Lentiviral vectors transduced ADA null BM cells at high efficiency. (A) Schematic representation of the lentiviral vector construct used in this study. Expression of the human ADA cDNA was driven by the PGK promoter. R and U5 indicate long terminal repeat (LTR) regions; ΔU3, deletion of the 3 LTR; SD and SA, splice donor and acceptor sites; ψ encapsidation signal including the 5′ portion of the gag gene (GA); RRE, Rev-response element; cPPT, central polypurine tract; and Wpre, woodchuck hepatitis virus posttranscription regulatory element. (B) ADA expression level in ADA^–/– BM cells following lentiviral gene delivery. Cells were transduced for 24 hours in the presence of cytokines with either PGK-ADA LV (p24, 0.4 μg/10⁶ cells) (ADA) or PGK-GFP control vector (GFP) (MOI, 50). At 5 to 7 days after transduction, ADA expression was analyzed on protein extracts by Western blot. As controls, ADA expression in cultured murine ADA^+/+ BM cells (mADA^+/+) and human mononuclear cells (hADA^+/+) is shown. GAPDH was included as loading control. UT indicates untransduced. (C) ADA activity was measured by HPCE-based analysis in 5- to 7-day cultured BM cells derived from ADA^–/– mice, either mock-transduced or transduced with LV-ADA (n = 9), or from ADA^+/+ mice (n = 7). Values are shown as mean ± standard deviation (SD).