A tetracycline-regulated adenoviral expression system for in vivo delivery of transgenes to lung and liver.
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-expressing adenovirus resulted in a further increase of LacZ and sPLA(2) expression. Transgene expression in vitro was eliminated by the addition 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.
Tietge
Uwe J F
UJ
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA.
Kozarsky
Karen F
KF
Donahee
Mary H
MH
Rader
Daniel J
DJ
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D054501:Group II Phospholipases A2; D006367:HeLa Cells; D006801:Humans; D007763:Lac Operon / Q000502:physiology; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000502:physiology; D019076:Transgenes
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D013752:Tetracycline