688. Supplemental Immune Suppression Is Required for AAV-F8 ITI in Hemophilia A Mice with Pre-Existing Inhibitors
Authors: David Markusic, Brett Palaschak, Irene Zolotukhin, Roland Herzog
Journal: Molecular Therapy (2016)

Hemophilia is an inherited coagulation disorder resulting in the loss of functional clotting factors VIII (hemophilia A) or IX (hemophilia B). Patients with the severe hemophilia (less than 1% residual clotting factor activity) develop spontaneous bleeds into the joints and closed spaces and have a high incidence of morbidity if not treated. Current treatment involves frequent infusion of short-lived exogenous plasma derived or recombinant clotting factors. A major complication with protein therapy is the development of inhibitory antibodies in 25-30% hemophilia A and 3-5% hemophilia B patients that prevent normalization of hemostasis with continued protein therapy. Inhibitor patients are treated with either bypassing agents or immune tolerance induction (ITI) therapy requiring daily infusions (months to years) of supraphysiological levels of clotting factor protein. ITI therapy is effective in up to 70% of hemophilia A patients, while it often fails in hemophilia B patients due to the development of pathogenic antibodies that induce anaphylaxis and nephrotic syndrome. Both treatment options for inhibitor patients have high associated financial and health related costs for the patient. We have previously shown that hemophilia B mice with pathogenic inhibitory antibodies are effectively treated with a novel immune tolerance induction protocol using a liver directed adeno-associated virus expressing the gene encoding for human factor IX protein (AAV8-F9). AAV8-F9 ITI rapidly eliminates pre-existing inhibitors and provided sustained therapeutic levels of FIX protein even following repeated exposure to recombinant FIX protein. Based on our success with hemophilia B, we sought to develop a complementary protocol for hemophilia A mice with pre-existing inhibitors. Our initial studies were conducted using a codon-optimized F8 (coF8) gene, which has been shown to enhance the levels of transcribed FVIII protein. In the first set of experiments we tested two different vector doses (1×1011 and 1×1012 vg) of an AAV8-cohF8 vector for factor VIII expression levels and tolerance induction in hemophilia A mice on two different genetic backgrounds (BALB/c-F8−/Y and 129/BL6-F8−/Y). Since BALB/c-F8−/Y mice develop lower titer FVIII inhibitors following intravenous hFVIII protein challenge compared to 129/BL6-F8−/Y mice we expected better outcomes with AAV8-cohF8 vector treated BALB/c-F8−/Y mice. Surprisingly BALB/c-F8−/Y mice spontaneously developed FVIII inhibitors approximately 4 weeks following vector delivery. In contrast,129/BL6-F8−/Y mice expressed vector dose dependent therapeutic levels FVIII protein without inhibitors and 75% of the mice remained inhibitor free following four weekly IV FVIII protein challenges. Subsequent studies for AAV8-cohF8 ITI were conducted in 129/BL6-F8−/Y mice. We generated a group of inhibitor mice and treated a subset of these mice with 1×1012 vg AAV8-coF8. AAV8-cohF8 ITI alone was not sufficient to eliminate inhibitors. Therefore we designed two additional studies to determine if 1×1012 vg AAV8-coF8 ITI would be more effective if combined with transient immune suppression using oral rapamycin (4mg/kg) or anti-CD20 (10mg/kg) mediated B cell depletion. While rapamycin adjunct therapy failed to lower inhibitors, preliminary data show that anti-CD20 combined with AAV8-coF8 ITI leads to a sharp reduction in high titer inhibitors. Given that FVIII protein invokes a stronger immune response in mice and patients, our data suggests that successful gene therapy for hemophilia A may require supraphysiological levels of FVIII protein to prevent inhibitor formation and transient immune suppression to be effective as an ITI therapy.