Development of Multigene and Regulated Lentivirus Vectors

Reiser, Jakob; Lai, Zhennan; Zhang, Xian-Yang; Brady, Roscoe O.

doi:10.1128/jvi.74.22.10589-10599.2000

Cited by 110 publications

(83 citation statements)

References 71 publications

(54 reference statements)

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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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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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“…In some cases, the two components of the tet system have been cloned into separate viruses, which requires coinfection of the target cells by both viruses to obtain regulatable transgene expression. [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.…”

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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“…The method is derived from earlier work by Reiser et al, 23 who demonstrated that lentiviral cotransduction does work efficiently without affecting the transfer efficiency of either of the vectors.…”

Section: Lentiviruses Construction Production and Transductionmentioning

confidence: 99%

Development of an inducible suicide gene system based on human caspase 8

et al. 2005

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Suicide gene-therapy strategies are promising approaches in treating various diseases such as cancers, atherosclerosis, and graft-versushost-disease. Here, we describe the development of a new effector gene based on inducing functional caspase 8, the initiator caspase in the death-receptor pathway. We constructed vectors encoding a constitutively active form of human caspase 8 (CC8), and demonstrated the efficient killing of a variety of cell types in transfection and lentivirus-transduction assays. We then analyzed the ability to control the apoptotic activity of a caspase 8-derived construct through the ARIADt homodimerization system (FKC8), a system shown to be extremely effective in several cellular models upon retroviral and lentiviral gene transfer. Similarly, two transcription-regulation systems, muristerone-regulated and Tet-On, were tested to control the expression of CC8. The homodimerization-regulated system FKC8 was shown to be the most efficient system with low background activity in noninduced conditions. In the presence of a dimerizer, it was as active as the activated Tet-On system. From our data, we conclude that the dimerizer-dependent human caspase 8 represents a highly inducible and very powerful system to eradicate transduced cell populations. In addition to its application in experimental gene therapy, this variant may be highly useful for mechanistic research related to apoptosis.

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Development of Multigene and Regulated Lentivirus Vectors

Cited by 110 publications

References 71 publications

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

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

Development of an inducible suicide gene system based on human caspase 8

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