The ability to transfer genes into human hematopoietic stem cells is a long sought-after goal of hematologists and other interested in curing stem cells disorders. Although successful stem cell gene transfer in murine models is now feasible, translation of this success to humans has been difficult. The central thesis of this project is that significant advances in the technology of gene transfer in humans are most likely to come from a multidisciplinary approach involving retrovirology, cell biology, and clinical experimentation. Project 3 is the interface between basic scientists engaged in designing new vectors, murine cell biologists improving efficiency of gene transfer, and clinical scientists engaged in pilot gene transfer studies in human. Overall, we propose to 1) analyze the efficiency of various vectors and infection conditions to transfer genes to human hematopoietic cells in vitro, working closely with Projects 1 and 2; 2) develop techniques to assay expression of transferred genes suitable for analysis of clinical samples; 3) provide the laboratory support necessary for the conduct of the clinical trials in Project 6; 4) develop optimum gene transfer protocols for human use through analysis of gene transfer efficiency in cancer patients, and 5) compare in vitro assays of human stem cells with outcome of gene transfer in vivo. In collaboration with Project 6, we propose to initiate clinical trials in which the MDR-1 gene is transferred to stem cells in cancer patients undergoing allogeneic or autologous transplantation for high risk malignancies. Pilot studies will investigate safety and efficacy of a series of potential technical advances in human gene transfer. Ultimately, gene transfer protocols which are the most promising will be compared directly using competitive repopulation in humans. Therefore, the long term goals of this project are to develop safe, effective protocols to transfer genes into human stem cells. At that point, we will initiate trials aimed at gene replacement therapy in patients with inherited gene defects. In anticipation of those trials we will aim to develop assays of transduction and expression efficiency of these genes suitable for clinical samples and will compare various transduction protocols for in vitro efficacy. This project is an essential link between Projects 1 and 2 with Project 6, and is likewise dependent on those projects for its successful completion.
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