GENE THERAPY APPROACHES FOR BLOOD AND VASCULAR DISEASES - Perhaps one of the most significant findings within the past 2-3 decades has been the demonstration that many human diseases, including those affecting the blood and vascular systems, are the result of abnormal gene expression. In this proposal, we will exploit the inherent ability of viruses to efficiently infect cells, and use them to introduce potentially therapeutic transgenes into hematopoietic progenitors, vascular, and muscle cells. We will focus upon recombinant adeno-associated virus (AAV) vectors, which possess several important characteristics which make them promising for potential gene therapy including non-pathogenicity, wide host range, ability to transduce important quiescent cell populations, low immunogenicity, and potential for stable integration into the host genome, thereby promoting long term transgene expression. In Project 1, exciting studies supporting rAAV mediated gene transfer to human hematopoietic cells will be extended. Quiescent CD34+/CD38- human hematopoietic progenitors, a population enriched for stem cells and relat5ively recalcitrant to retroviral transduction, will be transduced with rAAV vectors, and vector integration and transgene expression will be examined after transplantation into a NOD/SCID animal model to determine whether true progenitor will be examined after transplantation into a NOD/SCID animal model to determine whether true progenitor populations can be genetically modified. In Project 2, rAAV vectors encoding wild type apo A-I, a proven anti-atherogenic agent which promotes transport of cholesterol from arterial walls to the liver, and the potentially even more potent apo A-I Milano mutant will be studied for their ability to inhibit systemic atherosclerotic plaque formation in the apo E knockout mouse. In addition, the unique ability of rAAV vectors to mediate long term (.6-12 months) transgene expression following intramuscular infection will be assessed. Finally, in Project 3, the overall frequency and sites of integration of rep negative rAAV vectors within primary human cells will be examined,, and methods designed to provide Rep functions in trans, which could potentially enhance integration and promote site specific integration, will be evaluated. This research will be supported by a Vector Core (Core B), for production of qualified, high titer vectors, and an Administrative Core (Core A) which will foster intensive scientific interactions and collaborations, and insure a successful outcome. Results from these studies will provide invaluable information for the development of gene therapy approaches to blood and vascular disease.
