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. 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 have been used in a recently 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 demonstrated that the fibronectin fragment CH-296 coated on culture vessel surfaces will greatly augment the gene transfer correction of stem cells from patients with CGD. More recently in collaboration with a group at St. Jude Hospital Medical Center in Memphis we have developed a version of our CGD corrective vector that is pseudotyped with the FLYDR (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 have begun a collaboration with a group from the University of Pennsylvania who have a dog model of XSCID in which we will test 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, and this system will be applied to the in vivo animal model. We have also begun to explore the use of 3rd generation modified self-inactivating lentivirus vectors that are derived from the Human Immunodeficiency Virus (HIV)-1 genome. These vectors appear to have greater potential to target the most primitive stem cells which are not dividing. 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. Our initial studies were in mice, but in collaboration with investigators at the University of Maryland and the New England Primate Center (Harvard University, we have demonstrated high levels of engraftment of gene marked cells in primate 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.
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