Persistence of plasmid-mediated expression of transgenes in human mesenchymal stem cells depends primarily on CpG levels of both vector and transgene.

BACKGROUND

Gene therapy and cell modification for clinical applications using plasmid vectors are considered to be a safe and promising strategy. One of the major problems with plasmid vector-based constructs is a rapid decline of transgene expression in cells in vitro and in vivo. An important role of CpG motifs or bacterial vector backbone in expression silencing has been suggested.

METHODS

To address the effects of CpG motifs on transgene expression maintenance in stem cells in vitro, we constructed a novel pMBR2 plasmid vector containing 13 CpG motifs only. pMBR2 constructs with CpG-free and CpG-replete firefly luciferase inserts were introduced into cultured human adipose-derived mesenchymal stem (MSCs) by electroporation, and luciferase expression levels were monitored for 3 weeks.

RESULTS

The pMBR2 vector with CpG-free luciferase insert demonstrated the highest persistence of expression, whereas the wild-type luciferase insert containing 97 CpG motifs demonstrated lower expression maintenance in the same vector. In comparison, the same inserts in the CpG-replete pCDNA3 vector demonstrated significantly lower expression levels and only a minimal persistence of expression. β-galactosidase and enhanced green fluorescent protein genes inserted into pMBR2 vector also demonstrated higher expression levels and better maintenance compared to the same genes in pCDNA3 vector.

CONCLUSIONS

The persistence of plasmid vector expression in human MSCs is determined primarily by CpG content of both vector and transgene. The data obtained in the present study indicate that the pMBR2 vector with a minimized number of CpG motifs is appropriate for extended plasmid-mediated expression of transgenes in MSCs and possibly other types of stem cells.