Gaucher's disease is a debilitating sphingolipid storage disorder affecting approximately 30,000 patients in the United States. The goal of this project is to develop improved gene transduction methods which would allow to cure this disease by expressing functional levels of glucocerebrosidase (GC) in macrophages of Gaucher's disease patients by transducing their hematopoietic repopulating cells with a human GC gene. We want to develop more efficient in vitro transduction methods for hematopoietic stem cells, improve conditioning and ex vivo and in vivo selection strategies in the dog model and finally confirm our results in a small number of primates before proceeding to a clinical application in humans. The experiments outlined will be the basis for an ensuing clinical study designed not only to cure Gaucher's disease, but also to serve as a model for gene therapy of other human diseases. It can be estimated that at least ten percent of a Gaucher patient's macrophages must express the GC gene in order to obtain a GC activity sufficient to improve disease manifestations. For that reason, retroviral transduction and engraftment of transduced hematopoietic repopulating cells have to be made more efficient. To achieve both, we first propose to improve the in vitro maintenance, expansion and transduction of hematopoietic repopulating cells. This will involve strategies to determine optimal exposure times to retrovirus vector supernatant and to develop optimal ex vivo growth conditions for stem cells to proliferate and integrate efficiently vector DNA without loss of self-renewal capacity. Second, we propose to generate safe, high-titer retrovirus GC vectors with high affinity for canine and primate cells. Specifically, we have produced a single-gene-expressing GC vector (PG13/LgGC) and will generate two-gene-expressing GC vectors that coexpress selectable genes. Third, we propose studies in dogs on how to increase the percentage of transduced repopulating cell clones that engraft. To accomplish this aim, we will compare different conditioning regimens that reduce the amount of endogenous repopulating cells prior to the infusion of transduced cells, and we will develop ex vivo and in vivo selection strategies for cells expressing a selectable marker gene in order to reduce the number of non- transduced cells. Finally, in preparation for treating patients, we propose to test in a nonhuman primate model the transduction and transplantation strategies found to be most effective. The outlined studies will address basic questions of applied gene therapy and stem cell biology and accelerate the application of these disciplines towards the treatment of disease.
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