Phenotypic correction of hemophilia after in vivo HSC transduction of hCD46^+/+/F8^−/−mice. (A) Schematic of the experiment. hCD46^+/+/F8^−/− mice were mobilized and in vivo transduced as described in Figure 2. Mice received 4 rounds of O⁶BG/BCNU treatment and were euthanized at week 16 after in vivo transduction. (B) Plasma anti-ET3 antibody titers in representative mice of the 4 experimental settings tested. Black curves, representative antibody titers in hCD46-transgenic mice after in vivo HSC transduction (see Figure 2). Green curve, representative antibody titers in hCD46^+/+/F8^−/− mice after in vivo transduction; red curves, antibody titers in hCD46^+/+/F8^−/− mice after in vivo transduction, whereby mice received recombinant ET3 protein injections to control bleeding during blood sampling; blue curves, representative antibody titers in hCD46^+/+/F8^−/− mice after ex vivo transduction. (C) ET3 mRNA in peripheral blood RBCs measured by qRT-PCR relative to mouse mRPL10 mRNA levels. (D) ET3 mRNA levels in bone marrow mononuclear cells at week 16 measured by qRT-PCR relative to mouse mRPL10 mRNA levels. (E) Immunoprecipitation/western blot with lysates from total blood cells (ie, mostly erythrocytes). The specific ET3 band appears ∼168 kDa. (F) 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. (G) ET3 mRNA in bone marrow erythroid Ter119⁺ and nonerythroid Ter119⁻ cells at week 16 relative to mouse mRPL10 mRNA levels. (H) VCN per cell in bone marrow MNCs at week 16 after in vivo HSC transduction. In vivo HSC transduced mice were kept alive without side effects for 16 weeks.