Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.

Platelet-targeted gene therapy induces immune tolerance in hemophilia and beyond.

Blood platelets have unique storage and delivery capabilities. Platelets play fundamental roles in hemostasis, inflammatory reactions, and immune responses. Beyond their functions, platelets have been used as a target for gene therapy. Platelet-targeted gene therapy aims to deliver a sustained expression of neo-protein in vivo by genetically modifying the target cells, resulting in a cure for the disease. Even though there has been substantial progress in the field of gene therapy, the potential development of immune responses to transgene products or vectors remains a significant concern. Of note, multiple preclinical studies using platelet-specific lentiviral gene delivery to hematopoietic stem cells in hemophilia have demonstrated promising results with therapeutic levels of neo-protein that rescue the hemorrhagic bleeding phenotype and induce antigen-specific immune tolerance. Further studies using ovalbumin as a surrogate protein for platelet gene therapy have shown robust antigen-specific immune tolerance induced via peripheral clonal deletions of antigen-specific CD4- and CD8-T effector cells and induction of antigen-specific regulatory T (Treg) cells. This review discusses platelet-targeted gene therapy, focusing on immune tolerance induction.

Desialylated Platelet Clearance in the Liver is a Novel Mechanism of Systemic Immunosuppression.

Platelets are small, versatile blood cells that are critical for hemostasis/thrombosis. Local platelet accumulation is a known contributor to proinflammation in various disease states. However, the anti-inflammatory/immunosuppressive potential of platelets has been poorly explored. Here, we uncovered, unexpectedly, desialylated platelets (dPLTs) down-regulated immune responses against both platelet-associated and -independent antigen challenges. Utilizing multispectral photoacoustic tomography, we tracked dPLT trafficking to gut vasculature and an exclusive Kupffer cell-mediated dPLT clearance in the liver, a process that we identified to be synergistically dependent on platelet glycoprotein Ibα and hepatic Ashwell-Morell receptor. Mechanistically, Kupffer cell clearance of dPLT potentiated a systemic immunosuppressive state with increased anti-inflammatory cytokines and circulating CD4+ regulatory T cells, abolishable by Kupffer cell depletion. Last, in a clinically relevant model of hemophilia A, presensitization with dPLT attenuated anti-factor VIII antibody production after factor VIII ( infusion. As platelet desialylation commonly occurs in daily-aged and activated platelets, these findings open new avenues toward understanding immune homeostasis and potentiate the therapeutic potential of dPLT and engineered dPLT transfusions in controlling autoimmune and alloimmune diseases.

PACSIN2 regulates platelet integrin ß1 hemostatic function.

BACKGROUND: Upon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins ß1 and ß3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. OBJECTIVES: Here we investigated the role of PACSIN2 in platelet function. METHODS: Platelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin ß1. RESULTS: Pacsin2-/- mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2-/- platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2-/- platelets had hyperactive integrin ß1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2ß1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin ß1. By contrast, Pacsin2-/- platelets had normal integrin αIIbß3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin ß1 in Pacsin2-/- mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin ß7, a model for integrin ß-subunits. CONCLUSIONS: Pacsin2-/- mice displayed severe thrombus formation defects due to hyperactive platelet integrin ß1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin ß1 hemostatic function.

Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells.

BACKGROUND: We previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIIInull mice transplanted with 2bF8-transduced Sca-1+ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost. OBJECTIVE: In this study, we aimed to describe the mechanism of this platelet-FVIII loss. METHODS: We monitored platelet-FVIII expression in FVIIInull mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1+ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell-deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity. RESULTS: Platelet-FVIII expression was sustained in 2bF8Tg bone marrow transplantation but not in 2bF8Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8Tg-Sca-1+ cells and secreted interferon gamma, but were not stimulated by 2bF8Tg platelets/megakaryocytes, suggesting that 2bF8Tg-Sca-1+ cells are targets for FVIII-primed CD8 T cells. When 2bF8Tg-Sca-1+ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice. CONCLUSION: Pre-existng immunity can alter the engraftment of 2bF8Tg-Sca-1+ cells through the cytotoxic CD8 T-cell-mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.

Hemophilia a patients with inhibitors: Mechanistic insights and novel therapeutic implications.

The development of coagulation factor VIII (FVIII) inhibitory antibodies is a serious complication in hemophilia A (HA) patients after FVIII replacement therapy. Inhibitors render regular prophylaxis ineffective and increase the risk of morbidity and mortality. Immune tolerance induction (ITI) regimens have become the only clinically proven therapy for eradicating these inhibitors. However, this is a lengthy and costly strategy. For HA patients with high titer inhibitors, bypassing or new hemostatic agents must be used in clinical prophylaxis due to the ineffective ITI regimens. Since multiple genetic and environmental factors are involved in the pathogenesis of inhibitor generation, understanding the mechanisms by which inhibitors develop could help identify critical targets that can be exploited to prevent or eradicate inhibitors. In this review, we provide a comprehensive overview of the recent advances related to mechanistic insights into anti-FVIII antibody development and discuss novel therapeutic approaches for HA patients with inhibitors.

