Persistent expression of canine factor IX in hemophilia B canines

We previously demonstrated that direct intramuscular injection of recombina nt adeno-associated virus (rAAV) carrying the human FIX(hFIX) cDNA can safe ly be administered to hemophilic B canines and express human factor IX prot ein; however the functional activity of the hFIX protein could not be asses sed due to anti-human FIX antibody (inhibitor) formation. To test the thera peutic efficacy of rAAV in hemophilic dogs, rAAV type 2 (rAAV2) carrying ca nine FIX (cFIX) cDNA was injected into the skeletal muscle of two dogs at d oses of 10(12-13) particles. Circulating cFIX protein levels were maintaine d for 1 year at levels of 1-2% of normal. Hemostatic correction (WBCT and A PTT) paralleled plasma FIX antigen levels. Both dogs still required plasma infusion for spontaneous and traumatic bleeding events. inhibitors to cFIX protein were not detected in either animal by Bethesda assay. Neutralizing I antibodies directed against AAV-2 capsid were pronounced and persistent V ector DNA and mRNA transcripts were defected only at the injected skeletal muscle tissue. Analysis of both high and low molecular weight DNA identifie d both replicative episomal and integrated AAV species. These results demon strate that persistent secretion of the FIX transgene protein, necessary fo r successful gene therapy of hemophilia B, can be achieved using the parvov irus-based rAAV vector.