One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the defective human parvovirus adeno-associated virus (AAV) in hopes of developing a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co- infection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, wild type (wt) AAV genome integrates via its ends into the host chromosome in a site-specific manner and resides there in a latent state until the cell is infected with helper virus. The interest in AAV as a eukaryotic vector has centered around the biology of this virus. In addition to this unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, etc.), it is ubiquitous in humans, and is completely nonpathogenic integrating virus. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for hemopoietic cells. We initiated studies testing both human globin genes as well as the Fanconi anemia C gene (FAC). Previous efforts utilizing the globin locus control region (LCR) sequence has met with difficulty using other vector systems. We have demonstrated high level, tissue specific expression of human globin genes in human erythroid cells, where expression and globin protein was detected. Although globin expression was detected after rAAV infection, transduction was low in vitro. In continued analysis of this system, we demonstrated expression and correction of lymphoblast cells from a Fanconi patient using an AAV vector carrying the Fanconi anemia gene (FAC). Utilization of this vector in patient CD34 enriched bone marrow cells demonstrated gene transduction and selective growth advantage in colony forming assay. These studies were extended to non-human primates bone marrow cells with preliminary results suggesting gene marking out to 3 months. This last observation and data from in vitro systems suggests a rate limiting step in efficient transduction of rAVV in hematopoietic progenitor cells is the lack of stable integration. The overall objective of the proposed work is to study integration in bone marrow stem cells using the well characterized wt AAv site-specific integration system and a new animal model we have developed. The long range goal is to better understand these molecular step in primary bone marrow stem cells with the ultimate goal of developing specific viral vectors with efficient transducing capability as a result of targeted integration.
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