TARGETED GENE THERAPY FOR HEMOPHILIA A

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Hemophilia A (congenital deficiency of coagulation factor VIII) is the most common (serious) congenital bleeding disorder worldwide. Hemophilia A is a model system for definitive genetic therapy for the following reasons: (I) it is caused by a single- gene defect, (ii) its biological processing and synthesis have been extensively studied, (iii) therapeutic levels of plasmatic clotting factors ameliorate hemorrhagic episodes, and (iv) physiologic levels in human are low (100 - 200 ng/mL), with clinical manifestations closely paralleling circulating fVIII levels such that therapeutic benefit can be reached with 5 percent fVIII activity (5 - 10 ng/mL). As a definitive means of genetic treatment, this laboratory has focused on adeno- associated virus (AAV) for fVIII gene therapy strategies, as AAV infection is not associated with human diseases, and cells carrying proviruses fail to express novel cell-surface antigens, thereby failing to serve as immunological targets. If a helper virus is not present during an AAV infection, the AAV genome integrates in a site-specific region (AAVS1) on human chromosome 19q, a property that is dependent on AAV rep68/78 and inverted terminal repeats (TR's). By virtue of being produced primarily in the liver, the hepatocyte is the logical site for gene replacement strategies for hemophilia A, although vascular endothelial cells remain an attractive target as a means of regulated delivery by co-expression with Von Willebrand factor. In this proposal, we will continue to develop novel viral vectors for hemophilia A gene therapy, viruses which collectively share the unique integrating properties of the AAV terminal repeats, as a means of definitive treatment for hemophilia A. To date, we have been the only laboratory that has successfully generated a fVIII/rAAV virus, serving as proof-of-principle for further research in this direction. Three viruses will be characterized for their ability to deliver B-domain-deleted factor VIII recombinant AAV, adeno/adeno-associated hybrid (Ad/AAV) virus, and mini-adenovirus (mAD) containing the AAV TR's, generated as a unique transcriptional byproduct of parental Ad/AAV hybrid viruses. Additional aims will be directed at establishing the utility of vascular endothelial cells as targets for fVIII delivery, using both in vitro and in vivo models for targeted delivery into these cellular types. Optimal viral vectors will be studied in murine and canine models of hemophilia A. The proposed work is designed to lay the foundation for anticipated trials in humans.