Fitusiran reduces bleeding in Factor X-deficient mice.

Factor X (FX)-deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aimed to explore the use of fitusiran (an investigational siRNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX-deficiency. We therefore developed a novel model of inducible FX-deficiency, generating mice expressing <1% FX activity and antigen (f10low-mice). Compared to control f10WT-mice, f10low-mice had 6- and 4-fold prolonged clotting times in Prothrombin Time- and activated Partial Prothrombin Time-assays, respectively (p<0.001). Thrombin generation was severely reduced, irrespective whether tissue factor or factor XIa was used as initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury-model. Furthermore, in two distinct bleeding models, f10low-mice displayed an increased bleeding tendency compared to f10WT-mice. In the tail-clip assay blood loss was increased from 12±16 microliter to 590±335 microliter (p<0.0001). In the saphenous vein puncture (SVP)-model, the number of clots generated was reduced from 19±5 clots/30 min for f10WT-mice to 2±2 clots/30 min (p<0.0001) for f10low-mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low-mice with fitusiran (2x10 mg/kg at one-week interval) resulted in 17±6% residual antithrombin activity and increased thrombin generation (4-fold and 2-3-fold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP-model, the number of clots was increased to 8±6 clots/30 min (p=0.0029). Altogether, we demonstrate that reduction of antithrombin levels is associated with improved hemostatic activity under conditions of FX-deficiency.

von Willebrand factor increases in experimental cerebral malaria but is not essential for late-stage pathogenesis in mice.

BACKGROUND: Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. OBJECTIVES: To investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). METHODS: Both Vwf+/+ and Vwf-/- mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. RESULTS: Plasma VWF levels significantly increased upon PbANKA infection in Vwf+/+ animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end-stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf-/- mice compared to Vwf+/+ mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf-/- mice manifested overall clinical ECM features similar to those observed in Vwf+/+ animals. At day 8.5 post-infection, however, clinical ECM features in Vwf-/- mice were slightly more beneficial than in Vwf+/+ animals. Despite these minor differences, overall survival was not different between Vwf-/- and Vwf+/+ mice. Similarly, PbANKA-induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. CONCLUSIONS: Our study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late-stage ECM pathogenesis.

Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors.

Novel therapies for hemophilia, including non-factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama-derived single-domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII-deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi-paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long-term expression of the bi-paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor.

A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

von Willebrand factor in experimental malaria-associated acute respiratory distress syndrome.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a lethal complication of severe malaria, characterized by marked pulmonary inflammation. Patient studies have suggested a link between von Willebrand factor (VWF) and malaria severity. OBJECTIVES: To investigate the role of VWF in the pathogenesis of experimental MA-ARDS. METHODS: Plasmodium berghei NK65-E (PbNK65) parasites were injected in Vwf+/+ and Vwf-/- mice. Pathological parameters were assessed following infection. RESULTS: In accordance with patients with severe malaria, plasma VWF levels were increased and ADAMTS13 activity levels were reduced in experimental MA-ARDS. ADAMTS13- and plasmin-independent reductions of high molecular weight VWF multimers were observed at the end stage of disease. Thrombocytopenia was VWF-independent because it was observed in both Vwf+/+ and Vwf-/- mice. Interestingly, Vwf-/- mice had a shorter survival time compared with Vwf+/+ controls following PbNK65 infection. Lung edema could not explain this shortened survival because alveolar protein levels in Vwf-/- mice were approximately two times lower than in Vwf+/+ controls. Parasite load, on the other hand, was significantly increased in Vwf-/- mice compared with Vwf+/+ mice in both peripheral blood and lung tissue. In addition, anemia was only observed in PbNK65-infected Vwf-/- mice. Of note, Vwf-/- mice presented with two times more reticulocytes, a preferential target of the parasites. CONCLUSIONS: This study suggests that parasite load together with malarial anemia, rather than alveolar leakage, might contribute to shortened survival in PbNK65-infected Vwf-/- mice. VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially explains the increase in parasite load.

Improved molecular platform for the gene therapy of rare diseases by liver protein secretion.

