Phenotypic correction of hemophilia after ex vivo HSC transduction of hCD46^+/−/F8^−/−Lin⁻cells and transplantation into hemophilia A F8^−/−mice. (A) Schematic of the experiment: bone marrow was harvested from hCD46^+/−/F8^−/− mice, and lineage-negative cells (Lin⁻) were isolated by MACS. Lin⁻ cells were transduced with HDAd-LCR-ET3/mgmt and HDAd-SB alone at a total multiplicity of infection of 500 vp per cell. After 1 day in culture, 1 × 10⁶ transduced cells per mouse were transplanted into lethally irradiated F8^−/− mice. At week 4, O⁶BG/BCNU treatment was started and repeated 4 times every 2 weeks. With each cycle, the BCNU concentration was increased from 5 mg/kg, to 7.5 mg/kg, to 10 mg/kg (twice). At week 18, mice were euthanized. Before and after in vivo selection, mice received immunosuppressive drugs. BM, bone marrow. (B) ET3 activity measured in plasma by Coamatic FVIII assay. Activities of 100% and 5% are indicated by dotted lines. (C) ET3 mRNA levels in peripheral blood RBCs measured by qRT-PCR relative to mouse mRPL10 mRNA levels. (D) Bleeding after tail clipping. Blood was collected in saline over 45 minutes, and the hemoglobin amount was determined and expressed as milligrams normalized to body weight. (E) Concentrations of TAT complexes in plasma. Plasma from hCD46-transgenic mice served as control for physiological levels. Mice transplanted with ex vivo transduced HSCs were kept alive without side effects for 18 weeks. *P < .05. n.s., not significant.