Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

Abstract

Gene therapy for hemophilia using adeno-associated virus (AAV) vectors allows long-term coagulation factor expression. We examined the potential of a novel engineered liver-tropic AAV3B-based vector AAV.GT5 for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, while in vivo transduction efficacy into the liver and the increase in coagulation factor IX (FIX) antigen following intravenous injection of these vectors were similar in PXB mice (chimeric mice with a humanized liver) and macaques. The discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the original AAV3B. The intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced vector transduction into the liver and reduced vector distribution to the kidney in pigs. In macaques, the intra-hepatic artery injection of AAV.GT5 yielded a comparable increase in FIX antigen with a one-third dosage of peripheral venous administration. Two of four macaques who received AAV.GT5 intravenously did not develop neutralizing antibodies (NAbs) against AAV.GT5, while AAV-Spark100 induced serotype-specific NAbs in all four macaques. The NAb produced after the administration was relatively specific to the serotype and less responsive to the other serotype. As a result, the administration of AAV.GT5 successfully boosted FIX expression in one animal previously given AAV-Spark100. Thus, AAV.GT5 has different biodistribution and immunogenic characteristics compared with AAV-Spark100, and the intra-hepatic vascular administration may lessen the vector dose and avoid vector distribution to other organs.

Successful liver transduction by re‐administration of different adeno‐associated virus vector serotypes in mice

“…Finally, we examined changes in NAb titer and specificity after AAV vector administration in non‐human primates. We utilized serum from previous experiments comparing two engineered AAV vectors, AAV3B‐based AAV.GT5 and AAV8‐related AAV‐Spark100, in cynomolgus monkeys 23 . We analyzed the prevalence of NAbs against AAV3B, AAV5, and AAV8 in serum samples of these animals.…”

“…We utilized serum from previous experiments comparing two engineered AAV vectors, AAV3B-based AAV.GT5 and AAV8-related AAV-Spark100, in cynomolgus monkeys. 23 We analyzed the prevalence of NAbs against AAV3B, AAV5, and AAV8 in serum samples of these animals. As shown in Table 3, despite the administration of prednisolone for immune suppression, more than half of the animals developed NAbs for the closely related AAV.…”