This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy 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 gene therapy 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 a system of gene therapy 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 an ongoing 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 will be 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's 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. This vector will serve as the basis for the next generation clinical trial of gene therapy in the planning stage. We have also begun to explore the use of highly modified lentivirus vectors in the laboratory. These vectors may 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 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. In other studies we have contributed to genetic studies of patients with the inherited disorder known as Hyper IgE-Recurrent Infection Syndrome. These studies involved the delineation of the clinical features and the delineation of potential sites of the gene(s) causing this disorder.
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