A major problem in liver-directed gene therapy is the development of an immune response to the therapeutic transgene. Previously, we have found that mouse CMV-driven factor VIII cDNA delivered in a first-generation adenovirus to hemophilia A mice provokes a substantial immune response to both factor VIII and adenoviral proteins. This response can be blunted by suppression of T-cells with anti-CD4. Over the past year, it has become clear that adeno-associated virus (AAV) is a safer and perhaps more effective delivery vehicle than adenovirus. In this project, we aim to carry out long-term correction of hemophilic mice and dogs by delivery of FVIII cDNA to liver in an AAV vector. Our goal is to devise the means to deliver the FVIII cDNA without encountering an immune response. We have recently cloned a short SQ version of the mouse FVIII cDNA driven by a small liver-specific promoter (human a1-antitrypsin promoter) in an AAV vector. This and other vectors will be tested for preliminarily for therapeutic effect in vitro and in vivo; then in immunosuppressed, FVIII-deficient mice; and finally in immunocompetent hemophilic mice. The total size of the mouse FVIII-SQ cDNA in this vector is under 4.4 kb, leaving roughly 380 bp for promoter/enhancer combinations. In immunocompetent mice, we will measure FVIII activity, FVIII antigen, and both cellular and humoral immune responses to FVIII. Using the information gained from mice experiments, we will attempt correction of hemophilia A dogs using canine FVIII-SQ cDNA via liver directed expression. We hope to overcome any immune response to FVIII and provide successful long-term treatment of these animal models. These studies are critical precursors to clinical trials of liver-directed therapy of hemophilia A using AAV vectors.