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 i n 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 di rect muscle injection. For the current study, rAAV with the human fact or IX (hF.IX) cDNA under the control of the cytomegalovirus (CMV) imme diate-early promoter was constructed and rAAV/hF.IX proved capable of transducing hemophilic dog primary fibroblast cultures in a dose-depen dent fashion. Direct intramuscular injection of 2.5 x 10(12) rAAV/hF.I X virus particles into the hindlimbs of a hemophilia B dog was tolerat ed without bleeding or systemic reaction, and the animal was asymptoma tic throughout the entire study. Transient reduction in the whole bloo d clotting time (WBCT) occurred during the first week, with the antici pated development of an antihuman F.IX inhibitor antibody which corres ponded with the loss of coagulation correction. At 10 weeks after vect or administration, immunohistochemical analysis of injected muscle con firmed continued hF.IX expression. Limited areas oi focal lymphocytic infiltration and myofiber pathology were detected which directly corre lated with positive antibody staining for helper adenovirus contaminat ion. 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 c ontinued development of this approach for gene therapy of human diseas es, including hemophilia B.