Figure 5.

LVsh5/C46 vector does not induce deleterious effect on cells. PBMCs from three to five healthy donors were transduced with LVsh5/C46 (sh5/C46) at MOIs of 1 or 10 or and were compared to untransduced cells and to cells transduced with a control lentivirus vector encoding eGFP (enhanced green fluorescent protein) at MOI of 1. In different experiments, transduction efficiency was determined by flow cytometry to be 15–44% at an MOI of 1 and 36–64% at an MOI of 10. Cells were assessed for apoptosis, proliferation, and inflammation; results were normalized to untransduced cells; and Student’s t-test was performed. (a) Apoptosis was not induced in LVsh5/C46-transduced PBMCs. Caspase 3/7 activities were assessed 7 days posttransduction by luminescence assay (n = 3). Staurosporine was used as a positive control. (b) LVsh5/C46 did not alter viability or proliferative capacity of modified PBMCs. Cells were cultured for 7 days, and proliferation rate was assessed (n = 5). IL-2 was used as a positive control. (c–e) Transduced PBMCs were cultured for 8 days, and the level of the cytokines (c) interferon-γ (n = 3), (d) interleukin-6 (n = 4), and (e) TNF-α (n = 4) was determined. Cells treated with PHA were used as positive controls. *P < 0.05 compared to untransduced cells. **P < 0.01 compared to untransduced cells. MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor.