Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia.

A recombinant adeno-associated virus (rAAV) vector carrying the human factor IX cDNA was tested for safety and therapeutic gene expression in a canine model of human hemophilia B. Intramuscular delivery of rAAV was chosen based on our previous work which described long-term (> 1.5 years) reporter gene expression in immunocompetent mice following direct muscle injection. For the current study, rAAV with the human factor IX (hF.IX) cDNA under the control of the cytomegalovirus (CMV) immediate-early promoter was constructed, and rAAV/hF.IX proved capable of transducing hemophilic dog primary fibroblast cultures in a dose-dependent fashion. Direct intramuscular injection of 2.5 x 10(12) rAAV/hF.IX virus particles into the hindlimbs of a hemophilia B dog was tolerated without bleeding or systemic reaction, and the animal was asymptomatic throughout the entire study. Transient reduction in the whole blood clotting time (WBCT) occurred during the first week, with the anticipated development of an antihuman F.IX inhibitor antibody which corresponded with the loss of coagulation correction. At 10 weeks after vector administration, immunohistochemical analysis of injected muscle confirmed continued hF.IX expression. Limited areas of focal lymphocytic infiltration and myofiber pathology were detected which directly correlated with positive antibody staining for helper adenovirus contamination. PCR tissue analysis revealed rAAV/hF.IX DNA solely in injected muscle tissue and adjacent lymph node, without dissemination to other organs (including gonads). This first large animal study suggests that intramuscular gene delivery using rAAV vectors is safe and supports continued development of this approach for gene therapy of human diseases, including hemophilia B.