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 high transduction efficiencies in a wide variety of cells including hematopoietic progenitors, stably integrate into cellular DNA often as multicopy tandem repeats, 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 mature peripheral blood T-lymphocytes and hematopoietic stem cells, highly attractive targets for gene therapy for AIDS, are normally quiescent. In this proposal, we will exploit the advantages of AAV-based vectors developed within our laboratory, and use them to transfer ribozymes specifically designed to cleave transcripts requisite for HIV productive infection to primary human CD34+ peripheral blood stem cells (PBSCs). Evidence that AAV vectors are capable of stable, nontoxic gene transfer into primary normal human marrow-derived cells with efficiencies of greater than 70% will be presented. Initial studies will be performed using ribozymes which have already been demonstrated to cleave the first exon of tat and the common exon of tat and rev (tat-rev), regions that are highly conserved within diverse HIV strains and whose products have been demonstrated to be requisite for HIV productive replication. Importantly, these ribozymes have been shown to protect human CD4 lymphocyte lines from HIV challenge. We will determine stability of AAV vector-mediated anti- HIV ribozyme expression, characterize vector integration sites, and determine potential effects of ribozyme expression upon multilineage development (colony formation) of human progenitors in vitro. We will also examine potential effects of constitutive anti-HIV ribozyme expression upon hematopoietic reconstitution in a murine transplant model in vivo. Finally, we will employ ribozyme encoding AAV vectors to genetically """"""""mark"""""""" a portion of hum CD34+ PBSCs in the context of nonmyeloablated autologous PBSC transplantation in HIV-seropositive individuals, and monitor for survival of ribozyme expressing cells (versus control vector transduced cells), effects upon hematopoietic multilineage development, and long-term transgene expression in vivo. Information obtained from this work will foster the development of methodologies for optimized gene transfer to human PBSCs and their progeny both in vitro and in vivo, will provide critical information about stem cell transplantation in HIV-infected patients and potential effects of anti-HIV ribozyme expression within hematopoietic cells, will facilitate AAV-based vector development for clinical gene therapy, and could provide the foundation for a gene therapeutic approach to the treatment of AIDS.
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