244. Factor VIII Immunogenicity Is a Barrier to AAV Gene Therapy for Hemophilia A
Authors: Allison M. Lytle, Harrison C. Brown, H. Trent Spencer, Christopher B. Doering
Journal: Molecular Therapy (2015)

Liver-directed adeno-associated virus (AAV) gene therapy utilizing a bioengineered, high-expression coagulation factor VIII (FVIII) transgene (ET3) has been demonstrated by our laboratory to produce therapeutic FVIII levels in a naïve, immune-competent murine model of hemophilia A (HA). Recently, other groups have shown that similar liver directed gene therapy approaches also have the potential to reverse pre-existing immunity, including inhibitors, to coagulation factor IX (FIX) in murine models of hemophilia B (HB). In the current study, we sought to determine if, similarly, ET3-AAV8 could reverse pre-existing FVIII immunity in HA mice. Cohorts of mice were immunized with either recombinant ET3 or human FVIII (hFVIII). Robust anti-FVIII immune responses were elicited as expected and characterized by ELISA (total IgG) and Bethesda (inhibitor) titers ranging from 6000-15,400 arbitrary units (AU) and 22-286 BU, respectively, for ET3 immunized mice, while hFVIII immunized mice showed respective ELISA and Bethesda titers ranging from 460-1480 AU and 23-63 BU. Subsequently, the mice were administered ET3-AAV8 at a dose (4e12 vp/kg) previously shown to produce complete correction of the FVIII deficiency (>50% normal human FVIII activity) and monitored for 16 weeks. No significant change was observed in either ELISA or Bethesda titers for either cohort indicating that the pre-existing immunity was not modulated substantively by FVIII-AAV8 (p>0.5). As Arruda and colleagues previously showed that liver-directed FVIII-AAV8 can eradicate pre-existing anti-FVIII antibodies in a canine model of HA harboring low Bethesda titers (£12 BU), we hypothesized that the capacity for ET3-AAV8 to eliminate pre-existing antibodies may exist under a certain immune threshold quantitatively linked to the inhibitor titer. To test this hypothesis, cohorts of HA mice were established with pre-existing Bethesda titers ranging from 0-25 BU (n=10). Following administration of ET3-AAV8, no significant changes in titers have been observed to date. A corollary question then arose regarding the immune modulation potential of FVIII-AAV8 in naïve HA mice, which has been studied extensively for FIX-AAV in HB mice. Specifically, we tested if a mid (4e12vp/kg) or high (2e13vp/kg) dose of ET3-AAV8 could tolerize naive HA mice to recombinant FVIII infusions. FVIII production was dose dependent with the mid cohort averaging 70% normal human levels (0.7 IU/ml) and the high dose cohort averaging 200% normal levels (2 IU/ml) over 5 months. Mice were then challenged with weekly intravenous injections of ET3. All mice treated with the mid AAV dose developed anti-FVIII ELISA and Bethesda titers similar to challenged controls with a concomitant loss of detectable plasma FVIII activity. However, mice in the high dose cohort maintained FVIII activity indicating a possible antigen production requirement for immune modulation consistent with current models of active tolerance. Collectively, these preclinical data indicate that current clinical FIX-AAV gene therapy data may not predict clinical outcomes for similar FVIII-AAV products, especially as trials progress into previously untreated and inhibitor populations.