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

被引:18
作者
Tietge, UJF [1 ]
Kozarsky, KF [1 ]
Donahee, MH [1 ]
Rader, DJ [1 ]
机构
[1] Univ Penn, Sch Med, Dept Med, Philadelphia, PA 19104 USA
关键词
gene transfer; adenovirus; tetracycline; secretory phospholipase A(2);
D O I
10.1002/jgm.384
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
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.hsPLA2) 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 Adtetoff.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. Copyright (C) 2003 John Wiley Sons, Ltd.
引用
收藏
页码:567 / 575
页数:9
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[1]  
ALI M, 1994, GENE THER, V1, P367
[2]   Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2 [J].
Balsinde, J ;
Balboa, MA ;
Dennis, EA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (14) :7951-7956
[3]  
BARR E, 1994, GENE THER, V1, P51
[4]   Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis [J].
Cormier, RT ;
Hong, KH ;
Halberg, RB ;
Hawkins, TL ;
Richardson, P ;
Mulherkar, R ;
Dove, WF ;
Lander, ES .
NATURE GENETICS, 1997, 17 (01) :88-91
[5]   Pharmacologically regulated gene expression in the retina following transduction with viral vectors [J].
Dejneka, NS ;
Auricchio, A ;
Maguire, AM ;
Ye, X ;
Gao, GP ;
Wilson, JM ;
Bennett, J .
GENE THERAPY, 2001, 8 (06) :442-446
[6]   TEMPORAL CONTROL OF GENE-EXPRESSION IN TRANSGENIC MICE BY A TETRACYCLINE-RESPONSIVE PROMOTER [J].
FURTH, PA ;
STONGE, L ;
BOGER, H ;
GRUSS, P ;
GOSSEN, M ;
KISTNER, A ;
BUJARD, H ;
HENNIGHAUSEN, L .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (20) :9302-9306
[7]   Highly controlled gene expression using combinations of a tissue-specific promoter recombinant adenovirus and a tetracycline-regulatable transcription factor [J].
Ghersa, P ;
Gobert, RP ;
Sattonnet-Roche, P ;
Richards, CA ;
Pich, EM ;
van Huijsduijnen, RH .
GENE THERAPY, 1998, 5 (09) :1213-1220
[8]   TIGHT CONTROL OF GENE-EXPRESSION IN MAMMALIAN-CELLS BY TETRACYCLINE-RESPONSIVE PROMOTERS [J].
GOSSEN, M ;
BUJARD, H .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (12) :5547-5551
[9]   A role for secretory phospholipase A(2) and C-reactive protein in the removal of injured cells [J].
Hack, CE ;
Wolbink, GJ ;
Schalkwijk, C ;
Speijer, H ;
Hermens, WT ;
vandenBosch, H .
IMMUNOLOGY TODAY, 1997, 18 (03) :111-115
[10]   Switching transgene expression in the brain using an adenoviral tetracycline-regulatable system [J].
Harding, TC ;
Geddes, BJ ;
Murphy, D ;
Knight, D ;
Uney, JB .
NATURE BIOTECHNOLOGY, 1998, 16 (06) :553-555