A comparative study of PiggyBac-mediated gene insertion and ubiquitous chromatin opening element incorporation to enhance Darbepoetin alfa expression

Stable and robust production of recombinant therapeutic proteins in mammalian cells remains challenging due to position effects and transcriptional silencing of the transgene following random integration. We investigated whether semi-targeted integration and UCOE-incorporation could enhance Darbepoetin alfa (DPO) transgene expression. A PiggyBac-mediated Darbepoetin alfa-secreting cell pool (PB-DPO) was established by semi-targeted insertion of a DPO-containing expression cassette using pB513B-1, a PiggyBac transposon-derived vector, into CHO DG44 host cells. A UCOE-incorporated Darbepoetin alfa-secreting cell pool (UCOE-DPO) was also used to compare DPO production rates between the two groups and the control (pOptiVEC-DPO) cell pool. DPO expression at both transcript and protein levels was compared across DPO-producing cell pools using real-time quantitative PCR, Western blotting, and ELISA. The PB-DPO showed ~ 1.96- and ~ 1.79-fold increases in mRNA and protein levels, respectively, whereas the UCOE-DPO exhibited even stronger enhancement, with ~ 2.94- and ~ 4.53-fold increases compared with the control. In conclusion, PiggyBac-mediated transgene transposition generated a cell population with increased transgene expression, likely through multiple insertions and preferential integration near transcriptionally active regions. Although both modified pools demonstrated increased DPO transcript and protein yields, UCOE-DPO was more effective at overcoming integration-associated silencing. Compared with the semi-targeted transposition method, UCOE incorporation further increased Darbepoetin alfa production by ~ 2.5-fold.