cFIX expression and anti-cFIX humoral responses in HB dogs following liver delivery of AAV-cFIX-Padua. Two University of Alabama HB dogs with no previous history of inhibitors were assessed for (A,D) cFIX antigen and activity, (B,E) anti-cFIX IgG2 and Bethesda units, and (C,F) whole blood clotting times after AAV8-cFIX-Padua administration. (A-C) Trex received 3 × 10¹² vg/kg and (D-F) Wick received 1 × 10¹² vg/kg. Black line, closed circles, cFIX activity and cFIX Bethesda units; gray line, open circles, cFIX antigen and anti-cFIX IgG2; horizontal dashed lines, positive levels of Bethesda units and IgG2; vertical dashed lines, time of challenge with recombinant cFIX; dark gray bar, WBCT of hemophiliac dogs (>45 minutes); light gray bar, WBCT of normal dogs (<8 minutes).