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Milwaukee, WI, United States

Chen Y.,Blood Research Institute | Chen Y.,Fujian Medical University | Schroeder J.A.,Blood Research Institute | Schroeder J.A.,Medical College of Wisconsin | And 9 more authors.
Molecular Therapy | Year: 2014

Here, we developed a clinically translatable platelet gene therapy approach for hemophilia B. Platelet-targeted FIX (2bF9) expression was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). Sustained therapeutic levels of platelet-FIX expression were obtained in FIX null mice that received 2bF9 LV-transduced HSCs. Approximately 6-39% of the platelets expressed FIX in the transduced recipients, which was sufficient to rescue the bleeding diathesis in FIX null mice in tail clipping models. Sequential bone marrow transplantation demonstrated that platelet-FIX expression in the secondary recipients was sustained, leading to phenotypic correction. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. Only one of the nine recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) after challenge with rhFIX. These data suggest that platelet gene therapy can not only restore hemostasis but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. © The American Society of Gene & Cell Therapy. Source


Shi Q.,Medical College of Wisconsin | Shi Q.,Blood Research Institute | Shi Q.,Childrens Research Institute | Shi Q.,Fund Research Center | And 9 more authors.
Journal of Thrombosis and Haemostasis | Year: 2015

Background: Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). Objective: To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. Methods: 2bF8 transgenic mice in the FVIII-/- background (2bF8tg+/-F8-/-) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. Results: Only 18% of 2bF8tg+/-F8-/-VWF-/- animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000BUmL-1. In contrast, 82% of 2bF8tg+/-F8-/-VWF+/+ mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50000BUmL-1. All 2bF8tg+/-F8-/-VWF-/- mice without inhibitors survived tail clipping and no VWF-/-F8-/- mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. Conclusion: VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors. © 2015 International Society on Thrombosis and Haemostasis. Source


Fang J.,Medical College of Wisconsin | Fang J.,Childrens Research Institute | Fang J.,Fund Research Center | Nurden P.,Plateforme Technologique et dInnovation Biomedicale | And 11 more authors.
Journal of Thrombosis and Haemostasis | Year: 2013

Background and objectives: β3-Deficient megakaryocytes were modified by human β3-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ3 amino acid substitution, for investigation of how an activated αIIbβ3 conformation affects platelets in vivo in mice. Patient/Methods: As in our previous report of an R560β3 mutation in a patient with Glanzmann thrombasthenia, R560β3 murine platelets spontaneously bound antibody that only recognizes activated αIIbβ3 bound to its ligand, fibrinogen. Results: With this murine model, we showed that αIIb-R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β3 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560β3 'wild-type' mice remained alive. Pathologic examination revealed that R560β3 mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β3 megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β3 washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. Conclusions: The results demonstrate that continuous occupancy of αIIbβ3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state. © 2013 International Society on Thrombosis and Haemostasis. Source


Schroeder J.A.,Medical College of Wisconsin | Schroeder J.A.,Blood Research Institute | Schroeder J.A.,Childrens Research Institute | Chen Y.,Blood Research Institute | And 11 more authors.
Journal of Thrombosis and Haemostasis | Year: 2014

Background: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A. Objective: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities. Methods: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMTP140K cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O6-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis. Results: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 108 platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge. Conclusion: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy. © 2014 International Society on Thrombosis and Haemostasis. Source


Du L.M.,Medical College of Wisconsin | Du L.M.,Childrens Research Institute | Du L.M.,Fund Research Center | Nurden P.,Plateforme Technologique et dInnovation Biomedicale | And 29 more authors.
Nature Communications | Year: 2013

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into a-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. © 2013 Macmillan Publishers Limited. All rights reserved. Source

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