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. 2015 Dec 8;1:15040. doi: 10.1038/celldisc.2015.40

Figure 2.

Figure 2

Synthesis and delivery of modified TERT mRNA transiently increased telomerase activity and extended telomeres length in CAR T cells. (a) Schematic of modified mRNA comprising the coding sequence of the full-length functional form of TERT or a CI form of TERT, flanked by HBB UTRs and a 151 nt poly-A tail, synthesized using modified nucleotides pseudouridine and 5-methylcytidine. (b) Transfection efficiency of T cells treated with modified mRNA encoding GFP at 12 h post transfection. (c) The transfection efficiency of T cells treated with modified mRNA encoding GFP measured using flow cytometry at 24 h post transfection exceeded 93.61%, and 62.6% of the cells were GFP positive after 48 h, whereas only 10% remained after 72 h. (d,e) The levels of hTERT mRNA were measured using RT-PCR and RT-qPCR. Treatment of CAR T cells with the same concentration of exogenous TERT mRNA or CI-TERT mRNA resulted in internalization of similar amounts of mRNA. (f) Detection of telomerase activity in T cells transfected with modified TERT mRNA using the TRAP. Telomerase activity indicated using a gel-based TRAP assay was detected in T cells treated with 2.0 μg TERT mRNA but not in controls, including cells treated with up to 2.0 μg of CI-TERT mRNA. White bands at the bottom of the gels represent positive controls for the PCR reaction. The sporadic bands in the vehicle lanes were artifacts due to primer dimers. (g, h) Telomere length was extended following modified TERT mRNA delivery. T cells were treated with 2.0 μg of TERT mRNA or CI-TERT mRNA three times every 24 h. Cells were collected for telomere length measurement at 7–10 day, and replicates were used for growth curve measurement, and in some cases, additional treatment at a later time point. *P<0.05, ***P<0.001.