Loss of HLA-A expression on primary OKT3-propagated T cells after genetic editing with ZFNs. (A). Loss of cell surface expression of HLA-A2 after electro-transfer of mRNA species encoding ZFN-L and ZFN-R targeting HLA-A2 (top). T cells were harvested 6 days after initial stimulation with γ-irradiated aAPC. Five million T cells were premixed with ZFN mRNA in 100 μL Human T-cell Nucleofector solution and electroporated in a cuvette using a Nucleofector II device with program T-20. Coexpressions of HLA-A2, CD4, and CD8 were analyzed 4 days after electro-transfer of graded doses of the mRNA species encoding ZFN-L and ZFN-R. Flow cytometry data were gated on the propidium iodide-negative, live cell population. Numbers in the lower-right quadrant indicate the percentage of CD4 and CD8⁺ T cells that are HLA-A^neg. Improved disruption of HLA-A expression by cold shock (bottom). Data were collected 4 days after electro-transfer of graded doses of the mRNA species encoding ZFN-L and ZFN-R. Cells were cultured at 30°C from days 1 to 3 after electro-transfer of ZFNs, returned to 37°C, and cultured for 1 additional day before analysis. (B) Improved efficiency of HLA-A disruption by ZFN-L and ZFN-R fused to the heterodimeric FokI domain variants. mRNA species encoding the ZFN-L and ZFN-R heterodimeric FokI mutants EL:KK targeting HLA-A were electro-transferred into primary T cells. HLA-A2 expression was analyzed after culturing the cells for 4 days at 37°C or 3 days at 30°C followed by 37°C for 1 day. X-axis represents CD4 and CD8 expression and y-axis represents HLA-A2 expression.