Many rare monogenic diseases are treated by protein replacement therapy, in which the missing protein is repetitively administered to the patient. However, in several cases, the missing protein is required at a high and sustained level, which renders protein therapy far from being adequate. As an alternative, a gene therapy treatment ensuring a sustained effectiveness would be particularly valuable. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy.

Long-Term Prevention of Congenital Thrombotic Thrombocytopenic Purpura in ADAMTS13 Knockout Mice by Sleeping Beauty Transposon-Mediated Gene Therapy.

OBJECTIVE: Severe deficiency in the von Willebrand factor-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) because of mutations in the ADAMTS13 gene can lead to acute episodes of congenital thrombotic thrombocytopenic purpura (TTP), requiring prompt treatment. Current treatment consists of therapeutic or prophylactic infusions of fresh frozen plasma. However, lifelong treatment with plasma products is a stressful therapy for TTP patients. Here, we describe the use of the nonviral sleeping beauty (SB) transposon system as a gene therapeutic approach to realize lifelong expression of ADAMTS13 and subsequent protection against congenital TTP. APPROACH AND RESULTS: We demonstrated that hydrodynamic tail vein injection of the SB100X system expressing murine ADAMTS13 in Adamts13-/- mice resulted in long-term expression of supraphysiological levels of transgene ADAMTS13 over a period of 25 weeks. Stably expressed ADAMTS13 efficiently removed the prothrombotic ultralarge von Willebrand factor multimers present in the circulation of Adamts13-/- mice. Moreover, mice stably expressing ADAMTS13 were protected against TTP. The treated mice did not develop severe thrombocytopenia or did organ damage occur when triggered with recombinant von Willebrand factor, and this up to 20 weeks after gene transfer. CONCLUSIONS: These data demonstrate the feasibility of using SB100X-mediated gene therapy to achieve sustained expression of transgene ADAMTS13 and long-term prophylaxis against TTP in Adamts13-/- mice.

ADAMTS13 and anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura - current perspectives and new treatment strategies.

A deficiency in ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type-1 repeats, member 13) is associated with thrombotic thrombocytopenic purpura (TTP). Congenital TTP is caused by a defect in the ADAMTS13 gene resulting in decreased or absent enzyme activity; acquired TTP results from autoantibodies that either inhibit the activity or increase the clearance of ADAMTS13. Despite major progress in recent years in our understanding of the disease, many aspects around the pathophysiology of TTP are still unclear. Newer studies expanded the TTP field from ADAMTS13 and inhibitory antibodies to immune complexes, cloned autoantibodies, and a possible involvement of other proteases. Additionally, several new treatment strategies supplementing plasma-exchange and infusion are under investigation for a better and more specific treatment of TTP patients. In this review, we discuss the recent insights in TTP pathophysiology and describe upcoming therapeutic opportunities.

Platelet-derived VWF is not essential for normal thrombosis and hemostasis but fosters ischemic stroke injury in mice.

Von Willebrand factor (VWF) is a key hemostatic protein synthesized in both endothelial cells and megakaryocytes. Megakaryocyte-derived VWF is stored in α-granules of platelets and is enriched in hyperactive "ultra-large" VWF multimers. To elucidate the specific contribution of platelet VWF in hemostasis and thrombosis, we performed crossed bone marrow transplantations between C57BL/6J and Vwf(-/-) mice to generate chimeric mice. Chimeric mice specifically lacking platelet VWF showed normal tail bleeding and carotid artery thrombosis, similar to wild-type mice. Chimeric mice with VWF present only in platelets were not able to support normal thrombosis and hemostasis. However, using a mouse model of transient middle cerebral artery occlusion, we observed that cerebral infarct sizes and fibrin(ogen) deposition in chimeric mice with only platelet VWF were significantly increased compared with Vwf(-/-) mice (P < .01). Blocking of the platelet VWF-glycoprotein (GP)Ib interaction abrogated this platelet VWF-mediated injury. These data suggest that whereas platelet-derived VWF does not play a crucial role in hemostasis and arterial thrombosis, it aggravates thrombo-inflammatory diseases such as stroke via a GPIb-dependent mechanism.

Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases. However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome.

RATIONALE: Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. OBJECTIVES: Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. METHODS: C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. MEASUREMENTS AND MAIN RESULTS: In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. CONCLUSIONS: We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.