Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression...

Gene delivery to esophageal tissue could provide novel treatments for diseases, such as cancer. The Sleeping Beauty (SB) transposon system, as a natural and non-viral tool, is efficient at transferring transgene into the human genome for human cell genetic engineering. The plasmid-based SB transposon can insert into chromosomes through an accurate recombinase-mediated mechanism, providing long-term expression of transgene integrated into the target cells. In this study, we aimed to investigate the activity of ED-L2 tissue-specific promoter that was engineered from the Epstein-Barr Virus (EBV) and combined with the hyperactive SB100X transposase to achieve the stable expression of T2-Onc3 transposon in esophageal squamous epithelial cells. Here we constructed an SB transposon-based plasmid system to obtain the stable expression of transposon upon introduction of a hyperactive SB transposase under the control of tissue-specific ED-L2 promoter via the lipid-based delivery method in the cultured esophageal squamous cell carcinoma cells. Among established human and mouse cell lines, the (ED-L2)-SB100X transposase was active only in human esophageal stratified squamous epithelial and differentiated keratinocytes derived from skin (KYSE-30 and HaCaT cell lines), where it revealed high promoter activity. Data offered that the 782 bp sequence of ED-L2 promoter has a key role in its activity in vitro. The (ED-L2)-SB100X transposase mediated stable integration of T2-Onc3 in KYSE-30 cells, thereby providing further evidence of the tissue specificity of ED-L2 promoter. The KYSE-30 cells modified with the SB system integrate on average 187 copies of the T2-Onc3 transposon in its genome. In aggregate, the (ED-L2)-SB100X transposase can be efficiently applied for the tissue-specific stable expression of a transgene in human KYSE-30 cells using SB transposon.