks fibrin to stabilize clots and increase red blood cell retention during clot contraction, and therefore, clot weight; these functions have been specifically associated with fibrin alpha-chain crosslinking and production of high molecular weight (HMW) crosslinked fibrin species. Although pilot studies have indicated that in hemophilia co-administration of FXIII with hemostatic agents improves hemostasis, the biochemical mechanisms evoked during co-administration have yet to be elucidated. We hypothesize that co-administration of FXIII with hemostatic agents enhances hemostasis in hemophilia by accelerating FXIII activation, increasing fibrin crosslinking, and improving the composition of contracted clots.We first analyzed in vitrotissue factor-activated coagulation in FVIII-deficient plasmas (n=5) with or without buffer (normal saline with 0.75% bovine serum albumin), plasma-derived FXIII (2 U/mL), and/or recombinant human FVIII (1 U/mL). Reactions were performed at 37°C and quenched with urea/EDTA at varying time points. Clot lysates were separated on 10% Tris-glycine gels, blotted, probed for FXIII(a) and fibrin(ogen), and analyzed by densitometry. Compared to buffer, FXIII, or FVIII-treated hemophilic plasma, FVIII+FXIII co-treatment increased the formation rate and level of FXIIIa. Furthermore, compared to buffer or FXIII-treated hemophilic plasma, both FVIII-treatment and FVIII+FXIII co-treatment increased the formation rate and level of crosslinked fibrin species (gamma chain and HMW species). Notably, compared to FVIII-treatment (alone), FVIII+FXIII co-treatment increased the formation rate and level of HMW crosslinked fibrin species.Next, we analyzed clot formation in whole blood obtained from untreated FVIII-deficient (n=2) or FVIII-inhibitor (n=6) patients. Citrated whole blood was pre-incubated at 37°C for 30 minutes with or without buffer, FXIII (2 U/mL), rFVIIa (25 nM), FEIBA (1 U/mL), or rpFVIII (1 U/mL), alone and in combination with FXIII, and clot formation was triggered with tissue factor and recalcification. In thrombelastography assays, compared to buffer or FXIII-treated hemophilic whole blood, rFVIIa, FEIBA, and rpFVIII treatments alone or with FXIII co-treatment shortened the time to clot formation (clot time [R]). Compared to buffer, FXIII, FEIBA or rpFVIII-treated hemophilic whole blood, rFVIIa-treatment (alone) and co-treatment with rFVIIa+FXIII or FEIBA+FXIIII increased clot stability (area under the elastic curve). In whole blood clot contraction assays, compared to buffer, FXIII, rFVIIa, FEIBA, or rpFVIII-treated hemophilic whole blood, co-treatment with rFVIIa+FXIII, FEIBA+FXIII, or rpFVIII+FXIII significantly increased clot weight.Our data show that in hemophilic plasmas, co-administration of FXIII with hemostatic agents accelerates FXIIIa activation, resulting in accelerated and increased HMW species formation. Furthermore, in hemophilic whole blood, co-administration of FXIII with hemostatic agents improved clot biophysical characteristics and increased clot weight. Collectively, these data suggest co-administration of FXIII with conventional hemostatic agents may promote clot stability and improve the composition of contracted clots by enhancing fibrin alpha-chain crosslinking. DisclosuresNo relevant conflicts of interest to declare.

The Impact of VWF on FVIII Immune Responses in Hemophilia a Mice with Pre-Existing Anti-FVIII Immunity

