Abstract: Hemophilia is a particularly attractive model for developing a gene transfer approach for the treatment of disease. The protein is very well characterized, the genes are cloned and available, and there are large and small animal models of the disease. Moreover, in contrast to many diseases, there is no requirement for a specific target tissue for gene delivery, and the gene product itself does not require precise regulation of expression. Earlier efforts to establish a gene transfer approach to the treatment of hemophilia had failed to achieve the twin goals of long‐term expression at levels that were adequate to result in phenotypic improvement of the disease. We have exploited advances in vector development that occurred in the mid‐1990s to establish an experimental basis for an AAV (adeno‐associated viral vector)‐mediated gene transfer approach to the treatment of hemophilia B. Based on the observation that introduction of an AAV vector into skeletal muscle could result in sustained expression of β‐galactosidase, we engineered an AAV vector expressing human factor IX and demonstrated in immunodeficient mice that intramuscular injection of the vector resulted in long‐term expression of the secreted transgene product factor IX. Subsequently, we generated an AAV vector expressing canine factor IX; intramuscular injection into dogs with severe hemophilia B resulted in a dose‐dependent increase in circulating levels of factor IX. The animal treated at the highest dose showed prolonged expression (>3 years and still under observation) at a level (70 ng/ml, 1.4% of normal circulating levels of factor IX) likely to result in phenotypic improvement in humans. Detailed studies in tissue culture using human myotubes have shown that muscle cells are capable of executing the posttranslational modifications required for activity of factor IX, and that the specific activity of myotube‐synthesized factor IX is similar to that of hepatocyte‐synthesized material, although some details of posttranslational processing differ. Based on these and other safety and efficacy studies, a clinical trial of AAV‐mediated, muscle‐directed gene transfer for hemophilia B has been initiated. The study has a dose‐escalation design, with three subjects to be enrolled in three dose cohorts beginning with a dose of 2 × 10¹¹ vg/kg. Results in the initial dose cohort showed no evidence of toxicity associated with vector administration or transgene expression. Analysis of muscle biopsies done on injected tissue showed clear evidence of gene transfer by PCR and Southern blot and of gene expression by immunocytochemistry. The general characteristics of muscle transduction appear similar in humans and in other animal models. The goal of dose escalation is to find a dose that is nontoxic but that results in circulating levels of factor IX >1% in all patients.