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 dysregulation 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. We are exploring new methods to reduce graft versus host disease based on extracorporeal photopheresis and upon the use of agonists of the adenosine A2 receptor which reduce cytokine mediated inflammation reponses. The specific goals relating to gene therapy 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). Specifically, we developed a retrovirus vector producer cell line that secretes high titers of the MFGS vectors containing the gp91phox cDNA and recently developed new methods to concentrate that virus to high titer that will be used in a new clinical trial. We have also developed novel lentivirus vectors based on Simian Immunodeficiency Virus of the macaque type (SIV-mac) that very efficiently targets human, non-human primate and dog hematopoietic stem cells. We have conducted and are in the process of conducting large animal studies with these gamma retrovirus and lentivirus vectors. 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 10-20% of human neutrophils arising in this model from the mobilized peripheral blood stem cells of CGD patients transduced with SIV-gp91phox vector. This unprecedented level of gene correction in this model provides the basis for using this lentivector in a future clinical gene therapy trials for CGD and other immune deficiencies. We have also developed RD114 pseudotyped SIV-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 both our MFGS-gc and SIV-gc vectors to cure this disorder with in vivo gene therapy in this animal model. In vitro we have achieved levels of over 80% marking of dog stem cells using these vector. Specifically, we have 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, a number of dog treated with either the gamma retrovirus-gc or SIV-gc vectors have achieved long term high level restoration of the immune system. This is a novel and unprecedented demonstration of the feasibility of in vivo gene correction using direct injection of gene therapy vector. 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 achieving 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 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%. 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 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.
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