Development of inhibitory antibodies (inhibitors) against FVIII is the significant complication in protein replacement therapy for hemophilia A (HA). Currently, immune tolerance induction (ITI) with aggressive infusion of high-dose FVIII represents the only effective therapeutic approach for eradication of FVIII inhibitors and results in restoration of normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing VWF will affect the efficacy of the ITI is still a debated issue in the treatment of inhibitor patients. In this study, we explored the impact of VWF on FVIII immune responses in HA with pre-existing anti-F8 immunity using both in vitro and in vivomodels.Since the FVIII immune response is CD4+ T cell dependent, we first investigated how VWF affects FVIII-primed CD4+ T cells in response to FVIII restimulation. To address this question, we used a T cell proliferation assay. FVIIInull (F8null) mice were immunized with recombinant human FVIII (rhF8) to induce inhibitor development. Splenocytes from primed mice were labeled with CellTrace™ Violet and cultured with rhF8 with or without rhVWF. Four days later, cells were analyzed by FACS to assess the daughter (proliferated) cell population. The percentage of daughter CD4+ T cells (14.0±7.5%) in the condition cultured with 1 U/ml of rhF8 was significantly higher than without rhF8 (3.7±1.7%, n=6). The daughter cells further increased to 21.5±10.3% when cells were incubated with 10 U/ml of rhF8. However, when rhVWF was added to the culture media in addition to rhF8, percentages of daughter CD4+ T cells were significantly decreased in both the 1 U/ml and 10 U/ml rhF8 treatment groups (10.4±7.1% and 15. 8±8.4%, respectively). To further explore how VWF affects the FVIII immune response, we analyzed cytokine profiles in T cell culture supernants using a multiplex ELISA assay. The levels of IFNg and IL10 in the groups cultured with rhF8 in the presence of rhVWF were significantly lower than in the groups cultured with rhF8 only. The levels of TNFa, IL4, IL5, and IL12 in the groups cultured with rhF8 together with rhVWF were not significantly different than those in rhF8 groups without VWF. These results demonstrated that VWF significantly suppresses rhF8-primed CD4+ T cell proliferation in response to rhF8 restimulation and the inhibition is via the Th1 pathway.In a setting of pre-existing anti-F8 immunity, how FVIII-specific memory B cells respond to FVIII-restimulation and mature to antibody secreting cells (ASCs) is the critical pathway in terms of the clinical efficacy of FVIII infusion. To investigate how VWF affects memory B cell maturation upon FVIII restimulation, we used ELISPOT-based assay. Splenocytes from rhF8-primed HA mice were used as the source to prepare F8-specific memory B cell pools. CD138+ cells were depleted and the remaining cells were used as a pool of memory B cells. To stimulate the maturation of F8-specific memory B cells into ASCs, memory B cell pools from primed F8null mice were cultured with rhF8 with or without rhVWF for 6 days. After culture, newly formed ASCs were assessed by the ELISPOT assay. There were 54.4±19.5 ASCs/106 cells when cells from memory B cell pool were cultured with 0.05 U/ml rhF8. In contrast, there was only 15.6±1.6 ASCs/106cells after the cells were cultured with rhF8 together with rhVWF, indicating that memory B cell maturation is suppressed in the presence of rhVWF.We then used an in vivo model to further evaluate the impact of VWF on the immunogenicity of FVIII in HA with pre-existing immunity. Since we are unable to mimic the human ITI in F8null mice, we transferred memory B cells from rhF8-primed F8null splenocytes into immunocompromised F8null mice followed by rhF8 immunization in the presence or absence of rhVWF. Blood samples were collected one week after immunization for analysis. The inhibitor titer in animals that received rhF8-primed memory B cell pool followed by rhF8 immunization was 45.9±63.0 BU/ml (n=11), which was significantly higher than the titer in animals immunized with rhF8 together with rhVWF (23.9±38.4, P<.01). These results demonstrate that VWF suppressed the anti-F8 memory response in vivo.In summary, our ex vivo and in vivo data demonstrated that VWF attenuates F8-primed CD4+T cells and memory B cells in response to rhF8 restimulation, suggesting that infusion of FVIII together with VWF might reduce anti-F8 memory responses in HA with inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Effects of Joint Blood Volume and Hemostasis Correction on Vascular Changes in the Joint after Induced Hemarthrosis in Hypocoagulable Mice

