Eucaryotic viral vectors based upon the nonpathogenic parvovirus, adeno- associated virus (AAV) have recently emerged as promising vehicles for efficient gene transfer to a wide variety of cellular targets. AAV-based vectors are noncytopathic, possess wide species and tissue tropism, have high transduction efficiencies, stably integrate into host chromosomal DNA, and, unlike murine retrovirus vectors, efficiently transduce cells regardless of proliferating status. This latter characteristic is of critical importance for potential in vivo gene therapy, as subpopulations of mononuclear peripheral blood and pluripotential hematopoietic stem cells, highly attractive targets for gene therapy for a variety of diseases including AIDS, are normally quiescent. In this proposal, we will exploit the advantages of AAV-based vectors developed within our laboratory, and analyze gene transfer to both normal and malignant human marrow derived cells with efficiencies of greater than 70% will be presented. In addition, we will extend these results to purified subpopulations of primary marrow-derived cells, determine stability of vector-mediated transgene expression, characterize vector integration sites, and determine potential effects of transduction upon cellular function and multilineage development in vitro. In addition, we will directly compare AAV and retroviral vector-mediated transduction and transgene expression within these critical cellular populations. Finally, we will employ both AAV and retrovirus-based vectors encoding the neomycin phosphotransferase gene to genetically """"""""mark"""""""" a portion of human CD34+ peripheral blood stem cells (PBSCs) in the context of combination high dose chemoradiotherapy with autologous transplantation for indolent B-cell non- Hodgkin's lymphoma, and monitor for relapse with genetically marked cells. Such double labeling experiments within the same individual will allow a comparison of vector transduction efficiencies, effects upon hematopoietic multilineage development, and long term transgene expression in vivo. Information obtained from this work will further our understanding of and help to derive methodologies for optimal gene transfer to and stability of transgene expression within primary human marrow-derived cells both in vitro and in vivo, will provide important information about patterns of engraftment following PBSC transplantation, sites of relapse, the adequacy of """"""""conditioning"""""""" and stem cell purging regimens following autologous transplantation for hematologic malignancies, could facilitate AAV-based vector development for clinical gene therapy, and could provide the foundation for a gene therapeutic approach to the treatment of hematologic malignancies and AIDS.
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