Figure 3.

Combination of Transduction Enhancers Increases Lentiviral CD34⁺ HSPC Transduction over Their Single Use

(A) Experimental timeline. CD34⁺ HSPCs from 3 HDs were pre-stimulated for 24 h and then transduced with the LV vector depicted in Figure 2A at MOIs of 20 and 10 in the presence or absence of selected transduction enhancer (TE) combinations in two sets of experiments. Cells were cultured in SCGM plus STF. Cells were washed after 24 h and analyzed by FCM 1 week post-transduction. Vector copy numbers (VCNs) were determined 2 weeks post-transduction (MOI 10 set of experiments). (B) Percentage of CD34⁺CD90⁺ HSPCs^prim 1 week after LV transduction at a MOI of 20. NTC, non-transduced control; -, no TE or vehicle; DMSO, vehicle only; PGE2, prostaglandin E2; PS, protamine sulfate; Stauro, staurosporine. Horizontal lines indicate baseline levels in the absence of TE treatment (“-” condition) for each donor. Numbers in boxes above the graph indicate the mean fold change from the three experiments, relative to the “no TE” transduced control. (C and D) Total EGFP expression (= normalized percentage of EGFP⁺ cells × median EGFP intensity within the EGFP⁺ fraction) in the live (C) and in the CD34⁺CD90⁺ HSPC^prim (D) fraction 1 week after LV HSPC transduction at a MOI of 20. Vertical bars represent the mean total expression. Numbers in boxes above bars indicate the mean fold increase (if ≥1.1) from the three experiments. (E) Total EGFP expression in live cells 1 week post-LV-HSPC-transduction at a MOI of 10. Vertical bars represent the mean total expression. Numbers above bars indicate the mean fold increase (if ≥1.1) from the three experiments. Note: for 2 (green and orange symbols) of the 3 donors, Stauro treatment was performed for 24 h, including the transduction period, instead of the 2-h pre-incubation period chosen in previous experiments. (F) Vector copy number (VCN) per diploid cell, determined by real-time qPCR detecting the post-transcriptional regulatory element (PRE) 2 weeks post-transduction at a MOI of 10. Vertical bars represent the mean of three experiments. Numbers in boxes above bars indicate the mean fold increase. Note that, for 2 (green and orange symbols) of the 3 donors, Stauro treatment was performed for 24 h, including the transduction period, instead of the 2-h pre-incubation period chosen in previous experiments.