Introduction:Hemophilia causes recurrent bleeds into 'target joints' despite clotting factor replacement. Vascular remodeling associated with hemarthrosis is thought to contribute to vessel "leakiness" and perpetuated bleeding, but the underlying mechanisms remain unknown. Here, we studied the effects of joint blood volume and timing of hemostasis correction on vascular changes after induced hemarthrosis in FVIII-deficient mice, and in BALB/c mice treated temporarily with warfarin and anti-FVIII to create reversible hemostasis suppression.Methods: Mice were injured by sub-patellar puncture of the right knee to induce hemarthrosis. FVIII-deficient mice were treated with two doses of recombinant human FVIII (rhFVIII) or saline with an 8-12 hour interval. The first dose of rhFVIII was given i) 2 hours before injury (prophylaxis), ii) 2 hours after injury (early), iii) 8 hours after injury (intermediate), or iv) 16 hours after injury (late). BALB/c mice were administered 10 µg/ml warfarin in drinking water for 1 week plus 0.25 mg/kg anti-FVIII 2 hours prior to injury. This dose of warfarin yielded a ~4-fold increase in prothrombin time that was reversed with 100 IU/kg 4-Factor prothrombin complex concentrate on day 2 post-injury in one group of mice, and maintained in another. Clearance of anti-FVIII within 4 days was indicated by normalization of blood loss in tail clip experiments. On day 2 after injury, the hematocrit was determined as a measure of joint bleeding and incapacitance was monitored by left:right weight bearing. Vascularity was assessed at baseline and 1-2 weeks post-injury by high resolution musculoskeletal ultrasound with Power Doppler (MSKUS/PD) and histology with Safranin-O-Fast Green staining. Statistical analyses were conducted by Mann-Whitney test.Results: In FVIII-deficient mice, knee injury significantly reduced the mean hematocrit from 47 % to ~25 % for mice receiving late or no rhFVIII treatment (p<0.001). Hematocrit remained at 47 % with prophylaxis, and decreased to 40 % (p=0.01) and 37 % (p<0.0001) with early and intermediate rhFVIII treatment, respectively. In the saline-only group, hemarthrosis caused a 3.1-fold increase in left:right weight bearing of the hind legs (p=0.008). Earlier treatments with rhFVIII alleviated this incapacitance and mice receiving rhFVIII prophylaxis had no deficit. In contrast, vascular changes were similar across treatment groups, regardless of hematoma size. Mean vessel number increased ~3-fold compared to baseline for all groups (p<0.05). This was accompanied by moderate increases in mean vessel diameter and percentage of vessels with a diameter ≥ 20 µm. Vascular perfusion measured by MSKUS/PD increased significantly (~2.5-fold) with early (p=0.01), intermediate, late or no rhFVIII treatment (p<0.001), but not with prophylaxis.Bleeding tendency and weight bearing deficits of the injured leg in BALB/c mice treated with warfarin and anti-FVIII were comparable to those observed in FVIII-deficient mice. Mean hematocrit decreased to 28 % (p<0.0001) and left:right weight bearing ratio increased to 2.6 (p=0.003). Mean vessel number in injured knees increased to a similar extent in the warfarin continuation and warfarin reversal groups (~1.5-fold; p=0.02). In contrast, mean vessel diameter and percentage of vessels with a diameter ≥ 20 µm were elevated only in mice continued on warfarin (p=0.008 and p=0.02, respectively).Conclusions: FVIII prophylaxis or early treatment reduced blood loss into joints of FVIII-deficient mice and preserved joint function. However, neovascularization and vascular changes consistent with remodeling were not proportional to joint blood volume and were mostly unresponsive to short-term FVIII replacement, regardless of timing. Similarly, neovascularization in joints of hemostatically compromised BALB/c mice after induced hemarthrosis persisted despite normalization of hemostasis. Changes in vessel architecture, however, could be abrogated. This suggests that long-term hemostasis correction may protect against irreversible vascular remodeling in the joint, thought to underlie re-bleeding and development of hemophilic arthropathy. Further investigations are required to determine the effects of prolonged hemostasis correction on re-bleeding tendencies and joint health in hemophilia. DisclosuresMosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau. von Drygalski:Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau.

Visualization of haemophilic arthropathy in F8-/- rats by ultrasonography and micro-computed tomography.