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette. Platelet-MOG expression was introduced by lentivirus transduction of HSCs followed by transplantation. MOG protein was detected on the cell surface of platelets only in full-length MOG-transduced recipients, but MOG was detected in transmembrane-domain-less MOG1-157-transduced platelets intracellularly. We found that targeting MOG expression to platelets could prevent EAE development and attenuate disease severity, including the loss of bladder control in transduced recipients. Elimination of the transmembrane domains of MOG significantly enhanced the clinical efficacy in preventing the onset and development of the disease and induced CD4+Foxp3+ Treg cells in the EAE model. Together, our data demonstrated that targeting transmembrane domain-deleted MOG expression to platelets is an effective strategy to induce immune tolerance in EAE, which could be a promising approach for the treatment of patients with MS autoimmune disease.

Association of Platelet Desialylation and Circulating Follicular Helper T Cells in Patients With Thrombocytopenia.

Thrombocytopenia is a multifactorial condition that frequently involves concomitant defects in platelet production and clearance. The physiopathology of low platelet count in thrombocytopenia remains unclear. Sialylation on platelet membrane glycoprotein and follicular helper T cells (TFHs) are thought to be the novel platelet clearance pathways. The aim of this study was to clarify the roles of platelet desialylation and circulating TFHs in patients with immune thrombocytopenia (ITP) and non-ITP thrombocytopenia. We enrolled 190 patients with ITP and 94 patients with non-ITP related thrombocytopenia including case of aplastic anemia (AA) and myelodysplastic syndromes (MDS). One hundred and ten healthy volunteers were included as controls. We found significantly increased desialylated platelets in patients with ITP or thrombocytopenia in the context of AA and MDS. Platelet desialylation was negatively correlated with platelet count. Meanwhile, the circulating TFH levels in patients with thrombocytopenia were significantly higher than those of normal controls, and were positively correlated with desialylated platelet levels. Moreover, TFHs-related chemokine CXCL13 and apoptotic platelet levels were abnormally high in ITP patients. The upregulation of pro-apoptotic proteins and the activation of the MAPK/mTOR pathway were observed in the same cohort. These findings suggested that platelet desialylation and circulating TFHs may become the potential biomarkers for evaluating the disease process associated with thrombocytopenia in patients with ITP and non-ITP.

A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD.

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. Bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N mice, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

Thromboelastometry assessment of hemostatic properties in various murine models with coagulopathy and the effect of factor VIII therapeutics.

BACKGROUND: Rotational thromboelastometry (ROTEM) has been commonly used to assess the viscoelastic properties of the blood clotting process in the clinic for patients with a hemostatic or prothrombotic disorder. OBJECTIVE: To evaluate the capability of ROTEM in assessing hemostatic properties in whole blood from various mouse models with genetic bleeding or clotting disease and the effect of factor VIII (FVIII) therapeutics in FVIIInull mice. METHODS: Mice with a genetic deficiency in either a coagulation factor or a platelet glycoprotein were used in this study. The properties of platelet- or plasma-FVIII were also assessed. Citrated blood from mice was recalcified and used for ROTEM analysis. RESULTS: We found that blood collected from the vena cava could generate reliable results from ROTEM analysis, but not blood collected from the tail vein, retro-orbital plexus, or submandibular vein. Age and sex did not significantly affect the hemostatic properties determined by ROTEM analysis. Clotting time (CT) and clot formation time (CFT) were significantly prolonged in FVIIInull (5- and 9-fold, respectively) and FIXnull (4- and 5.7-fold, respectively) mice compared to wild-type (WT)-C57BL/6J mice. Platelet glycoprotein (GP)IIIanull mice had significantly prolonged CFT (8.4-fold) compared to WT-C57BL/6J mice. CT and CFT in factor V (FV) Leiden mice were significantly shortened with an increased α-angle compared to WT-C57BL/6J mice. Using ROTEM analysis, we showed that FVIII expressed in platelets or infused into whole blood restored hemostasis of FVIIInull mice in a dose-dependent manner. CONCLUSION: ROTEM is a reliable and sensitive assay for assessing therapeutics on hemostatic properties in mouse models with a bleeding or clotting disorder.

Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity.

Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)-mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.

Platelet gene therapy induces robust immune tolerance even in a primed model via peripheral clonal deletion of antigen-specific T cells.

