Phenotypic correction was achieved in transiently immunosuppressed HemA mice after IO infusion of G-F8/N6K12RH-LV. (A) F8/N6K12RH was cloned into a lentiviral transgene backbone plasmid controlled under a ubiquitous promoter EF1α (pEF1α-F8/N6K12RH). HemA mice were hydrodynamically injected with pEF1α-F8/N6K12RH (n = 3; 50 μg per animal) or pEF1α-F8/N6 (n = 5; 50 μg per animal) or sterile phosphate-buffered saline (PBS; n = 3; mock; 2 mL per animal). Plasma samples were collected on day 4 postinjection, and hFVIII activity was measured by activated partial thromboplastin time (aPTT) assay. (B) HemA mice were pretreated with drugs (Dex 4 times + anti-CD8α mAb 5 times) after IO infusion of G-F8/N6K12RH-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 mice treated with G-F8/N6K12RH-LV plus drugs, G-F8/N6K12RH-LV only, or G-F8/N6-LV only and mock mice were measured by enzyme-linked immunosorbent assay on day 84. (C) Plasma samples were collected from the treated or mock mice on day 84. hFVIII activity and anti-FVIII antibodies were measured by aPTT and Bethesda assays, respectively. (D) HemA phenotypic correction in mice treated with G-F8/N6-LV only or G-F8/N6K12RH-LV plus drugs was also evaluated by measuring carotid artery blood flow rate on day 84. Each symbol represents an individual animal. Data are expressed as mean ± standard deviation of the mean. **P < .01. ns, not significant.