Correction of the SCD mutation by ex vivo HSC transduction. (A) Schematic of the experiment. BM Lin⁻ cells were harvested from CD46/Townes mice and transduced with HDAd-PE5max at an MOI of 500 vp/cell. Cells were then either cultured for 3 days or transplanted into lethally irradiated C57BL/6 mice at 24 hours after transduction. The mice were followed for 16 weeks. For further evaluation of long-term repopulating cells, BM Lin⁻ cells from these mice were then used for secondary transplantation and these mice were monitored for another 16 weeks. (B and C) Analyses of day 3 cultures and colonies. (B) Target base conversions and indel frequency measured using NGS. Lin⁻ cells isolated from 3 independent donors were analyzed (n = 3 animals). (C) Allelic analysis in single Lin^– cell–derived progenitor colonies (n = 30). Editing was measured using Sanger sequencing at day 11 after plating of transduced Lin^– cells derived from a representative mouse. (D-H) Analyses of transplanted primary mice. (D) Engraftment of HDAd-PE5max–transduced HSCs measured using flow cytometry of human CD46 expression in PBMCs. (E) Analysis of target site editing (T>A, sickle site and G>A, silent PAM site) using Sanger sequencing in PBMCs at different time points after transplantation (n = 5 animals). (F) Editing (Sanger) at week 16 in PBMCs, splenocytes, BM MNCs, BM Lin^– cells and pooled progenitor colonies from plated Lin^– cells (Pooled CFU). (G) Target base conversions and indel frequency in BM MNCs (week 16) measured using NGS. (H) Allelic analysis in progenitor colonies derived from week-16 BM Lin^– cells (n = 36). Colonies from 3 individual mice were analyzed using Sanger sequencing (n = 12 for each mouse). All colonies had a 100% editing rate. (I-K). Analyses of secondary recipients similar to panels D-H. For panels C, E, F, H, J, and K, editing was measured using Sanger sequencing. For panels D-G, I-K, each symbol represents an individual mouse.