Long-term hFVIII expression in SB-treated hemophilia A mice that were tolerized as neonates. (A) Neonatal hemophilia A mice were tolerized within 24 hours of birth by a facial vein injection of 0.1 U g^–1 recombinant hFVIII. Pups were aged for 8 to 12 weeks and given 2 high-pressure tail-vein injections of 200 μg pSBT/F8 with 80 μg UB-SB10 (▦) or a luciferase plasmid (▴) (initial n = 4/group; 2 mice formed inhibitors and were dropped, leaving n = 3/group). Arrow shows time of each injection. Plasma was subsequently assayed for hFVIII activity by COATEST. Without SB transposase, hFVIII rapidly declined, whereas animals co-administered an SB-encoding plasmid exhibited significantly higher hFVIII activity (*P ≤ .05 by Student t test; error ± SD). (B) Same as in panel A, except all mice (n = 3) received transposase (80 μg pUB-SB10) and were assayed for 180 days for hFVIII activity by COATEST. (C) Bethesda assays on mice from Figure 4A-B. Anti-hFVIII inhibitory antibodies were detected in 2 of 11 mice injected with 0.1 U g^–1 recombinant hFVIII in the facial vein within 24 hours of birth. Inhibitors were assayed for 3 weeks after the first plasmid injection and in both cases (2 of 11) coincided with undetectable hFVIII activity by COATEST (not shown). Thus, 2 of 11 mice were presumably not tolerized to hFVIII (one with and one without pUB-SB10 co-injection, both from Figure 4A). The majority of plasmid-treated mice (9 of 11) were tolerized, as they did not form detectable inhibitors to hFVIII and had sustained hFVIII activity assayed by COATEST (panels A and B).