Hematopoietic stem cells (HSC) and endothelial precursor cells (EPC) provide an invaluable source of cells for autologous and allogeneic transplantation as well as gene therapy for congenital hematological and vascular disorders. The focus of this proposal is the challenge to identify and deliver factors that will allow for efficient in vivo expansion and mobilization for adequate pluripotent HSC and EPC that could be used for transplantation and gene therapy. The strategy is to capitalize on the robust, albeit transient expression mediated by adenovirus (Ad) gene transfer to express stem-cell active chemokines and angiogenic factors that promote extramedullary mobilization of both HSC and EPC. The preliminary data shows that Ad vector mediated in vivo expression of stem cell active cytokines and angiogenic factors with chemotactic potential such as vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF-1) and Angiopoietin-1 in the peripheral circulation can induce mobilization of HSCs and EPCs. Based on these studies, we hypothesize that regional and temporal expression of secreted and membrane bound angiogenic factors and stem cell active chemocytokines by Ad gene delivery will promote in vivo expansion and mobilization of marrow derived EPCs and HSCs to the peripheral circulation. These mobilized puripotent stem cells may be used for autologous or allogeneic transplantation or gene therapy. On the basis, this project seeks this strategy in the context of moving in to human application. First, we plan to determine whether regional delivery of Ad vectors expressing stem active chemocytokines induced in vivo expansion and mobilization of HSC and EPCs. Studies will be carried out to: 1) investigate whether sufficient, soluble and membrane bound Kit-ligand (Skl, Mkl) AND Flk- 2, alone or in combination can be delivered to marrow using Ad vectors to promote expansion and mobilization of HSCs; and 2) to determine whether sufficient angiogenic factors including soluble VEGF/121, VEGF/165, and matrix bound VEGF/189, placental growth factor (PLGF), Angiopoietin-1 and Angiopoietin-2 could be delivered and produced by Ad vectors to induce proliferation and mobilization of EPCs. Second, we plan to define the mechanism whereby chemocytokines induce mobilization of HSC and EPCs. The studies are planned to 1) evaluate the significance of chemokine-induced metalloproteinase (MMP) activation in the mobilization of stem cells by Ad vectors expressing SDF-1, VEGF isoforms in MMP (MMP-9) knock out mice; and 2) examine the role of endothelial specific adhesion molecules in the regulation of stem cell mobilization by Ad vectors expressing SDF-1, VEGF and angiopoietins in ICAM1, E-selectin and P-selectin knock out mice; and 3) assess the role of chemocytokine modulation of stem cell cycle in the mobilization of HSC and EPCs. Third, assess the efficacy of a novel approach of transplantation ex vivo AD vector transduced hematopoietic cells over-expressing chemocytokines, in mobilization HSC and EPCs. To evaluate this, studies have vector transduced hematopoietic cells over-expressing chemocytokines in mobilization HSC and EPCs. To evaluate this, studies have been designed to: 1) examine whether sufficient hematopoietic cells over-expressing Mkl, Flk-2, SDF- 1, VEGF/165 and VEGF/189 can be transplanted and delivered to the marrow to induce expansion of HSC and EPCs; and 2) assess whether sufficient chemokines can be delivered to the marrow environment by transduced hematopoietic cells to induce mobilization of HSC and EPCs.
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