Co-expression of p21Waf1/Cip1 in adenovirus vectors improves expression of a second transgene

• Published in: Gene therapy Vol. 16; no. 4; pp. 574 - 578
• Main Authors: Schumacher, A, Horvat, S, Woischwill, C, Wolff, G, Witt, C
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2009; Nature Publishing Group
• Subjects: Adenoviruses; Bcl-2 protein; Biomedical and Life Sciences; Biomedicine; Cell Biology; Cell cycle; Cell lines; Cyclin-dependent kinase inhibitor p21; Cytotoxicity; Enzyme-linked immunosorbent assay; Epithelial cells; Expression vectors; Flow cytometry; Gene Expression; Gene Therapy; Gene transfer; Hepatocytes; Human Genetics; Nanotechnology; short-communication; Toxicity; Transgenes; Viruses; vector design; gene expression; p21; adenovirus
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2009.2

First-generation adenoviral (Ad) vectors are frequently used vectors for experimental and clinical gene transfer. Earlier it has been shown that parallel overexpression of the cell cycle regulator p21 Waf1/Cip1 (p21) or antiapoptotic bcl-2 from a second vector reduces cytotoxicity and improves transgene expression. Here, we investigate whether the co-expression of p21 and α 1 -antitrypsin from a single vector improves vector safety and α 1 -antitrypsin expression. Cell lines (A549 and HeLa) and primary cells (small airway epithelial cells and hepatocytes) were infected with adenovirus vectors transducing α 1 -antitrypsin with (AdCMV.p21-RSV.hAAT) or without (AdRSV.hAAT) p21. α 1 -Antitrypsin expression and cytotoxicity were analyzed using western blot/ELISA and LDH/ALT/AST assays, respectively. Cell cycle profiles were determined by flow cytometry. Co-expression of p21 strongly increased the α 1 -antitrypsin expression in all cell types and at all doses tested. No changes in ALT/AST from hepatocytes and only minor increases in the LDH release in A549 and HeLa were observed with either vector. Cell cycle profiles were also not affected adversely. Incorporation of p21 in Ad vectors together with a gene of interest improves the vector performance; such vectors will allow the application of lower doses and thereby reduce immunological side effects.