title: An improved Tet-On regulatable FasL-adenovirus vector system for lung cancer therapy.
abstract: Gene therapy is a new therapeutic approach for the treatment of human cancers. Gene expression systems that can be regulated by drugs have been developed to improve the safety and efficacy of therapeutic transgene delivery. One of the most promising systems is the tetracycline (Tet)-responsive system in the Tet-On configuration. A major problem of the Tet-On system if used in viral vectors is the high basal activity of the Tet response element (TRE) promoter leading to leaky expression of transgenes under uninduced conditions. We therefore evaluated novel TRE promoters for controlling gene expression in an adenovirus vector (AdV) Tet-On system and further investigated them for expression of the pro-apoptotic CD95/Fas ligand (FasL) in human epithelial carcinoma cell line (HeLa) and lung cancer cells. Plasmid-based reporter gene assays showed that modifications within the tetO (7) and minimal immediate early cytomegalovirus promoter (CMV)(min) sequence of the TRE promoter reduced its leakiness and led to a markedly improved regulatability by doxycycline. Among several TRE promoters tested, a new construct (TRE-Tight1) containing modifications of both the tetO (7) sequence and the CMV(min) showed 11-fold reduced leakiness and 1.5-fold increased absolute transgene expression levels after induction, as compared to the original TRE. Under induced conditions, a TRE-Tight1 promoter-dependent AdV expressing the pro-apoptotic CD95L/FasL induced apoptosis and cell lysis in HeLa cells as efficiently as an AdV containing the original TRE promoter. In contrast to the latter, however, the vector with the modified TRE promoter left cells totally unaffected in the absence of the inducer. Stringently regulated induction of apoptosis and cell death by TRE-Tight1-AdV was also demonstrated in three human lung cancer cell lines. These data show that the novel TRE-Tight1 promoter has a high potential for closely controlled and efficient expression of cytotoxic genes in AdV-based anti-cancer approaches.
authors.affiliation: Department of Cardiology and Pneumology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin Germany.
authors.forename: Isaac
authors.initials: I
authors.lastname: Sipo
authors.forename: Almudena
authors.initials: A
authors.lastname: Hurtado Picó
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Wang
authors.forename: Jürgen
authors.initials: J
authors.lastname: Eberle
authors.forename: Iver
authors.initials: I
authors.lastname: Petersen
authors.forename: Stefan
authors.initials: S
authors.lastname: Weger
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Poller
authors.forename: Henry
authors.initials: H
authors.lastname: Fechner
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D053222:Fas Ligand Protein; D005786:Gene Expression Regulation / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000628:therapy; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000494:pharmacology; D018675:Toxicity Tests; D014407:Tumor Cells, Cultured; D048069:Tumor Necrosis Factors / Q000235:genetics*
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D048069:Tumor Necrosis Factors; D013752:Tetracycline; D004318:Doxycycline