A tetracycline-regulated adenoviral expression system for in vivo delivery of transgenes to lung and liver

摘要

Background Recombinant adenoviruses are an established tool for somatic gene transfer to multiple cell types in animals as well as in tissue culture. However, generation of adenoviruses expressing transgenes that are potentially toxic to the host cell line represents a practical problem. The aim of this study was to construct an adenoviral expression system that prevents transgene expression during the generation and propagation of the virus, and allows efficient gene transfer to lung and liver, major target organs of gene therapy. Methods Using the tet-off system we constructed tetracycline (tet) regulatable recombinant adenoviruses expressing the marker gene LacZ (Adtet-off.LacZ) as well as a secretory protein, human group IIA secretory phospholipase A_2 (Adtet-off.hsPLA_2). Expression (Western blot, activity assay) was tested in vitro (HeLa cells), and in vivo by gene transfer to lung and liver. Results Without addition of tetracycline we demonstrated expression of LacZ (Adtet-off.LacZ) and sPLA_2 (Adtet-off.hsPLA_2) in HeLa cells. Providing additional tet-transactivator (tTA) protein either by stable transfection or coinfection with a tTA-expression adenovirus resulted in a further increase of LacZ and sPLA_2 expression. Transgene expression in vitro was eliminated by the additional of tetracycline to the culture medium. Adtet-off.LacZ and Adtet-off.hsPLA_2 allowed successful gene transfer in vivo to lung and liver. While the expression was highly efficient within the lungs, however, additional tTA was necessary to achieve high-level expression within liver. Conclusions Tet-regulatable adenoviral expression systems may facilitate the construction of recombinant adenoviruses encoding potentially toxic transgenes and permit regulated transgene expression.