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. We have developed avian sarcoma leukocytosis virus (ASLV) vectors and site-specific non-viral vectors based on phage for hematopoietic target cell applications, due to more favorable insertion site profiles. ASLV can transduce rhesus long-term repopulating cells, as first demonstrated this year in our in vivo autologous transplantation moedl. We have continued to utilized the LAM-PCR technology to identify and track clonal contributions to peripheral blood populations following transplantation of CD34+ tranduced progenitor cells. 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 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. Surprisingly, we have found that lineage-restricted or lineage skewed progenitors contribute for at least three years in primates, with populations of clones contributing to T cells versus granulocytes that have little overlap. 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. 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. This past year one animal transplanted over 5 years ago with a CD34+ cells transduced with a standard retroviral vector developed a form of acute myeloid leukemia. The insertion site in the abnormal clone was found to be within an anti-apoptotic gene BCL2-A1. Expression vectors have been constructed expressing both BCL2A1 and MDS1/EVI1 to investigate the mechanism of immortalization or transformation. 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. Using the same model we have found improved immune reconstitution in animals treated with keratinocyte growth factor.
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