IntroductionHemophilia A, a congenital deficiency or dysfunction of factor VIII (FVIII), is the most common severe inherited bleeding disorder in humans. Severe hemophilia A patients have less than 1% of normal FVIII activity, and suffer from spontaneous or traumatic joint and muscle hemorrhage, leading to a chronic painful and disabling arthropathy. Bleeding into body cavities or the brain can result in significant morbidity and mortality if not treated aggressively. 1 Current treatment in the developed world, FVIII protein replacement, has established that restoring circulating FVIII levels above 1% of normal prevents most spontaneous bleeding, and levels above 5% are sufficient to improve the disease from a severe to a mild form. However, the limited worldwide supplies of both plasma-derived and recombinant FVIII, its short half-life in vivo (ϳ 12 hours), and the high cost of treatment (Ͼ $150 000 per year) make gene therapy an attractive alternative to better manage and cure the disease.Previously, we have shown that gene therapy with an AAV2 vector encoding a B-domain-deleted (BDD) canine FVIII (cFVIII) cDNA under the control of a liver-specific promoter resulted in an average of 2% to 3% of normal canine FVIII activity in 2 hemophilia A dogs, 2 providing preliminary support for the feasibility of this approach in humans. In order to further improve the efficacy of liver-targeted AAV-cFVIII, we explored the possibility of using alternative serotypes of AAV. We also assessed the duration of therapeutic benefit following a single injection of AAV-cFVIII in hemophilia A dogs.Since the isolation of AAV2, many different AAV serotypes have been isolated from human and nonhuman primate tissues. 3 In comparison with the prototypic AAV2, AAV vectors pseudotyped with other serotypes show superior transduction efficiency in various tissues: AAV1 in muscle, 4 pancreatic islets, 5 heart, 6 vascular endothelium, 7 brain and central nervous system (CNS), 8,9 and liver 10 ; AAV3 in Cochlear inner hair cells 11 ; AAV4 in brain 12 ; AAV5 in brain and CNS, 8,13 lung, 14-16 eye, 17,18 arthritic joints, 19 and liver 20 ; AAV6 in muscle, 21,22 heart, 23 and airway epithelium 24 ; AAV7 in muscle 4 ; and AAV8 in muscle, 4,25 pancreas, 26 heart, 25 and liver. [27][28][29][30][31] The tissue tropism of different AAV serotypes may permit targeting of AAV vectors to human disease. However, as most of these tissue-specific tropisms have been reported in the rodent, it is important to evaluate cross-species fidelity of differential targeting among serotypes in larger animal modelsIn this report, we have compared the efficacy, gene transfer efficiency, and biodistribution of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 delivered by portal-vein injection in hemophilia A mice. Furthermore, since prior studies have demonstrated that the hemophilia dog model, compared with the mouse model, more accurately predicts the therapeutic outcomes in humans and other primates, 32,33 we have determined the long-term efficacy and safety of AAV2-cFVI...