This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy or for use in allogeneic transplantation in the treatment of inherited diseases affecting cells of the immune system. This project also studies CD34 positive hematopoietic adult human stem cells from patients with inherited immune deficiencies with the ultimate goal of developing hematopoietic stem cell based therapies for these disorders. We have developed new methods and materials which improve our ability to get new genes into human blood stem cells. We are also exploring the potential of busulfan as a more stem cell specific and immune system sparing conditioning regimen for transplant so we need to understand better the effect of such agents on human CD34 positive hematopoietic adult human stem cells. Evidence from human and animal studies of gene therapy suggest that providing an in vivo growth or survival advantage to genetically corrected blood cells can improve the outcome of gene therapy by increasing the percent of corrected cells in the body. One approach to this is to co-express the therapeutic gene (such as the corrective gene for X linked CGD) with a gene that allows for selective enrichment. In studies with collaborators we have explored the use of the methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU in a non-human primate model achieving marking rates of up to 20%. Thus, we need to understand better how to culture and subject to selection human stem cells. As noted, a major goal of this project is to examine in CD34 positive hematopoietic adult human stem cells the role of the CXCR4 chemokine receptor (ligand is SDF-1) on engraftment in marrow. We showed that overexpression of CXCR4 in human CD34 hematopoietic stem cells enhanced engraftment of these cells in the NOD/SCID mouse xenotransplant model. The immunodeficiency, WHIM (warts, hypogammaglobulinemia, infections, myelokathexis apoptosis of neutrophils), is caused by truncations in the C-terminus of CXCR4. We created gene transfer vectors to over express the WHIM type mutant CXCR4 in CD34 stem cells and showed that this resulted in increased migration, adhesion and intracellular calcium flux in response to SDF-1. We showed that this was caused by a failure to downregulate or to internalize the mutant receptor providing a biochemical basis for the dominant hyperfunction abnormality of CXCR4 activity associated with WHIM. We also find that the mutant CXCR4 enhances engraftment of cells expressing this mutant receptor and it may be a useful tool to enhance engraftment. CD26 is a protease expressed on bone marrow stroma and also on some CD34 positive hematopoietic adult human stem cells. CD26 is a type IV dipeptide proteinase that can cleave and inactive SDF-1. We show that treatment of NOD/SCID mice with Diprotin A, an inhibitor of CD26 on marrow stroma, markedly enhances engraftment of CD34 positive hematopoietic adult human stem cells in this xenograft model. We believe that this could be an important therapeutic method to enhance engraftment.
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