The development of inhibitory antibodies to exogenous FVIII is an important complication of FVIII infusion in hemophilia A patients. Generation of such inhibitors might have the potential to preclude gene therapy for hemophilia A. In this project, we propose to investigate a novel approach for gene therapy of hemophilia A and hemophilia A with inhibitors based on the hypothesis that targeting the production of FVIII to a regulated secretory cell type (platelet) that activates at the site where FVIII is needed could overcome the presence of inhibitory antibodies. We have recently developed a vector (2bF8) that targets human FVIII expression to platelets under control of the platelet-specific 1IIb gene promoter. Using this construct, we can correct the murine hemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. Another study using 2bF8 lentivirus-mediated bone marrow (BM) transduction and syngeneic transplantation resulted in therapeutic levels of FVIII expression in platelets in hemophilic mice with no antibody development. Our most recent studies have demonstrated that syngeneic transplantation of hematopoietic stem cells (HSC) from 2bF8 transgenic mice can efficiently restore hemostasis to hemophilic mice with inhibitors. In this R01 grant application, we propose to further explore whether 2bF8 lentiviral-mediated gene transfer can introduce sufficient platelet-FVIII expression and rescue the hemorrhagic phenotype in hemophilic mice and hemophilic sheep with inhibitors. To accomplish these, we first propose to transduce HSC from immunized FVIII null mice with 2bF8 lentivirus and transplant into immunized littermates. After BM reconstitution, the recipients will be analyzed by PCR, real-time PCR, LAM-PCR, confocal microscopy, FVIII activity assays, and inhibitor assays. Phenotypic correction will be assessed by tail clip survival test and in vivo thrombus formation. Secondly, we will transduce HSC from hemophilic sheep with or without inhibitors using 2bF8 lentivirus and then transplant the 2bF8-transduced autologous cells back into the animals. The treated animals will be analyzed by the assays as described above, plus whole blood clotting time, thrombin generation assay and thromboelastography assay. Treated animals will be closely monitored to determine if the hemorrhagic phenotype has been improved. Lastly, we will transplant the 2bF8-transduced sheep HSC into hemophilic sheep fetuses to investigate the efficacy and safety of fetal gene therapy using 2bF8 lentiviral vector. These studies should allow us to address the efficacy and safety of our lentivirus-mediated platelet-specific gene therapy approach in animal models with anti-FVIII specific immunity, with the potential to develop a long- term strategy for gene therapy of hemophilia A patients and patients with inhibitors.

Public Health Relevance

Hemophilia A is the most common severe bleeding disorder resulting from hereditary deficiency of the blood clotting protein, factor VIII (FVIII). The development of inhibitory antibodies to exogenous FVIII is considered a severe and important complication of FVIII infusion in hemophilia A patients. In this project, we will develop a gene therapy approach by targeting the synthesis of FVIII to blood platelets, that might cure hemophilia A patients as well as patients with inhibitors.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Link, Rebecca P
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Medical College of Wisconsin
Schools of Medicine
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Fahs, Scot A; Hille, Matthew T; Shi, Qizhen et al. (2014) A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII. Blood 123:3706-13
Shi, Qizhen; Kuether, Erin L; Chen, Yingyu et al. (2014) Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells. Blood 123:395-403
Schroeder, J A; Chen, Y; Fang, J et al. (2014) In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection. J Thromb Haemost 12:1283-93
Kanaji, S; Fahs, S A; Ware, J et al. (2014) Non-myeloablative conditioning with busulfan before hematopoietic stem cell transplantation leads to phenotypic correction of murine Bernard-Soulier syndrome. J Thromb Haemost 12:1726-32
Chen, Yingyu; Schroeder, Jocelyn A; Kuether, Erin L et al. (2014) Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(null) mice. Mol Ther 22:169-77
Kanaji, Sachiko; Kuether, Erin L; Fahs, Scot A et al. (2012) Correction of murine Bernard-Soulier syndrome by lentivirus-mediated gene therapy. Mol Ther 20:625-32
Montgomery, Robert R; Shi, Qizhen (2010) Alternative strategies for gene therapy of hemophilia. Hematology Am Soc Hematol Educ Program 2010:197-202
Shi, Qizhen; Montgomery, Robert R (2010) Platelets as delivery systems for disease treatments. Adv Drug Deliv Rev 62:1196-203
Shi, Qizhen; Fahs, Scot A; Kuether, Erin L et al. (2010) Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A. Blood 116:3049-57