Figure 7.

Cytotoxicity of HDAds expressing BEs vs CRISPR/Cas9. Human CD34⁺ HSPCs were transduced with the CRISPR vector HDAd-HBG-CRISPR, the BE vector HDAd-ABE-sgHBG-2, or a control vector HDAd-GFP at an MOI of 3000 vp per cell. Twenty-four hours after transduction, the cells were used for CFU assay, in vitro erythroid differentiation, and in vivo engraftment studies. (A) Colonies with more than 100 cells (including CFU-E, CFU-G, CFU-GM, BFU-E, CFU-M, and CFU-GEMM) were scored 14 days after plating the transduced cells in methylcellulose supplemented with cytokines and growth factors. The ratio of each colony type was similar among different treatments. Experiments were performed in triplicate. Representative data (mean with SD) of 3 different donors are shown. (B) Target site editing was measured by a T7EI assay at 4 days after transduction (4dpt), various time points after differentiation (Diff), and 8 weeks after transplantation. (C) Cells were IV infused into irradiated NOD-scid IL2rγ^null mice at 5 × 10⁵ cells per mouse (n = 3 per treatment). Nontransduced cells or HSPCs transduced with a GFP-expressing vector (HDAd-GFP) were used as controls. Engraftment reflected by percentage of human CD45⁺ cells in PBMCs at the indicated weeks after infusion was measured by flow cytometry. Each dot represents 1 animal. *P < .05. ns, not significant.