IO infusion of G-F8X10K12-LV induced formation of inhibitory antibodies in HemA mice. (A) FVIII expression from new FVIII variant F8X10K12 in mice and cell culture. Both BDDF8X10K12 and F8/N6 were cloned into a lentiviral transgene backbone plasmid controlled under a ubiquitous promoter EF1α (pEF1α-F8X10K12 and pEF1α-F8/N6, respectively). HemA mice were hydrodynamically injected with pEF1α-F8X10K12 (n = 3) or pEF1α-F8/N6 (n = 9; 50 μg per animal) or sterile phosphate-buffered saline (PBS; mock; 2 mL per animal). Plasma samples were collected on day 4 postinjection, and hFVIII activity was measured by activated partial thromboplastin time assay (left). E-F8X10K12-LV and E-F8/N6-LV were generated to transduce 293T cells (multiplicity of infection, 100) on day 0. On day 5, hFVIII expression levels in 293T cells were detected by flow cytometry (right). (B) HemA mice were treated with IO infusion of G-F8X10K12-LV (2.2 × 10⁶ ifu per animal) or G-F8/N6-LV (2.2 × 10⁶ ifu per animal) or sterile PBS (mock; 20 μL per animal) on day 0. hFVIII levels in platelet lysates in G-F8X10K12-LV– or G-F8/N6-LV–treated or mock mice were measured by enzyme-linked immunosorbent assay on day 90. (C) Anti-FVIII antibodies in the plasma samples collected from the treated and mock mice were measured by Bethesda assay on day 120. Each symbol represents an individual animal. Data are expressed as mean ± standard deviation of the mean.