Figure 3.
Reactivation of γ-globin in β-YAC mice after in vivo HSPC transduction and selection. (A) Experimental procedure. β-YAC/CD46 mice (n = 9) were mobilized by G-CSF/AMD3100 and in vivo transduced with HDAd-ABE-sgHBG-2+HDAd-SB. Four rounds of selection by O6BG/BCNU were performed at 4, 6, 8, and 10 weeks after transduction. The mice were killed at week 16. Lin− cells were isolated from bone marrow and IV injected into lethally irradiated C57BL/6J mice. The secondary transplanted mice were observed for another 16 weeks. (B) GFP markers in PBMCs at various time points after transduction. Each symbol represents 1 animal. (C) Flow cytometry showing GFP expression in PBMCs in 1 animal. (D) γ-Globin expression in peripheral RBCs measured by flow cytometry. (E) Flow cytometry charts showing γ-globin expression in total blood cells in a representative animal. (F) γ-Globin expression measured by flow cytometry in erythroid Ter-119+ and nonerythroid Ter-119– cells in blood. (G) Human γ-globin chain levels in RBC lysates measured by HPLC. Data are percentages of γ-globin chain levels over mouse α- or β-globin or human β-globin. (H) γ-Globin expression at mRNA level measured by reverse transcription-PCR. Data are expressed as fold change over mouse HBA or HBB or over human HBB mRNA. (I) Vector copy number (VCN) in total bone marrow cells. Primers specific to human mgmtP140K were used.