Human F.IX expression as a function of time and anti-F.IX antibody measurements. (A) Transgenic mice tolerant to human wild-type F.IX received AAV vectors encoding F.IX-WT (▴) or variants (K5A [], K5A/V10K [▪], R338A [×]) at a dose of 1 × 10¹² vg/kg by intramuscular injections. Skeletal muscle-derived F.IX levels determined by ELISA are detectable in plasma samples collected 2 weeks after injection and throughout the duration of the experiment. Immunodeficient mice (^*; CD4 knock-out mice) as a control group presented similar increase in circulating F.IX after intramuscular injection of AAV-F.IX-WT. When the same vector was administered to normal C57BL/6 (nontolerant [•]) mice, no F.IX was detected after 4 weeks of injection. (B) Detection of specific IgG1 antibodies to F.IX-WT (▪) or variants K5A (▦) or R338A (□) at 4 weeks after vector injection were restricted to nontolerant C57Bl6 mice. The numbers of animals tested are indicated. Data shown are mean values ± SD.