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. A new component of this research which we have incorporated into this project during the past year is an initiative to establish from patients with primary immune deficiencies induced pleuripotent stem cells (iPS cells) with the primitive properties of embryonic stem cells capable of differentiating into hematopoietic stem cells, and into mature neutrophils and other cells of the immune system. 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 use of those gene transfer systems to correct the genes and gene defects causing primary immune deficiencies in iPS cells derived from patients with PIDs. 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 and upon more primitive stem cells capable of differentiating into CD34 positive hematopoietic 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. In the same mouse NOD/SCID xenograft system, we have during the past year demonstrated that forcing excess expression of the growth regulatory element HOXB4 in human hematopoietic stem cells will enhance engraftment of those human stem cells in the NOD/SCID mouse xenograft model. We did this by transferring the gene for an element upstream of HOXB4 regulation rather than by just overexpressing HOXB4 directly. These important experiments are ongoing, but we hope that similar to the observations of others, the overexpression of HOXB4 could be a means of increasing the number of primitive human hematopoietic stem cells that engraft. As noted above, we have together with our collaborator, Dr. Linzhao Cheng at Johns Hopkins University initiated a project to develop iPS cells from patients with primary immune deficiencies, focussing initially on patients with X-linked chronic granulomatous disease (X-CGD) (a PID caused by a defect of microbicidal hydrogen peroxide production by blood neutrophils). We have successfully completed the first stage of this project by establishing X-CGD iPS cells from bone marrow mesenchymal cells. Our next goal is to determine the conditions to cause differentiation of X-CGD iPS cells into mature neutrophils. This will establish a model that will allow exploration of the potential of applying methods of gene repair to the X-CGD iPS cells to correct the CGD phenotype in neutrophils that arise from the gene corrected iPS cells.
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