Anti-hFIX IgG antibodies in normal mice after protein injection or neonatal gene transfer. (A) Anti-hFIX antibody levels after protein injections. Mice of the indicated strain that did not receive gene transfer began to receive weekly IP injections of 30 IU/kg hFIX at 2 to 4 months after birth, for a total of 10 doses. The relative levels of anti-hFIX IgG antibody in milligrams per milliliter were determined by immunoassay and are plotted versus the time in weeks after the first dose of protein. Each line represents a single animal. For the BALB/c and C57BL/6 mice, 2 and 3 mice, respectively, failed to make antibodies (plotted as 0.001 mg/mL on this semilog scale) at any time of evaluation, as indicated by the N near the line at the bottom. (B) Anti-hFIX antibody levels in mice after neonatal gene transfer. These are the same C3H and BALB/c:129S mice that received neonatal injection of 1 × 10¹⁰ TU/kg of hAAT-hFIX-WPRE as described in Figure 1B. At 4.5 months after transduction, mice began to receive weekly injections of hFIX without adjuvant for a total of 10 doses, as indicated by the short black arrows. At 7 and 7.75 months after transduction, mice received hFIX in adjuvant, as indicated by the long open arrows. Anti-hFIX IgG antibody levels are shown at the indicated time in months after transduction. None of the C3H (N = 5) or BALB/c:129S (N = 3) mice made detectable antibodies at any time of evaluation.