Figure 4.

T3v4 PD-1 TALEN Is as Efficient as T3v1 PD-1 TALEN at Disrupting PDCD1 Gene Expression in Primary T Cells and Does Not Induce Mutagenesis at the Off-Site Sequences Tested

Four days after activation, 5 million T cells were transfected with 10 μg of each mRNA encoding the left and right arms of TALEN T3v2 PD-1 (A) or TALEN T3v1, T3v2, T3v3, or T3v4 (B and C) in combination with 10 μg of each mRNA encoding the left and right arms of TALEN TRAC. (A) The efficiency of TALEN-mediated gene processing was analyzed by high-throughput DNA sequencing analysis of engineered T cell genomic DNA harvested 6 days after transfection. Potential in-site and off-site targets were carefully evaluated for these loci. The off-site OS9, OS10, OS11, and OS12 have identical potential TALEN binding sites as the OS8. The PCR products generated for deep-sequencing analysis for OS8, OS9, OS10, OS11, and OS12 are more than 99.8% identical. Thus, OS8 represents the pooled data of the off-sites mentioned above. (B) Nine days after transfection, T cells were reactivated using Dynabeads human T activator CD3/CD28. Two days later, the mean fluorescence intensity (MFI) of surface PD-1 detection was assessed by flow cytometry on viable non-transfected T cells and transfected T cells using PD-1 mAb in combination with a live/dead cell marker. Histograms are representative of at least two independent experiments. (C) The efficiency of TALEN-mediated gene processing was analyzed by high-throughput DNA sequencing analysis of engineered T cell genomic DNA harvested 6 days after transfection. Potential in-site and off-site 9 and off-site 3 targets were carefully evaluated for these loci.