Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells

The leading FDA-approved drugs for epigenetic cancer therapy are small molecule compounds that activate silenced tumor suppressors by inhibiting enzymes that generate aberrant repressive chromatin. Although promising, this approach is limited because chromatin-modifying enzymes often target non-chromatin proteins and can serve dual functions as gene repressors and activators. Previously, we have demonstrated that a transgenically expressed synthetic polycomb-derived transcription factor (PcTF) could activate genes in silenced chromatin via specific interactions with histone H3 trimethylated at lysine 27 (H3K27me3). Efficient non-viral intracellular delivery remains a challenge for protein-based biologics. Herein, we report the delivery of cell-penetrating PcTF (CP-PcTF) to cultured cells. We expressed and purified recombinant PcTF that was fused in frame with a nuclear localization signal and a cell penetrating peptide tag (TAT). We demonstrated rapid and efficient uptake of soluble CP-PcTF by osteosarcoma U-2 OS cells grown in 2-D monolayers and 3-D spheroids. However, CP-PcTF had a modest effect on gene expression and cell proliferation compared to transgenically-expressed PcTF from our previous work. Overall, these results suggest that TAT is a very effective delivery vehicle for the recombinant transcriptional regulator PcTF, and that further technical development is needed to deliver functional PcTF into cell nuclei.