Figure 2.

Single Transduction Enhancers Increase Transduction of CD34⁺ HSPCs with Lentiviral and Alpharetroviral Vectors

(A) Schematic representation of the EGFP-encoding alpharetroviral (ARV) and lentiviral (LV) SIN vectors used in this study. R, repeat region; U5, unique 5; hPGK, human phosphoglycerate kinase promoter; PRE, post-transcriptional regulatory element; ΔU3, unique 3 region with self-inactivating deletion. (B) Experimental scheme. CD34⁺ HSPCs from 3 HDs were pre-stimulated for 24 h prior to transduction in the presence or absence of single transduction enhancers (TEs) in SCGM plus STF. Transduction was carried out at a MOI of 20, which is lower than the MOI used with most gene therapy protocols, allowing for a better observation of TE effects due to a lower transduction rate. Cells were washed after 24 h and analyzed by FCM 1 week post-transduction. Donor C was used in 2 independent experiments for selected conditions. (C and D) Percentage of CD34⁺CD90⁺ HSPCs^prim 1 week post-transduction with LV (C) and ARV (D) vectors at a MOI of 20. NTC, non-transduced control; -, no TE or vehicle; DMSO, vehicle only; PGE2, prostaglandin E2; PS, protamine sulfate; Stauro, staurosporine; OH-Stauro, 7-hydroxy-staurosporine. Horizontal lines indicate baseline levels in the absence of TE treatment (“-” condition) for each donor. (E and F) Total EGFP expression (= normalized percentage of EGFP⁺ cells × median EGFP intensity within the EGFP⁺ fraction) in live cells 1 week post-transduction with LV (E) and ARV (F) vectors 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 four experiments. (G and H) Total EGFP expression in the CD34⁺CD90⁺ HSPC^prim fraction 1 week post-transduction with LV (G) and ARV (H) vectors 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 four experiments.