The goal of the proposed research is to investigate the contribution of genetically marked autologous peripheral blood repopulating cells (PBRC) to long-term hematopoietic reconstitution after human autologous marrow transplantation and to determine whether and how the PBRC contribution is modified by pretreatment with different hematopoietic growth factors. Optimal methods for transduction of the neo marker gene into PBRC will be determined using cocultivation on vector-producing packaging cells and subsequent incubation in vector-containing stromal cell cultures, a method which has been shown to be effective in transducing canine hematopoietic repopulating cells. Peripheral blood and marrow cells will be analyzed repeatedly after transplantation by PCR and cell culture for the presence of the neo gene to determine: a) whether PBRC contribute to long-term hematopoietic reconstitution and b) whether and how the PBRC contribution is modified by pretreatment with different hematopoietic growth factors. These studies would also help determine the best strategies for transducing retrovirus vectors into human hematopoietic repopulating cells, the requisite next step before gene therapy in combination with marrow or PBRC transplantation can be used for treating human disease.
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