hFIX expression and anti-hFIX IgG antibody levels in HB mice. (A) hFIX levels in mice transduced as neonates. Neonatal 129S×C57BL/6 HB mice were injected IV with a high (1 × 10¹⁰ TU/kg), medium (1 × 10⁹ TU/kg), low (1 × 10⁸ TU/kg), or very low (1 × 10⁷ TU/kg) dose of hAAT-hFIX-WPRE at 2 or 3 days after birth. Average plasma hFIX antigen levels ± SEM are shown. (B) Anti-hFIX IgG antibody levels after hFIX protein injection. Adult HB mice that never received gene transfer began to receive weekly IP injections of 30 IU/kg hFIX without adjuvant at 2 to 4 months after birth and continued for 10 injections total, as indicated by the short vertical arrows in this and subsequent panels. Plasma anti-hFIX antibody levels were determined at the indicated time in weeks after the first dose of hFIX. Each line indicates an individual mouse. Values are plotted as 0.001 mg/mL for the 5 mice that failed to make antibodies at any time of evaluation. (C) Anti-hFIX IgG antibody levels after hFIX protein injection with adjuvant. Adult HB mice that never received gene transfer received 2 injections of 30 IU/kg hFIX in adjuvant separated by 3 weeks. Long open arrows indicate the time of injection of protein with adjuvant in this and subsequent panels. Plasma anti-hFIX IgG antibody levels are plotted versus the time after the first dose of hFIX. Each line indicates an individual mouse. (D-H) Anti-hFIX IgG antibody levels in HB mice that were transduced as neonates. Plasma from mice that were treated at birth with a high (D), medium (E), low (F), or very low (G-H) dose of hAAT-hFIX-WPRE and began to receive hFIX protein injections at 4.5 months after birth was tested for anti-hFIX–specific IgG antibodies at the indicated time after birth. These are the same animals whose hFIX levels are shown in panel A. For panels D and E, the line with open circles represents an individual mouse with low and transient levels of an antibody, whereas the line with closed circles represents 5 mice that did not have detectable antibodies at any time of evaluation. For panel F, the line with open circles represents an animal that developed an antibody after administration of 1 dose of hFIX in adjuvant. The line with closed triangles represents 3 mice that did not develop antibodies after 10 injections of hFIX without adjuvant. The line with closed circles represents 3 mice that did not develop antibodies after 10 injections of hFIX without adjuvant and 1 or 2 injections of hFIX with adjuvant. (G) Anti-hFIX IgG antibody levels in HB mice that were transduced with the very low dose of RV as neonates and were challenged with hFIX as indicated. Each line represents an individual animal. Neonatal mice were injected at birth with the very low dose of RV and were challenged at 2 and 2.75 months with hFIX in adjuvant. Each line indicates an individual animal.