Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering

Abstract

The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage. However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors. To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process. The human (HaCaT, A549) and rat (NBT II, MH1C1) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Adβ-gal adenovirus, encoding for the reporter gene E. coli β-galactosidase, in the presence of various polybrene concentrations. We determined the amount of β-gal positive cells by X-gal staining and the β-gal expression by ONPG-assay after 24 h. In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Adβ-gal resulted in a marked increase of β-gal positive cells and β-gal protein expression. The efficacy of polybrene showed a clear dose dependency. The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent. In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.