Figure 2.
In vitro studies with CD34+ cells from patients with SCD infected with HDAd-PE5max. (A) SCD CD34+ cells (from 3 donors) were infected with HDAd-PE5max or the control virus HDAd-mgmt/GFP at an MOI of 4000 vp/cell or left untransduced (UNTD). On day 3, cells were either plated in Methocult for progenitor colony assays or subjected to ED with or without O6BG/BCNU in vitro selection. (B) Editing rates measured using NGS at various time points after transduction and in vitro differentiation with or without selection. (C) DNA chromatograms from Sanger sequencing showing target site T>A conversion in cells of donor #3 after ED. (D) Percentage of reads with indels. (E) Number of progenitor colonies formed from 2000 plated CD34+ cells (counted at day 14 of culture). BFUE, blast-forming units-erythroid; CFU-GM, granulocyte-macrophage colony–forming unit. (F) Editing measured using Sanger sequencing in donor #3 progenitor colonies after HDAd-PE5max transduction and selection (n = 111). (G) Fold expansion of total cell at day 18 of ED vs day 7 of ED. N = 3 donors. (H and I) Flow cytometry for ROS at day 18 of ED of untransduced cells and cells transduced with HDAd-PE5max without and with O6BG/BCNU in vitro selection (sel). (H) Percentage of ROS-positive cells. (I) ROS mean fluorescence intensity. (J) Percentage of erythroid progenitors counted on cytospins from differentiated SCD CD34+ cells at days 14 and 18 of ED, untransduced (UNTD) and HDAd-PE5max–transduced without and with selection. Two scientists counted 5 random fields. (K) Smears of total blood cells subjected to a sodium metabisulfite sickling assay. Pictures shown here are from day 18 of ED. The scale bar is 25 μm. ∗P < .05. Statistical significance was assessed using one-way ANOVA with Šidák’s multiple comparisons test to calculate P values. Baso, basophilic erythroblasts; dpt, days post transduction; Diff, in vitro ED; mat. Ortho, maturing orthochromatic erythroblasts; Ortho, orthochromatic erythroblasts; Poly, polychromatic erythroblasts; Pro, proerythroblasts; Pyrcs, pyrenocytes; Retics, reticulocytes.

In vitro studies with CD34+ cells from patients with SCD infected with HDAd-PE5max. (A) SCD CD34+ cells (from 3 donors) were infected with HDAd-PE5max or the control virus HDAd-mgmt/GFP at an MOI of 4000 vp/cell or left untransduced (UNTD). On day 3, cells were either plated in Methocult for progenitor colony assays or subjected to ED with or without O6BG/BCNU in vitro selection. (B) Editing rates measured using NGS at various time points after transduction and in vitro differentiation with or without selection. (C) DNA chromatograms from Sanger sequencing showing target site T>A conversion in cells of donor #3 after ED. (D) Percentage of reads with indels. (E) Number of progenitor colonies formed from 2000 plated CD34+ cells (counted at day 14 of culture). BFUE, blast-forming units-erythroid; CFU-GM, granulocyte-macrophage colony–forming unit. (F) Editing measured using Sanger sequencing in donor #3 progenitor colonies after HDAd-PE5max transduction and selection (n = 111). (G) Fold expansion of total cell at day 18 of ED vs day 7 of ED. N = 3 donors. (H and I) Flow cytometry for ROS at day 18 of ED of untransduced cells and cells transduced with HDAd-PE5max without and with O6BG/BCNU in vitro selection (sel). (H) Percentage of ROS-positive cells. (I) ROS mean fluorescence intensity. (J) Percentage of erythroid progenitors counted on cytospins from differentiated SCD CD34+ cells at days 14 and 18 of ED, untransduced (UNTD) and HDAd-PE5max–transduced without and with selection. Two scientists counted 5 random fields. (K) Smears of total blood cells subjected to a sodium metabisulfite sickling assay. Pictures shown here are from day 18 of ED. The scale bar is 25 μm. ∗P < .05. Statistical significance was assessed using one-way ANOVA with Šidák’s multiple comparisons test to calculate P values. Baso, basophilic erythroblasts; dpt, days post transduction; Diff, in vitro ED; mat. Ortho, maturing orthochromatic erythroblasts; Ortho, orthochromatic erythroblasts; Poly, polychromatic erythroblasts; Pro, proerythroblasts; Pyrcs, pyrenocytes; Retics, reticulocytes.

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