Expression of hFIX and anti-hFIX IgG levels in normal dogs after gene transfer or protein injection. (A) hFIX antigen levels after neonatal gene transfer. Newborn normal dogs (N = 5) were injected intravenously with 3.2 × 10⁹ TU/kg hAAT-hFIX-WPRE at 2 days after birth, and plasma was tested for hFIX antigen levels at the indicated time in months after transduction. (B) hFIX antigen levels after gene transfer to young dogs. Two 8-week-old dogs (B85 and B90) were injected intravenously with 5 × 10⁸ TU/kg hAAT-hFIX-WPRE, whereas one 11-week-old dog (M1595) was injected intravenously with 2 × 10⁸ TU/kg. The plasma was tested for hFIX antigen levels at the indicated time in months after transduction. Antigen levels that were undetectable were plotted as 0.5 ng/mL on this semilog scale. (C-E) Anti-hFIX IgG antibody levels in dogs. Anti-hFIX IgG antibody levels were determined by immunoassay. If antibody was undetectable, it was plotted as 0.1 μg/mL on this semilog scale. (C) Plasma was from the dogs that were transduced as newborns and whose antigen levels are shown in panel A. Time of evaluation varied from 6 to 9 months after birth. (D) Plasma was from the dogs that were transduced as juveniles and whose antigen levels are shown in panel B, and the values are plotted at the indicated times after transduction. (E) Plasma was from dogs that began to receive weekly IV injections of 30 IU/kg hFIX at 8 weeks after birth, which was continued for 10 weeks, as indicated by the black arrows. Antibody levels are plotted versus the time after the first dose of hFIX protein.