1091. Successful Induction of Immune Tolerance to Canine Factor VIII after Lentiviral-Mediated Gene Therapy in Neonatal Hemophilia A Mice

Top of pageAbstract The development of neutralizing antibodies (inhibitors) directed against Factor VIII (FVIII) is currently the most significant complication associated with FVIII replacement therapy in hemophilia A patients and presents a considerable obstacle to successful gene therapy strategies. Induction of immunological tolerance (ITI) to FVIII involves frequent intravenous infusions of FVIII according to various therapeutic regimens, and has been successful in many inhibitor-positive patients. However, ITI is not successful in all patients and it requires frequent intravenous access and is very expensive. Alternative methods of inducing tolerance to the FVIII protein may prove beneficial especially if this can be achieved prior to inhibitor development. With a view to utilizing the potential of neonatal immune-privilege, we administered canine-FVIII expressing lentiviral vectors prior to FVIII protein challenge. The lentiviral vector, hAAT-cFVIII-LV, contained a B domain-deleted canine FVIII transgene under the control of a liver specific promoter. 1x108 Transducing Units (TU)/mouse were administrated to 0-1 day old Balb/c hemophilia A neonates either intravenously (IV), intraperitoneally (IP) or subcutaneously (SC). Neutralizing anti- canine FVIII antibodies developed in all mice treated with IP or SC injection, In contrast, FVIII activity (range <10-1586.7mU/mL) was detected for greater than 3 months in 8/13 mice treated intravenously and all mice failed to develop high-titer neutralizing anti-FVIII antibodies. To evaluate long-term tolerance induction following this protocol, 3 of these mice were challenged, 52 weeks post-hAAT- cFVIII-LV treatment, with four intravenous infusions of 0.2ug recombinant canine FVIII (rcFVIII) protein (80U/kg). Splenocytes were isolated, cultured and in vitro cytokine production and T cell proliferation, was assessed. Treated mice injected with rcFVIII remained inhibitor negative and this tolerance was accompanied by increased in vitro T cell production of IL-10 in parallel with decreased production of IL-6 and IFN-|[gamma]| in vitro. The splenocytes from these tolerant mice are capable of transferring tolerance to na|[iuml]|ve recipient hemophilia A Balb/c mice indicating that the mechanism underlying this phenomenon likely involves regulatory T lymphocytes.