Analyses of secondary recipients. Lin⁻ cells from in vivo transduced mice were transplanted into lethally irradiated C57Bl/6 mice (N = 7). The cells from 1 donor were injected into 1 recipient. (A) Engraftment based on human CD46 expression in PBMCs measured by flow cytometry. (B) Editing of target sites in PBMCs at the indicated weeks after transplantation measured by Sanger sequencing. (C) Editing measured by Sanger sequencing at week 16 after transplantation of PBMCs, splenocytes, BM MNCs, pooled CFU cells, and BM Lin⁻ cells. (D) Target base conversions T > A (sickle repair) and G > A (silent PAM) measured by NGS in week 16 BM MNCs. (E) Comparison of correction levels between the primary mice and secondary recipients. Individual primary (donor) and secondary (recipient) mice were matched using a paired Student's t test. ∗P ≤ .05. (F) Indel frequencies measured by NGS in week 16 BM MNCs of primary mice and secondary mice. (G) GFP expression in LT-HSCs was characterized by the following phenotype: CD150⁺/CD48^–/LSK. Left panel: schematic of the experiment. CD46-transgenic mice were either mobilized with G-CCSF + AMD3100 or WU-106/AMD3100 as described in Figure 1. HDAd-GFP was injected IV 45 and 75 minutes after AMD3100 (in 2 doses of each 4 × 10¹⁰ vp per mouse) for G-CSF/AMD3100 mobilized mice, or 2 hours and 2.5 hours after WU-106 AMD310 (also in 2 doses of each 4 × 10¹⁰ vp per mouse). Five days later, the mice were euthanized and BM MNCs were analyzed by flow cytometry for GFP expression in LT-HSCs. Right panel: percentage of GFP⁺ LT-HSC cells. One-way ANOVA with Bonferroni posttest for multiple comparisons was used. ∗P ≤ .05.