TARGETED GENE THERAPY FOR HEMOPHILIA A

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Hemophilia A is the second most common congenital bleeding disorder worldwide, accounting for approximately 90% of hemophilia. Adequate hemostasis requires a minimal plasma factor VIII (FVIII) concentration (10 - 50 ng/ml; < 0.25 nM), suggesting that even inefficient systemic delivery systems may be successful. The vector of choice in this proposal is adeno-associated virus (AAV), which in the absence of its helper virus integrates as a stable provirus into the human genome, independent of the cell's proliferative state. Except for the terminal repeats, integration of AAV into the genome requires no transcriptional regulatory elements, reducing the risk of oncogenesis and allowing foreign promoters inserted into these viruses to retain normal function. This grant will focus on the generation of appropriate FVIII deletional constructs for proposed rAAV/FVIII delivery systems. Based on previous work in these and other laboratories, the size of these constructs should not interfere with rAAV packaging and replication constraints, nor functional FVIII expression. A minimum-length cell-specific promoter will be characterized and used to drive cell-specific expression. Novel methods designed to overcome rAAV size constraints and modest viral titers will be explored. After initial in-vitro studies, animal models will be used to further study and develop in-vivo expression systems. Although this proposal specifically addresses methods for FVIII delivery, the results may be generally applicable to a wide variety of gene- targeting strategies.