A major complication of haemophilia is haemophilic arthropathy (HA), a debilitating disorder with an incompletely defined pathobiology. High-resolution imaging may provide new knowledge about onset and progression of HA, and thereby support identification of new treatment opportunities. Recently, a F8-/- rat model of HA was developed. The size of the rat allows for convenient and high resolution imaging of the joints, which could enable in vivo studies of HA development. To determine whether HA in the F8-/- rat can be visualized using ultrasonography (US) and micro-computed tomography (μCT). Sixty F8-/- and 20 wild-type rats were subjected to a single or two induced knee bleeds. F8-/- rats were treated with either recombinant human FVIII (rhFVIII) or vehicle before the induction of knee bleeds. Haemophilic arthropathy was visualized using in vivo US and ex vivo μCT, and the observations correlated with histological evaluation. US and μCT detected pathologies in the knee related to HA. There was a strong correlation between disease severity determined by μCT and histopathology. rhFVIII treatment reduced the pathology identified with both imaging techniques. US and μCT are suitable imaging techniques for detection of blood-induced joint disease in F8-/- rats and may be used for longitudinal studies of disease progression.

The Immunogenicity of Platelet-Derived FVIII in Hemophilia a Mice with or without Pre-Existing Anti-FVIII Immunity

Recent studies from our group and others have demonstrated that FVIII ectopically targeted to platelets under control of the platelet-specific αIIb promoter (2bF8) can efficiently restore hemostasis in hemophilia A mice even in the presence of high-titer inhibitory antibodies directed against FVIII (inhibitors). Our studies have demonstrated that platelet-targeted FVIII gene therapy can not only correct the hemophilic phenotype, but also induce FVIII-specific immune tolerance. In the platelet gene therapy model, hematopoietic stem cells (HSCs) are ex vivo transduced with lentivirus carrying 2bF8 and transplanted into the recipient. Sufficient preconditioning has to be employed to create space for therapeutic engraftment of the transduced HSCs. It is not clear whether preconditioning affects the potential for an immune response in the context of platelet-derived FVIII. Furthermore, if current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically, as potential transfusion alternative, in hemophilia A patients even with inhibitors. One important question that has not been explored, however, is the immunogenicity of platelet-derived FVIII. To investigate whether platelet-derived FVIII can act as an immunogen in hemophilia A mice, we infused transgenic mouse platelets with a level of platelet-FVIII of 6 mU/108 platelets into naïve FVIIInull mice without any preconditioning weekly for 8 weeks. These platelets were transfused to a level between 20 to 57% of total platelets upon infusion, and all animals survived the tail-clip survival test 13-hours after platelet infusion. The level of platelet-FVIII in the infused animals was 0.11 ± 0.01 mU/108 platelets (n = 6) even one week after infusion. Neither inhibitory nor non-inhibitory anti-FVIII antibodies were detected in the infused mice during the study course (n = 9). All animals developed inhibitors following further challenge with recombinant human FVIII (rhF8) at a dose of 50 U/kg by intravenous injection weekly for 4 weeks, indicating that infusion of platelets containing FVIII does not trigger an immune response in hemophilia A mice. We then explored whether platelets containing FVIII can act as an immunogen in FVIIInull mice with pre-existing anti-FVIII immunity. FVIIInull mice were immunized with rhF8 to induce anti-FVIII antibodies. Four week after the last immunization, 2bF8 transgenic platelets were transfused into rhF8-primed FVIIInull mice (n = 4) weekly for 4 weeks and anti-FVIII antibody titers were monitored. There was not significant augmentation of FVIII-specific antibodies as determined by Bethesda assay for inhibitory antibodies and ELISA assay for total anti-FVIII IgG, indicating that infusion of platelets containing FVIII does not stimulate an anti-FVIII memory response in the inhibitor model. To investigate whether preconditioning affects the anti-FVIII immune response, animals were pre-conditioned with a sub-lethal 660 cGy total body irradiation (TBI) followed by 2bF8 transgenic platelet infusion weekly for 8 weeks. No anti-FVIII antibodies were detected in recipients (n = 6) after 2bF8 transgenic platelet infusion. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower (75 ± 42 BU/ml) than in the naïve FVIIInull mice without preconditioning when the same infusion protocol was employed (270 ± 76 BU/ml), indicating that 660 cGy TBI plus 2bF8 transgenic platelet infusion may suppress anti-FVIII immune response. In conclusion, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in hemophilia A mice. DisclosuresNo relevant conflicts of interest to declare.

Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy

The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion.We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD.To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin.Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025).These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs.In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. DisclosuresNo relevant conflicts of interest to declare.