Improved Induction of Immune Tolerance to Factor IX by Hepatic AAV-8 Gene Transfer

Abstract

Gene therapy for hemophilia B has been shown to result in long-term expression and immune tolerance to factor IX (F.IX) after in vivo transduction of hepatocytes with adeno-associated viral (AAV-2) vectors in experimental animals. An optimized protocol was effective in several strains of mice with a factor 9 gene deletion (F9^−/−). However, immune responses against F.IX were repeatedly observed in C3H/HeJ F9^−/− mice. We sought to establish a gene transfer protocol that results in sustained expression without a requirement for additional manipulation of the immune system. Compared with AAV-2, AAV-8 was more efficient in transgene expression and induction of tolerance to F.IX in three different strains of wild-type mice. At equal vector doses, AAV-8 induced transgene product-specific regulatory CD4⁺CD25⁺FoxP3⁺ T cells at significantly higher frequency. Moreover, sustained correction of hemophilia B in C3H/HeJ F9^−/− mice without antibody formation was documented in all animals treated with ≥4 × 10¹¹ vector genomes (VG)/kg and in 80% of mice treated with 8 × 10¹⁰ VG/kg. Therefore, it is possible to develop a gene transfer protocol that reliably induces tolerance to F.IX largely independent of genetic factors. A comparison with other studies suggests that additional parameters besides plateau levels of F.IX expression contributed to the improved success rate of tolerance induction.