Comparison of differently mobilized CD34⁺ cells in terms of susceptibility to transduction, globin expression, and engraftment rates. (A) The mean frequency of vector positive colonies with vector copy number ≥0.45. The column on the left represents the average gene transfer efficiency achieved in all differently mobilized cells (n = 31). *P = .04 (unpaired Student t test). (B) Comparative vector copy number analysis among transduced CD34⁺ cells obtained by different mobilization approaches, depicted as single colony distribution. The mean vector copy achieved in all groups of mobilization is also shown on the left. ****P < .0001 (Kruskal-Wallis test). (C) The normalized vector-encoded β-globin expression per vector copy, overall (left bar) and per mobilization mode (HU+G-CSF: n = 6; G-CSF n = 5; Plerixafor: n = 12; Plerixafor+G-CSF: n = 4) in bulk erythroid cultures. *P = .05 (Kruskal-Wallis test). (D) The normalized vector-encoded β-globin expression per vector copy, overall (left bar) and per mobilization mode (HU+G-CSF: n = 2; G-CSF n = 3; Plerixafor: n = 6; Plerixafor+G-CSF: n = 3) in pools of BFU-E colonies. *P = .03 (1-way analysis of variance [ANOVA]). (E-H) Engraftment rates measured monthly by flow cytometry and expressed as a percentage of (E) human CD45⁺ cells, **P = .001 (1-way ANOVA); (F) human CD3⁺ cells, **P = .007 (1-way ANOVA); (G) human CD19⁺ cells, *P = .02 (1-way ANOVA), (H) human CD33⁺ cells, ***P = .0008 (1-way ANOVA), and (I) human CD235a⁺ cells in the peripheral blood of xenografted mice (HU+G-CSF: gray line; G-CSF: dashed line; Plerixafor: black line with clear squares; Plerixafor+G-CSF: black line with black circles). No differences, among the differently mobilized cells, were encountered in the erythroid lineage where the engraftment, as expected for a xenograft model, was overall poor.¹⁶  The mice surviving at each time point after transplantation with the TNS9.3.55-transduced mobilized CD34⁺ cells from individual patients (displayed in parentheses) are shown in the table provided at the bottom of the figure. Only cells from patient samples that provided sufficient numbers for transplantation were injected. Mortality allowed a small number of animals to be evaluated long-term (supplemental Results). Plrxfr: plerixafor; Pt, patient.