Six-Year Follow-Up of Inhibitor Prone Hemophilia B Dogs Treated with Muscle and Liver-Directed AAV2 Mediated Factor IX Gene Therapy.

Abstract

Hemophilia B, caused by factor IX (FIX) deficiency, is intensively studied as a model for gene therapy. Preclinical studies in mice, dogs and initial clinical trials have documented the feasibility of adeno-associated virus (AAV) mediated gene therapy for hemophilia B. The purpose of this study is to report 6 year follow-up of inhibitor prone hemophilia B dogs treated with muscle or liver directed AAV2 FIX gene therapy. Short term, one year follow-up of these dogs (n=6) has been previously reported. The dogs treated with liver directed AAV2 FIX have not had a single bleed requiring FIX replacement in the 6 years since vector administration. The dogs undergoing muscle directed gene therapy have continued to have a bleed frequency similar to untreated FIX deficient dogs. The whole blood clotting time (WBCT), activated clotting time (ACT), and activated partial thromboplastin time (APTT) have remained at the upper limits of the normal ranges for the six year follow-up in the two liver treated dogs. The FIX activity has remained stable between 4–10% in both liver treated dogs but undetectable in the dogs undergoing muscle directed therapy. The vector/FIX sequences have persisted in liver biopsies but were undetectable in WBC and sperm DNA. Administration of purified canine FIX protein resulted in an immune response to FIX in the muscle treated dogs but not the dogs treated with liver directed gene therapy, suggesting FIX tolerance had been established in the hemophilia B dogs with liver directed gene therapy. A complete clinical evaluation of the dogs undergoing liver directed gene therapy including CBC, serum chemistries, bile acid profile, hepatic MRI and CT scans and liver biopsy was normal with no evidence for tumor formation. These results demonstrate that AAV mediated liver directed gene therapy completely corrects the hemophilia phenotype without toxicity or inhibitor development in the inhibitor prone null mutation dogs for more than six years.