Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo.We have continued to further enhance gene transfer efficiency into rhesus engrafting cells, resulting in early levels of marked cells as high as 50-80%, with stable levels of 5-35% in all lineages, a range with clinical utility. These levels can be achieved with traditional amphotropic MLV vectors, as well as with novel SIV-based lentiviral vectors. The high levels have allowed us to continue to track clonal contributions to hematopoiesis for the first time in a large animal model. We have utilized a new technology that allows simultaneous assessment of multiple clonal contributions to peripheral blood populations. We have found a different population of engrafting cells that contribute for the first 1-2 months post-transplantation, that are then replaced by a very stable set of 30-100 clones that contribute to all lineages for over 6-7 years in some animals. We have investigated the impact of cytokine therapy, radiation, and chemotherapy on the in vivo behavior of stem cell clones, using this powerful methodology. Prolonged cytokine treatment with either G-CSF or SCF does not significantly alter the number of stem cell clones contributing to hematopoiesis, nor result in detectable clonal exhaustion or recruitment. In contrast, treatment with low dose total body irradiation or with busulfan results in a significant decrease in stem cell clones contributing to peripheral blood lineages. We have investigated the lineage contributions of individual stem and progenitor cell clones, asking whether clones contribute equally to each lineage such as granulocytes, T cells, B cells, dendritic cells and mast cells. Given the occurence of leukemia in two children receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells in France, we have performed large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with cells transduced either with MLV or SIV vectors. Thus far, no animal has developed leukemia or any hematologic disorder, in a cohort of 46 animals followed greater than 1 year, and for a median now of 4.5 years. The insertion site analysis shows non-random preference for insertions within genes for both MLV and SIV, with SIV insertions distributed evenly over the length of genes and particularly being found in highly gene rich chromosomal regions. MLV instead targets the region around transcriptional start sites. Over 49 ?common integration sites?, or genes or genomic areas with more than one integration event have been found. These highly non-random events indicate either a strong non-random preference for integration at these sites, or an in vivo engraftment or survival/proliferative advantage for these clones. 14 independent insertions were localized to the MDS1/EVI1 locus, an area previously implicated in spontaneous leukemias and in retroviral mutagenesis with replication competent viruses. These findings have important implications for future gene therapy clinical applications. We have continued to investigate the transforming properties of the novel rabaptin/PDGFRbeta fusion protein a murine model, and broadened our investigation of activated tyrosine kinases in myeloproliferative syndromes to patients with hypereosinophilic syndromes. We have also investigated the activity of the tyrosine kinase inhibitor imatinib on T lymphocytes. Using the non-human prrimate model, we have investigated immune reconstitution in detail following transplantation with purified CD34+ cells versus whole mobilized peripheral blood, as well as cells ex vivo expanded for 4 days in the presence of cytokines. We find significant improvements in immune reconstitution using ex vivo expanded cells.
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