ArticleGene therapy2004
Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.
Article in Gene therapy, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 36 citations in OpenAlex.
- Sterilization of Channel Catfish (Ictalurus punctatus) via Overexpression of bax Gene Regulated by a Tet-off System in the Primordial Germ Cells.Marine biotechnology (New York, N.Y.) · 2025Article
- Roles of miR-592-3p and Its Target Gene, TMEFF1, in the Nucleus Accumbens During Incubation of Morphine Craving.The international journal of neuropsychopharmacology · 2022Article
- Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD.Scientific reports · 2018Article
- A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting.British journal of clinical pharmacology · 2013Review
- Genetic therapy for the nervous system.Human molecular genetics · 2011Review
- Regulation of retinal function but nonrescue of vision in RPE65-deficient dogs treated with doxycycline-regulatable AAV vectors.Molecular therapy : the journal of the American Society of Gene Therapy · 2010Article
- Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors.Gene therapy · 2010Article
- Controlling brain tumor growth by intraventricular administration of an AAV vector encoding IFN-beta.Cancer gene therapy · 2009Article
- Construction and application of an inducible system for homogenous expression levels in bulk cell lines.PloS one · 2009Article
- In vivo gene regulation using tetracycline-regulatable systems.Advanced drug delivery reviews · 2009Review
- Molecular therapy of obesity and diabetes by a physiological autoregulatory approach.Nature medicine · 2009Article
- In vitro analysis of promoter activity in Müller cells.Molecular vision · 2008Article
- Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging.Gene therapy · 2008Article
- Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression in mammalian cells and mice.BMC biotechnology · 2007Article
- Regulatable gene expression systems for gene therapy.Current gene therapy · 2006Review
- The "perivascular pump" driven by arterial pulsation is a powerful mechanism for the distribution of therapeutic molecules within the brain.Molecular therapy : the journal of the American Society of Gene Therapy · 2006Article
- Gene therapy progress and prospects--vectorology: design and production of expression cassettes in AAV vectors.Gene therapy · 2006Review
- Long-term inducible expression in striatal neurons from helper virus-free HSV-1 vectors that contain the tetracycline-inducible promoter system.Brain research · 2006Article
- Current status of transcriptional regulation systems.Cytotechnology · 2006Article
- Viral-based modelling and correction of neurodegenerative diseases by RNA interference.Gene therapy · 2006Review
Corrections and comments
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
Abstract
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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