Low expression of LDL receptor on resting T cells, B cells, and CD34⁺cells limits VSV-G-LV binding, fusion, and transduction of these gene-therapy targets. (A) Unstimulated human T cells, B cells, and CD34⁺ cells (G0) or 24-hour prestimulated (stim) T cells (anti-CD3 + anti-CD28 + IL-2), B cells (SAC + IL-2), and hCD34⁺ cells (TPO + SCF + Flk-3L) were transduced with a GFP-encoding VSV-G-LV vector at an MOI = 50 (T and B cells) or MOI = 100 (CD34⁺ cells) and GFP⁺ cells were analyzed at day 3 posttransduction by FACS (see supplemental Methods, available on the Blood Web site); for LDL-R detection, freshly isolated or 24-hour prestimulated cells (see above) were incubated with the anti–LDL-R antibody (mouse mAb; R&D Systems) followed by staining with anti-mouse APC antibody (white open histograms), a control incubation with the latter antibody alone was performed (gray filled histogram); for fusion detection, freshly isolated or 24-hour prestimulated cells were incubated overnight with GFP gesicles⁷  at 4°C to allow only binding or at 37°C to allow binding followed by fusion. The cells were then treated with trypsin to remove the GFP gesicles at the cell surface that did not fuse with the cells. (B) Equivalent quantities of VSV-G-LV or LV particles without envelope (measured by p24 content) were incubated with freshly isolated or 24-hour prestimulated cells (2^E5 cells) for 1 hour at 4°C and then washed 4 times to remove unbound vector particles. The cells were pelleted and the cell-associated HIV capsid content (p24) was determined by ELISA (means ± SD; n = 3). The p24 signal for nonenveloped LVs was used as reference. (C) Entry through LDL-R was evaluated by blocking with a monoclonal antibody (C7, aLDL-R at 5 μg/mL; Santa Cruz Biotechnology) or by competition with soluble LDL receptor at 0.5 μg/mL (LDL-R 0.5; R&D Systems) or 5 μg/mL (LDL-R 5). A 1-hour preincubation of the prestimulated T cells, B cells, and CD34⁺ cells with either blocking agent was performed before transduction with GFP-encoding VSV-G-LVs (MOI 50 for T and B cells; MOI 100 for CD34⁺ cells) or MV-LVs (MOI of 10) for 48 hours, followed by FACS analysis for detection of GFP⁺ cells (means ± SD; n = 3). aLDL-R, anti-low density lipid receptor antibody; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter; IL, interleukin; mAb, monoclonal antibody; MOI, multiplicity of infection; SAC, staphylococcus aureus Cowan; SCF, stem cell factor; TPO, trombopoietin. Blood samples were obtained from healthy donors after informed consent and after local ethical committee approval in accordance with the Declaration of Helsinki.