Naked plasmid transfer of hFVIII plasmids into hemophilia A mice with or without immunomodulation (costimulation blockade). hFVIII levels and inhibitory antibody formation over time in hemophilia A mice after treatment with pBS-HCRHPI-hFVIIIA beginning on day 1. Transient immunomodulation included Ctla4-Ig on days 1 and 2(n = 8 total; A,B); MR1 on days -1, 1, 2, 7, and 14 (n = 9 total; C,D); and Ctla4-Ig with MR1 (n = 9 total; E,F) using the same combined schedule and dosages as with each individual agent. Fifty μg plasmid in 2 mL saline solution was injected into the tail vein of mice in 5 to 8 seconds. Two separate cohorts of animals (n = 4-5 per group) were used at separate times for each set of immunomodulation experiments; the data from 2 cohorts were combined and presented in the figure. Respective immunosuppressive drugs were administered intraperitoneally at indicated times. #Death of one treated mouse in panel C. Mice were bled at regular intervals. Circulating hFVIII activities and inhibitory antibody titers were evaluated as in Figure 1.