Human F.IX expression and anti–F.IX anti-body measurements as a function of dose and time in AAV-1–injected immunocompetent hemophilic mice. C57Bl/6 hemophilia B mice were injected at a high (1.6 × 10¹³ vg/kg, n = 5), medium (4 × 10¹² vg/kg, n = 2), or low (6.5 × 10¹¹ vg/kg, n = 3) dose at intramuscular sites. Panels A-D refer to medium- and high-dose cohorts, panel E to low-dose cohort. Each line represents one mouse. (A) Human F.IX levels were determined by ELISA. Levels in high-dose animals range from 300 ng/mL to 2200 ng/mL at 8 weeks, with levels of approximately 100 ng/mL at the same time point in medium-dose animals. After the initial lag, F.IX levels remain stable for the duration of the experiment. (B) Anti–F.IX-specific IgG1 antibodies. These were detected in all animals, peaked at 2 to 4 weeks after injection, markedly decreased by 8 weeks, and disappeared by 12 weeks after injection. (C) Bethesda titers. Inhibitory antibody is first detected at 2 weeks after injection, reaches a maximum at 4 weeks, then gradually diminishes. (D) Western blot to detect anti–F.IX antibody. Lanes 1 to 5 contain serum from the mice treated at a high dose, lanes 6 to 7 conatin serum from mice treated at a lower dose. At the 4-week time point, antibody was detected in 5 of 7 mice. At the 8-week time point it was detected in only 2 of 7 mice. (E) Left panel represents hF.IX levels determined by ELISA. Center and right panels represent antibodies to F.IX detected in all 3 mice by specific IgG to F.IX or by Bethesda assay, respectively. *Denotes the death of one animal after week 8.