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 the pathophysiology of inherited immune deficiencies with the ultimate goal of developing hematopoietic stem cell based therapies for these disorders. One of the main barriers to more extensive application of allogeneic hematopoietic stem cells transplantation for correction of inherited immune deficiency is chronic graft versus host disease. Therefore the study of the immune disregulation associated with chronic graft versus host disease, and the development of treatments to prevent or treat graft versus host disease also fall within the scope of this project. We have developed new methods and materials which improve our ability to get new genes into human blood stem cells. The specific goals were to develop the pre-clinical systems of gene therapy that could then be applied to correct the genetic defect in the X-linked genetic form of chronic granulomatous disease (CGD) and the X-linked form of severe combined immune deficiency (XSCID). Earlier results of this development program were used in a completed clinical trial of gene therapy for CGD and in the pre-clinical work required to set up a clinical trial for XSCID. That clinical trial for X-CGD and the results from that clinical trial are discussed in the report for Project Z01-AI-00645. Specifically, we developed a retrovirus vector producer cell line that secretes high titers of the MFGS vectors containing the gp91phox cDNA that will correct the functional defect in X-linked CGD neutrophils. We have developed a version of our CGD corrective vector that is pseudotyped with the RD114 retrovirus envelope resulting in extraordinarily high level of gene transfer into hematopoietic stem cells (routinely greater than 90%). The NOD/SCID immunodeficient mouse will accept grafts of human hematopoietic stem cells. Using the NOD/SCID mouse/human stem cell chimera we demonstrate the full functional correction of 20-30% of human neutrophils arising in this model from the mobilized peripheral blood stem cells of CGD patients transduced with RD114 pseudotyped MFGS-gp91phox vector. This unprecedented level of gene correction in this model provides the basis for using this approach in future clinical gene therapy trials for CGD and other immune deficiencies. We have also developed RD114 pseudotyped MFGS-common gamma chain (gc) vectors to treat XSCID. We also collaborated with a group from the University of Pennsylvania who have a dog model of XSCID in which we have tested the ability of these vectors to cure this disorder with gene therapy in this animal model. In vitro we have achieved levels of over 80% marking of dog stem cells using this vector. Treatment of XSCID dogs with ex vivo gene therapy of autologous marrow stem cells have demonstrated the appearance of gene corrected T lymphocytes. Although the majority of T lymphocytes in these treated animals express the corrective gene, the total number of circulating T-lymphocytes remains below normal, and there is no evidence for correction of B lymphocytes. Some marrow conditioning may be required to enhance engraftment of blood stem cells in these animals and thereby improve the production of corrected T lymphocytes. We have also used a novel approach for in vivo gene therapy in the XSCID dogs where 3 day old dogs are injected intravenously with corrected gene therapy vector. Using this method, two dogs have achieved long term high level restoration of the immune system. This is a novel and unprecedented demonstration of the feasiblity of in vivo gene corrected using direct injection of gene therapy vector. We are also developing 3rd generation modified self-inactivating lentivirus vectors that are derived from the Human Immunodeficiency Viruss (HIV)-1 genome as well as from the Simian Immunodeficiency Virus (SIV) for the treatment of CGD or XSCID. Our studies demonstrate that these lentivectors appear to have greater potential to target the most primitive stem cells which are not dividing and which can engraft in the NOD/SCID animal model. In other studies we are examining the role of different growth factors in stimulating CD34+ stem cells to divide and to determine the relationship between entry into the cell cycle, ability to transduce with retrovirus vectors, and the maintenance or loss of long term engrafting potential. These studies are essential to achhieving our goal of high levels of gene transfer into long term engrafting stem cell. In other studies we have studied the effects of low dose radiation or chemotherapy on the engraftment of stem cells in animal models. We have demonstrated high levels of engraftment of gene marked cells in mouse and non-human primate animal models using low intensity conditioning regimens consisting of non-ablative levels of total body radiation. Follow up studies are in progress looking at non-ablative chemotherapy regimens instead of using radiation, and preliminary studies suggested that the non-ablative combination of cyclophosphamide and fludarabine can achieve low level (0.3%) prolonged (greater than 1 year)gene transfer marking of blood cells in the primate model. We are now exploring the potential of busulfan as a more stem cell specific and immune system sparing conditioning regimen. 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 multiple drug resistence 1 (MDR1) gene. In more recent studies we have explored the use of methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU. We have created gene therapy vectors (both onco-retrovirus vectors and lentivirus vectors) that contain the CGD corrective gene together with a mutant MGMT that is resistant to inactivation by benzyl guanine. We have used these vectors to correct CD34+ cells from X-CGD patients; transplanted the corrected cells in the NOD/SCID mouse and then demonstrated that the percent of corrected human cells in the NOD/SCID mouse could be greatly increased by treatment of the NOD/SCID mouse with benzyl guanine plus BCNU. It may be possible to apply this approach to in vivo selective improvement of gene therapy in the clinical setting and a gene therapy trial for treatment of CGD using such a vector is in the planning stage. In other studies of human hematopoietic stem cells we have been exploring 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 gene transfer vectors to overexpress 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.
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