Analysis of secondary recipients (in vivo HSPC transduction approach). Bone marrow Lin⁻ cells harvested from in vivo–transduced β˗YAC/CD46 mice at week 12 after transduction were transplanted into lethally irradiated C57BL/6 mice. Secondary recipients were followed for 16 weeks. (A) Engraftment measured in blood samples at the indicated time points based on the percentage of human CD46⁺ cells in PBMCs. (B) Engraftment at week 16 based on the percentage of human CD46⁺ cells in mononuclear cells of blood, spleen, and bone marrow (BM). (C) Human γ-globin expression in secondary recipients. Shown is human γ-globin expression on RBCs from week 4 to week 16 after transplantation. (D) T7E1 assay on total blood and bone marrow mononuclear cells at week 16 posttransplantation. 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. For blood cells, the percentage of γ-globin⁺ cells in RBCs is also indicated. (E) Percentage of human γ-globin⁺ cells in erythroid (Ter119⁺) and nonerythroid (Ter119⁻) cells in blood and bone marrow (week 16 posttransplantation). (F) Percentage of HBG mRNA of human HBB mRNA. (G) HPLC data. Percentage of human Gγ- and Aγ-globin protein relative to human β-globin protein in RBCs from untransduced and HDAd-HBG-CRISPR mice (week 16 posttransplantation). (H) Cellular composition in blood (CD3⁺, CD19⁺, Gr-1⁺), spleen (CD3⁺, CD19⁺, Gr-1⁺), and bone marrow (CD3⁺, CD19⁺, Gr-1⁺, Ter119⁺, LSK) at week 16 after transplantation.