In vivo HDAd-HBG-CRISPR/mgmt transduction of mobilized HSPCs in β-YAC/CD46 mice. (A) Structure of HDAd-HBG-CRISPR/mgmt vector. In addition to the HBG CRISPR/Cas9 cassette, the vector contained a PGK promoter-driven mgmt^P140K gene. (B) Schematic diagram of the experiment. β-YAC/CD46 mice were mobilized by subcutaneous injections of G-CSF and AMD3100 and were subsequently injected intravenously with HDAd-HBG/CRISPR/mgmt. To avoid immune responses against the bacterial Cas9 and human mgmt^P140K proteins expressed from the episomal vector, mice received immunosuppressive drugs (IS) for 4 weeks. At weeks 4 and 6, mice were injected intraperitoneally with O⁶BG/BCNU at the indicated doses. Animals were euthanized at week 12 after in vivo transduction, and tissues were analyzed. Bone marrow Lin⁻ cells were transplanted into C57BL/6 mice. T7E1 assay on total blood (C) and bone marrow (D) mononuclear cells at week 12 posttransduction. Specific cleavage products are indicated by arrows. The numbers above the gels are ID tags of individual mice. The numbers below the gels are the percentages of target site cleavage. The percentage of γ-globin⁺ cells in RBCs is also indicated. (E) Percentage of human γ-globin⁺ cells in peripheral blood RBCs measured by flow cytometry in blood samples. (F) Percentage of human γ-globin⁺ cells in total bone marrow mononuclear cells. (G) Percentage of human γ-globin⁺ cells in erythroid (Ter119⁺) and nonerythroid (Ter119⁻) cells in blood and bone marrow. Ctrl, mock-transduced mice.