cFIX activity and antigen levels and cellular immune responses after peripheral transvenular delivery of AAV6-cFIX-Padua to skeletal muscle. Two adult inhibitor-prone UAB dogs ([A] U05, [B] U04) were treated with 3 × 10¹² vg/kg, and 1 UNC-CH dog ([C] O20) was treated with 9 × 10¹¹ vg/kg of AAV6-cFIX-Padua. cFIX antigen levels were measured by enzyme-linked immunosorbent assay (solid squares); cFIX activity levels were measured by activated partial thromboplastin time (solid circles). *Last dose of immunosuppression. Dashed arrows represent treatments with normal pooled canine plasma for bleeding; straight arrows represent immunogenic challenges with recombinant cFIX-WT. (D) Peripheral blood mononuclear cells collected 28 days after the last protein challenge were analyzed by interferon-γ ELISPOT analysis after stimulation with AAV6 capsid, cFIX-WT protein, or overlapping peptides spanning the 338 region (RATCLR/LSTKFTIYNM, LKVPVDRATCLR/LST). Phorbol myristate acetate plus ionomycin (PMA + iono) stimulation serves as the positive control. Bars indicate mean of 3 technical replicates and error bars are ± standard error of the mean.