Insulators coupled to a minimal bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain

Fitzsimons, Helen L.; McKenzie, J. M.; During, MJ

doi:10.1038/sj.gt.3301582

Cited by 64 publications

(59 citation statements)

References 27 publications

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“…The design of rAAVS3 might be further improved by the addition of insulators between the ITRs and the expression cassettes to further block any residual promoter activity of the ITRs. 24 QRT-PCR showed the background expression in the brain from the S3 vector was somewhat less than observed in vitro as determined by the hrGFP mRNA copy number normalized against b-actin. Interestingly, even though hGFP transgene expression could be turned off by 99.4% at the mRNA, this level was found to be about 10-fold higher than that of b-actin mRNA, which was used as the internal control for our experiments.…”

Section: Discussionmentioning

confidence: 92%

“…A more effective approach is to clone both expression cassettes into a single self-regulating vector. 24,26 In the self-regulating strategy, a tetcontrolled activator strongly activates transcription from a minimal CMV promoter in the absence of dox. All cells infected with a self-regulating vector will express both activator and transgene.…”

Section: Discussionmentioning

confidence: 99%

“…[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%

“…However, the limited insert size in rAAV for foreign genes makes it difficult to design a tet-regulatable vector with an insulator region between the two tet expression cassettes to minimize promoter activity originating from the ITRs which are necessary for rAAV packaging. 24,33 This limitation may result in leaky regulation in the context of vector backbone. In the present study, we constructed three vectors that represent all three possibilities for the orientations of the two tet expression units relative to each other and to the two ITRs.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…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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Bidirectional expression units enable streptogramin‐adjustable gene expression in mammalian cells

Fux

Fussenegger

2003

Biotech & Bioengineering

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Serious initiatives in gene therapy and tissue engineering require a sophisticated molecular toolbox combining DNA transfer technologies, human-compatible transcription control systems, as well as compact and robust expression configurations. We have designed several versatile bidirectional expression cassettes that enable coadjustable expression of two desired transgenes in response to clinically licensed antibiotics of the streptogramin class (pristinamycin, Pyostacin, Synercid). The bidirectional expression modules consist of a central operator (PIR) that is specific for the pristinamycin-dependent transactivator (PIT). Streptogramin-adjustable binding of PIT to PIR transactivates two divergently oriented promoters and initiates transcription of the desired transgenes. The bidirectional expression module can be equipped with different minimal promoters and configured for expression of (1) two functional effector genes, (2) one effector gene and a reporter gene, (3) PIT and an effector gene to form a highly compact one-vector expression arrangement. We have validated the streptogramin-adjustable bidirectional expression technology in different basic and autoregulated expression configurations in a variety of mammalian and human cell lines.

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Insulators coupled to a minimal bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain

Cited by 64 publications

References 27 publications

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

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

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

Bidirectional expression units enable streptogramin‐adjustable gene expression in mammalian cells

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