While platelet-specific gene therapy is effective in inducing immune tolerance to a targeted protein, how the reactivity of pre-existing immunity affects the efficacy, and whether CD8 T cells were involved in tolerization, is unclear. In this study, ovalbumin (OVA) was used as a surrogate protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either wild-type (WT)/CD45.2 or OT-II/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6 Gy of irradiation. Sustained platelet-OVA expression was achieved in >85% of OVA-primed recipients but abolished in animals with high-reactive pre-existing immunity. As confirmed by OVA rechallenge and skin graft transplantation, immune tolerance was achieved in 2bOVA-transduced recipients. We found that there is a negative correlation between platelet-OVA expression and the percentage of OVA-specific CD4 T cells and a positive correlation with the OVA-specific regulatory T (Treg) cells. Using the OT-I/WT model, we showed that antigen-specific CD8 T cells were partially deleted in recipients after platelet-targeted gene transfer. Taken together, our studies demonstrate that robust antigen-specific immune tolerance can be achieved through platelet-specific gene therapy via peripheral clonal deletion of antigen-specific CD4 and CD8 T effector cells and induction of antigen-specific Treg cells. There is an antagonistic dynamic process between immune responses and immune tolerance after platelet-targeted gene therapy.

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.

Platelet-Targeted FVIII Gene Therapy Restores Hemostasis and Induces Immune Tolerance for Hemophilia A.

Platelets are small anucleated blood components primarily described as playing a fundamental role in hemostasis and thrombosis. Over the last decades, increasing evidence has demonstrated the role of platelets in modulating inflammatory reactions and immune responses. Platelets harbor several specialized organelles: granules, endosomes, lysosomes, and mitochondria that can synthesize proteins with pre-stored mRNAs when needed. While the functions of platelets in the immune response are well-recognized, little is known about the potential role of platelets in immune tolerance. Recent studies demonstrate that platelet-specific FVIII gene therapy can restore hemostasis and induce immune tolerance in hemophilia A mice, even mice with preexisting anti-FVIII immunity. Here, we review the potential mechanisms by which platelet-targeted FVIII gene therapy restores hemostasis in the presence of anti-FVIII inhibitory antibodies and induces immune tolerance in hemophilia A.

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

Factor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell-mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, "semiprofessional" antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9.

BACKGROUND: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species-specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF-/-) rat by excision of all 52 exons of the VWF locus. METHODS: The entire VWF gene was eliminated in Sprague-Dawley (Crl:SD) rats via CRISPR/Cas9-mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF-/- rats were infused with FVIII-/- rat platelet poor plasma (PPP) to determine response of plasma FVIII. RESULTS: Breeding of VWF ± rats yielded VWF-/- offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF-/- rats. VWF-/- rats bled longer and more than VWF+/- and VWF+/+ rats when challenged. Transfusion of FVIII-deficient platelet-poor plasma induced a rapid rise in endogenous FVIII:C in VWF-/- rats. CONCLUSION: This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

The severe spontaneous bleeding phenotype in a novel hemophilia A rat model is rescued by platelet FVIII expression.

Previous studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII-/- rat model in a Dahl inbred background (Dahl-FVIII-/-) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII-/- rats. Sixty-one percent developed anti-FVIII inhibitors after ≥2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII-/- background, none of the resulting 2bF8tg+FVIII-/- rats (with platelet FVIII levels of 28.26 ± 7.69 mU/108 platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8tg bone marrow (BM) was transplanted into FVIII-/- rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII-/- rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8tg BM transplantation recipients were significantly improved compared with FVIII-/- control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8tg BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII-/- rats with no anti-FVIII antibody development.

Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice.

The development of neutralizing anti-FVIII antibodies (inhibitors) is a major complication of FVIII protein replacement therapy in patients with hemophilia A (HA). Although multiple lines of evidence indicate that the immune response against FVIII is CD4 T-cell-dependent and many FVIII-derived CD4 epitopes have already been discovered, the role of T follicular helper (TFH) cells in FVIII inhibitor development is unknown. TFH cells, a newly identified subset of CD4 T cells, are characterized by expression of the B-cell follicle-homing receptor CXCR5 and PD-1. In this study, we show for the first time that IV FVIII immunization induces activation and accumulation and/or expansion of PD-1+CXCR5+ TFH cells in the spleen of FVIII-deficient (FVIIInull) mice. FVIII inhibitor-producing mice showed increased germinal center (GC) formation and increased GC TFH cells in response to FVIII immunization. Emergence of TFH cells correlated with titers of anti-FVIII inhibitors. Rechallenge with FVIII antigen elicited recall responses of TFH cells. In vitro FVIII restimulation resulted in antigen-specific proliferation of splenic CD4+ T cells from FVIII-primed FVIIInull mice, and the proliferating cells expressed the TFH hallmark transcription factor BCL6. CXCR5+/+ TFH-cell-specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5+/+ TFH cells for the generation of FVIII-neutralizing antibodies. Together, our results demonstrate that the induction of activated TFH cells in FVIIInull mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH-cell activation may be a promising strategy for preventing anti-FVIII inhibitor formation in patients with HA.