Regulated and prolonged expression of mIFNα in immunocompetent mice mediated by a helper-dependent adenovirus vector

Aurisicchio, Luigi; Bujard, Hermann; Hillen, Wolfgang; Cortese, Riccardo; Ciliberto, Gennaro; Monica, Nicola La; Palombo, Fabio

doi:10.1038/sj.gt.3301596

Cited by 35 publications

(37 citation statements)

References 30 publications

(41 reference statements)

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“…18 Another interesting aspect emerging from the in vivo studies is the rapid return to base line after drug withdrawal. Differently to what we observed previously using the mIFN␣ as transgene, 12 the reversal of SEAP induction was slower. However, SEAP showed a longer half-life that requires 5 days to reach the basal level.…”

Section: Discussioncontrasting

confidence: 91%

“…12 Multiple viral passages were performed to reach a high titer which was monitored by TaqMan PCR measurements as previously described. 20 All the vectors were purified on a CsCl density gradient, and physical particles (pp) were measured by DNA optical density.…”

Section: Hd Vectorsmentioning

confidence: 99%

“…10 These vectors were successfully utilized in the liver both with the Tet system and with the RU486 based system. 11,12 However, these previous studies were not designed to establish general rules for achieving maximal level and tightest control of gene expression. Here we address this issue by comparing a set of HD adenovirus vectors carrying the same reporter gene but a variety of tet regulatory elements.…”

Section: Introductionmentioning

confidence: 99%

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Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2s-M2 tetracycline transactivator and repressor system

Salucci¹,

Scarito²,

Aurisicchio³

et al. 2002

Gene Ther

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Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2 s -M2) alone or in combination with Tet-repressor (tTS-Kid)

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Section: Discussioncontrasting

confidence: 91%

Section: Hd Vectorsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2s-M2 tetracycline transactivator and repressor system

Salucci¹,

Scarito²,

Aurisicchio³

et al. 2002

Gene Ther

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“…11 The Tet system is widely used to study gene function and to generate conditional mutants in cell lines and in transgenic animals (reviewed in: Baron and Bujard 12 and Gossen and Bujard 13 ). It has also been used to generate regulated gene delivery vectors based on adenoviruses, [14][15][16][17] adeno-associated virus, [18][19][20] retroviruses, 21,22 lentiviruses 23,24 and herpes simplex virus. 25 We therefore decided to use the Tet system to obtain a regulated, local expression of IFNs in the liver, and to study the effect on HBV replication in vivo in a transgenic mouse model.…”

Section: Introductionmentioning

confidence: 99%

Liver-specific expression of interferon γ following adenoviral gene transfer controls hepatitis B virus replication in mice

et al. 2005

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Interferons control viral replication and the growth of some malignant tumors. Since systemic application may cause severe adverse effects, tissue-specific expression is an attractive alternative. Liver-directed interferon gene therapy offers promising applications such as chronic viral hepatitis B or C or hepatocellular carcinoma and thus needs testing in vivo in suitable animal models. We therefore used the Tet-On system to regulate gene expression in adenoviral vectors, and studied the effect of liver-specific and regulated interferon g expression in a mouse model of chronic hepatitis B virus (HBV) infection. In a first generation adenoviral vector, genes encoding for firefly luciferase and interferons a, b or g, respectively, were coexpressed under control of the bidirectional tetracycline-regulated promoter P tet bi. Liverspecific promoters driving expression of the reverse tetracycline controlled transactivator ensured local expression in the livers of HBV transgenic mice. Following gene transfer, we demonstrated low background, tight regulation and a 1000-fold induction of gene expression by doxycycline. Both genes within the bidirectional transcription unit were expressed simultaneously, and in a liver-specific fashion in cell culture and in living mice. Doxycycline-dependent interferon g expression effectively controlled HBV replication in mice, but did not eliminate HBV transcripts. This system will help to study the effects of local cytokine expression in mouse disease models in detail. Gene Therapy (2005) 12, 668-677.

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“…[11][12][13][14][15][16] Another strategy is to combine both components into one self-regulating virus so that target cells only need to be infected by one virus to allow regulatable expression. 7,[17][18][19][20][21][22][23][24][25][26] Recombinant adeno-associated viral (rAAV) vectors have several properties that make them one of the most promising vehicles for gene delivery to the CNS. 27 These vectors have been reported to infect and transduce both dividing and nondividing cells, including neurons, with minimal cellular toxicity or host immune response.…”

Section: Introductionmentioning

confidence: 99%

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

et al. 2004

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Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

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Regulated and prolonged expression of mIFNα in immunocompetent mice mediated by a helper-dependent adenovirus vector

Cited by 35 publications

References 30 publications

Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2s-M2 tetracycline transactivator and repressor system

Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2s-M2 tetracycline transactivator and repressor system

Liver-specific expression of interferon γ following adenoviral gene transfer controls hepatitis B virus replication in mice

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

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