Platelets adhere, form a platelet plug and degranulate at sites of vascular injury. We are interested in testing whether ectopically expressed proteins during megakaryopoiesis would be stored within platelets and released at sites of injury, modulating the thrombotic process. Initial transgenic studies suggest that we can accomplish this process, delivering platelet-released (p) Factor VIII (FVIII) to a site of injury to improve the bleeding diathesis in FVIIInull mice. In the following specific aims, we propose to better understand and improve on the transgenic pFVIII model, and to extend these studies to a gene therapy approach.
Specific Aim I. Characterize clotting in the platelet delivery of FVIII model. Before human studies can be considered, the advantages and disadvantages of the delivery of FVIII via platelets needs to be better understood. Proposed studies should provide a better understanding of the details of the developing clot and define the efficacy of correcting the bleeding diathesis in FVIIInull mice in various bleeding models. We will address where and how pFVIII Is stored within platelets. Finally, we will test whether pFVIH's directed delivery to sites of injury may be more efficacious than plasma FVIII in the treatment of FVIIInull mice with inhibitors.
Specific Aim II. Enhancing the efficacy of platelet-based correction of the hemophilias. The highest level of pFVIII achieved so far in transgenic animals had the equivalent of an approximately 3% plasma activity level. We propose several strategies to enhance the present model including the addition of further gene regulatory elements, the expression of FVIII mutants that either enhance FVIII intracellular processing or resistance to inactivation, and the expression of activated FVII within the platelets.
Specific Aim III. Viral delivery of pFVIII. We have successfully extended these transgenic mice studies to bone marrow transplantation studies as a prelude to vector-based gene therapy. Using a lentiviral approach, we plan to examine lentiviral-based in vivo gene therapy, initially driving megakaryocyte-specific expression of eGFP and then FVIII in mice. If successful, these studies will be extended to FVIIInull dogs, where bleeds are more representative of those seen in patients. We believe that successful application of these ideas may provide new approaches for the management of Hemophilia A and perhaps other diseases affecting thrombus formation.
| French, Robert A; Samelson-Jones, Benjamin J; Niemeyer, Glenn P et al. (2018) Complete correction of hemophilia B phenotype by FIX-Padua skeletal muscle gene therapy in an inhibitor-prone dog model. Blood Adv 2:505-508 |
| George, Lindsey A; Sullivan, Spencer K; Giermasz, Adam et al. (2017) Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant. N Engl J Med 377:2215-2227 |
| Gollomp, Kandace; Lambert, Michele P; Poncz, Mortimer (2017) Current status of blood 'pharming': megakaryoctye transfusions as a source of platelets. Curr Opin Hematol 24:565-571 |
| High, Katherine A; Anguela, Xavier M (2016) Adeno-associated viral vectors for the treatment of hemophilia. Hum Mol Genet 25:R36-41 |
| Siner, Joshua I; Samelson-Jones, Benjamin J; Crudele, Julie M et al. (2016) Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models. JCI Insight 1:e89371 |
| Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R et al. (2016) CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes. Blood 127:675-80 |
| Sim, Xiuli; Poncz, Mortimer; Gadue, Paul et al. (2016) Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets. Blood 127:1227-33 |
| Marcos-Contreras, Oscar A; Smith, Shannon M; Bellinger, Dwight A et al. (2016) Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII. Blood 127:565-71 |
| Arruda, V R; Samelson-Jones, B J (2016) Gene therapy for immune tolerance induction in hemophilia with inhibitors. J Thromb Haemost 14:1121-34 |
| Lyde, R; Sabatino, D; Sullivan, S K et al. (2015) Platelet-delivered therapeutics. J Thromb Haemost 13 Suppl 1:S143-50 |
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