ADENOVECTORS FOR DELIVERY OF HEMATOPOIETIC GROWTH FACTORS AND RECEPTORS

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Adenovectors have proved highly efficient in transferring and expressing genes in murine and human marrow stromal and endothelial cells. At high multiplicities of infection, Ad vector expression can be obtained in immature hematopoietic cells (CD34+) and a variety of differentiated cell populations (erythroid, myeloid, dendritic, megakaryocyte). In vitro and in vivo murine studies will evaluate Ad vector delivery of cytokine genes (erythropoietin, G-CSF, GM-CSF c-kit ligand, Flk-2 ligand), singly or in combination, for augmentation of hematopoiesis and acceleration of recovery following chemotherapy, radiation, or bone marrow transplantation. Mutations at the 51 locus point to the importance of its product, the c-kit ligand and, specifically, the transmembrane isoform (KL-2) without a proteolytic cleavage site as important for stem cell migration, proliferation, and differentiation, and for optimal erythropoiesis. Soluble ligand administered systemically or produced in the Sl-Dicke mouse cannot substitute for the transmembrane form. Transmembrane isoforms of Flk-2 ligand may also play an important role in local regulation of stem cell self-renewal. Transmembrane forms of c-kit and Flk-2 ligands will be expressed by Ad vectors in stromal cells and marrow endothelium, in vitro or by locoregional administration in vivo, and compared to vectors expressing soluble ligand with emphasis on improved stem cell homing, stromal/endothelial adhesion and self-renewal. We have evidence that murine Notch expressed by primitive hematopoietic cells, and its ligand DLL1 expressed by stromal and endothelial cells, influence developmental fate decisions in hematopoiesis. This will be evaluated in hematopoietic systems where Ad vectors permit expression of high levels of Notch or its ligand. Transient overexpression of c-kit and Flk-2 on hematopoietic stem cells will be evaluated, and the impact on stem cell migration, adherence. self-renewal, proliferative status, and resistance to TGFbeta inhibition determined. Flk-l is expressed as a very primitive hematopoietic precursor (hemoblast/hemangioblast) that first appears in the yolk sac blood islands or their equivalent in embryonic stem cell differentiation, but is not expressed in later fetal or adult stem cells. We shall use Ad vectors to express Flk-l in adult stem cells and evaluate their proliferative response to VEGF alone or in combination with KL. Ad vector expression of the murine ecotropic retroviral receptor will be evaluated as a method for improving retroviral gene transduction of human stem cells. Introduction of a marker gene (mutated nerve growth factor) and of a methotrexate drug-resistance gene (mutated dihydrofolate reductase) will permit evaluation of stem cell transduction efficiency in a long-term culture assay and in vivo in SCID mice.