Enhancement of plasmid-mediated stable gene expression by woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in human embryonic kidney (HEK293) cells

Abstract

Influence of random integration site on the expression of transgene in mammalian cells makes it a major challenge to achieve high productivity of recombinant proteins. Optimization of expression vector is one of the most popular strategies to resolve this problem. Among this, woodchuck hepatitis virus  post-transcriptional regulatory element (WPRE) is a possible enhancer of gene expression in mammalian  cells that promotes efficient export of unspliced (RNA) into the cytoplasm, as has been proved in  transient transfection. In this study, WPRE was evaluated for enhancing stable gene expression levels in two industrial cell lines, human embryonic kidney (HEK293) and CHO-S, using the enhanced green fluorescent protein (EGFP), prourokinase (pro-UK) and protein C (PC) as the reporter gene. Based on the mean fluorescence intensity (MFI), WPRE exerted a clear positive effect on gene expression in HEK293 cells with an increase of EGFP expression level by approximately 2.5- to 3-fold independent of the  promoter used in plasmid vector. In contrast, in Chinese hamster ovary (CHO)-S cells, only a marginal effect on plasmid-mediated EGFP expression by WPRE was observed. The measurable increase of EGFP expression at the protein level was paralleled by an increase of EGFP RNA. Further test of the effect of WPRE on plasmid-mediated gene expression with two therapeutic proteins showed substantial increase of stable pro-UK and PC expression only in HEK293 by about 2.2-fold and 6.1-fold, respectively. The data of PC expression levels obtained from the random HEK293 cell clones transfected with WPRE-containing or lacking vector further demonstrated the enhancement of stable plasmid-mediated gene expression by WPRE in HEK293 cells. These results in stable transfectants show the positive effect of WPRE on transgene expression is cell-type dependent and promoter-independent, and provide valuable information to improve vectors for efficient and stable gene expression in HEK293 cells.

Key words: Mammalian cells, plasmid vector, stable gene expression, protein therapeutics, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).