250 Enhanced Amplitude and Functionality of T Cell Responses in Virally Infected Human Like Cmah Null Mice Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene[.]
IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY I – AAV Immunologic & Host Responses in Gene & Cell Therapy I – AAV 248 Tolerance Induction and Long-Term Correction in a Mouse Model of Hemophilia A with an AAV8 Serotype Carrying Codon-Optimized Human FVIII Brandon K Sack,1 Sherin M Merchant,1 Andrew M Davidoff,2 Roland W Herzog.1 Pediatrics, University of Florida, Gainesville, FL; 2Surgery, St Jude Children’s Research Hospital, Memphis, TN Treatment for hemophilia A relies on intravenous delivery of clotting factor VIII (FVIII) concentrate, which can be costly, invasive, and carries the risk of developing neutralizing antibodies to FVIII Liver-directed gene therapy offers the opportunity to both correct the disease long-term and offer tolerance to the FVIII transgene with one treatment However, studies in mouse models of hemophilia A have been limited by poor expression and low activity of human FVIII We produced AAV8 vectors encoding wild-type (WT-FVIII) or codon-optimized (coFVIII) cDNA encoding B domain-deleted human factor VIII under control of a liver-specific promoter These vectors were delivered intravenously at a dose of 1011vg/mouse to hemophilia A mice of either a mixed BL6-129/sv or a BALB/c background Mice receiving WT-FVIII gene therapy showed minimal improvement in clotting times as measured by aPTT (mean of 77 ±3 sec and 70±2 sec for BL/6-129/sv-HA and BALB/c-HA mice, respectively), correlating to ≤1% of normal levels AAV8-coFVIII resulted in much greater improvement in clotting times, with a peak average at weeks of 37±2 sec and 43±2 sec for BL/6-129/sv-HA and BALB/c-HA mice, respectively, which stabilized to 53±4 sec and 62±6 sec at 18 weeks post vector injection These clotting times correlated to a peak of 8-10% hFVIII activity, stabilizing to 2-4% Correction at this level would result in a significant improvement in quality of life for patients with severe hemophilia A but would necessitate occasional supplementation with FVIII protein To mimic this scenario, we gave weekly infusions of 1U FVIII Naïve control BL/6-129/sv-HA mice developed anti-FVIII IgG with a mean of 6±1.5 ug/mL, which correlated to a Bethesda titer of 154±44 BU Mice of this strain with prior AAV8-FVIII gene transfer developed a higher mean anti-hFVIII IgG titer of 7±0.9 ug/mL (336±88 BU) after protein challenge Consequently, clotting times in these mice increased to uncorrected levels In contrast, of BL/6-129/sv-HA mice that received AAV8-coFVIII were non-responsive to challenge and thus maintained correction A fourth mouse was hyporesponsive (1.4 BU) Naïve control BALB/c-HA mice developed 3±0.7 ug/mL anti-hFVIII IgG, corresponding to17±4 BU Interestingly, only 1/6 BALB/c-HA given AAV8-FVIII mice had detectable IgG response to hFVIII protein (with an inhibitor titer of 5.3 BU) However, clotting times of all mice rose after challenge, suggesting a low-grade immune response On the other hand, BALB/c-HA mice receiving AAV8coFVIII maintained correction after challenge, with 2/4 lacking anti-FVIII and 2/4 mice showing a low-titer antibody response of