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Symposium Report |
1 Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Bristol University, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK
Abstract
Inducible gene expression systems have typically encountered limitations, such as pleitropic effects of the inducer, basal leakiness, toxicity of inducing agents and low levels of expression. However, recently non-toxic, tightly regulated control of transgene expression has been reported for several systems, the most frequently cited being the tetracycline gene control system. We have found that the individual components of the Tet system [the Tet transactivators and tetracycline responsive element (TRE)] function optimally to control gene expression when they are incorporated into separate adenoviral vectors. Furthermore, incorporation of the Woodchuck hepatitis virus post-transcriptional enhancer (WPRE) allows a dual vector Tet-regulatable Ad system to be used at very low titres (2 x 104) that elicit a minimal inflammatory response, with no loss of transgene expression or ability to regulate transgene expression. This and similar regulatable systems will benefit studies investigating neuronal gene function and those seeking to develop effective neuronal gene therapy strategies.
(Received 6 October 2004;
accepted after revision 3 November 2004; first published online 12 November 2004)
Corresponding author J. B. Uney: Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (LINE), Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK. Email: James.uney{at}bris.ac.uk
The majority of gene transfer studies to date have used strong constitutive promoters to drive the expression of the transgene encoded within the vector (reviewed in Hermens & Verhaagen, 1998), such as the Rous Sarcoma virus (RSV) long terminal repeat (LTR) promoter and cytomegalovirus (CMV) immediate early promoter. These viral promoters are capable of producing high levels of protein expression in a wide range of host cells, including neurones. However, the study of gene function requires defined ‘physiological’ levels of gene expression that can also be temporally controlled effectively to mirror in vivo situations. Furthermore, it is essential in most gene therapy protocols that the expression of the transgene is within a ‘therapeutic window’ of concentration to maximize effects (Schmidt et al. 1995) and minimize the deleterious consequences of overexpression. For example, both an excess and a reduction of peripheral myelin protein 22 leads to abnormal peripheral myelination as observed in type-1 hereditary sensory-motor neuropathy and tomaculous neuropathy, respectively (Thomas & Harding, 1993). A number of inducible gene expression systems have therefore been developed in an attempt to meet the need for regulated gene expression (Yarranton, 1992). Initial attempts relied upon the use of heat shock protein (Schweinfest et al. 1998), metallothionine (Hu & Davidson, 1990) and steroid regulatory promoters (Ko et al. 1989). Although achieving a degree of regulation, these systems suffered from a number of limitations, including basal leakiness, toxicity of the inducing agents and low levels of expression (Yarranton, 1992). An additional disadvantage of these systems is the pleitropic effects of the inducing agent, for example heat shock (see below) will induce the co-ordinate induction of a variety of host genes, in addition to the gene under study, making experimental outcomes difficult to interpret. To overcome these restrictions a number of strategies have been adopted to allow transgene expression to be controlled in mammalian cells; these include the development of ecdysone-inducible (No et al. 1996), rapamycin-controlled (Rivera et al. 1999), progesterone receptor-based (Wang et al. 1994), oestrogen receptor ligand-based (Zerby et al. 2003) and tetracycline (Tet)-controlled inducible expression systems.
The Tet-regulated transactivation system was the first of these inducible systems not to suffer from significant basal leakiness and was highly inducible from the off state. It has therefore been highly developed since the original findings and in this paper we discuss how the Tet system has been used in adenoviral vectors to study neuronal gene function.
The original Tet gene control system
The tetracycline On/Off gene control system developed by Bujard and Gossen (Gossen M. & Bujard H., 1992; Gossen et al. 1995) is based on the Tn10 tetracycline (Tet) resistance operon of E. coli. In this organism expression of the Tet resistance genes are repressed in the absence of Tet, by the binding of the Tet repressor (TetR) to operator sequences (TetO) located within the promoter region upstream of the Tet operon. In the presence of Tet, TetR is inhibited from binding to TetO and transcription can then occur. By fusing TetR with the transcriptional transactivation domain of herpes simplex virus (HSV) virion protein 16 (VP16) a hybrid tetracycline-responsive transcriptional activator, tTA (Tet-Off), was created, which can then induce expression from a mammalian minimal promoter harbouring the TetO sequences (TRE). Induction of such a promoter by the Tet-Off transactivator is abolished in the presence of Tet, as the transactivator cannot bind to the promoter. In the Tet-On system (Gossen et al. 1995) the TetR has been modified by random mutagenesis (four amino-acid changes) such that it creates a reverse phenotype [the Tet-On reverse transactivator (rtTA)]. In direct contrast to the Tet-Off system this mutant transactivator requires tetracycline for the binding to TetO, and hence activation of transcription from TRE. The Tet-On transactivator protein is 100 times more sensitive to the Tet analogue, doxcycyline (Dox) and consequently Dox is used in the Tet-On system. The system also allows weak promoters to become much stronger due to the amplification obtained with the VP16 region on the transactivator (Yin et al. 1996) and it can greatly increase the levels of expression obtained from already powerful promoters (Gossen et al. 1995; Kistner et al. 1996; Liang et al. 1996). Finally, the pharmacological properties of the tetracyclines are also well documented and they are known to cross the blood/brain barrier readily (Sande & Mandell, 1975; Yim et al. 1985), which allows the application of the tetracycline-responsive systems to be used in the cells of the CNS (Mayford et al. 1996).
Adenoviral tetracyline-regulatable systems
To develop a system that could be used in neurones, the Ad transfection system was combined with the tetracycline regulatory system (Harding et al. 1997, 1998). This dual vector Ad–Tet system mediated the highly efficient transfection of neuronal cells in vitro and in vivo and also allowed the level of gene expression to be tightly regulated by Tet, or Dox. Indeed, transgene expression levels were directly related to Dox concentration in a linear manner in both neurones and HeLa cells (Harding et al. 1997). Furthermore, transgene expression could still be turned on in vivo months after the initial stereotaxic injection of the AdTet-On or AdTet-Off system simply by adding or removing Dox, although transgene expression was weaker (Harding et al. 1998). Importantly, the hybrid TRE-Pmin promoter and transactivator elements were incorporated onto separate viral vectors so that the TRE was not juxtaposed with the promoters used to drive transactivator expression. This avoided the promoter driving transactivator expression from non-specifically inducing expression from the TRE, and avoided background expression in the non-induced state. Furthermore, this strategy also allowed the transactivator to TRE promoter ratio to be altered; this is a particularly important characteristic because it was only possible to achieve the efficient regulation of transgene expression in vivo when the ratio of transactivator virus (AdrtTA and AdtTA) to reporter virus (AdTRE–EGFP) was reduced from 1 : 1 (the ratio used in primary cultures) to 1 : 20.
To achieve entirely neuronal or glial-specific regulatable targeting the Tet-off transactivator was placed under the control of the human synapsin-I promoter and the highly characterized glial fibrillary acidic protein (GFAP) promoter (Ralph et al. 2000). Transfection of mixed hippocampal cultures showed that the Ad-Synapsin-tTA (Tet-off) virus only allowed EGFP expression in neuronal cells, whereas the Ad-GFAP-tTA virus restricted expression to glial cells. Stereotaxic injection of the same vectors into adult rat hippocampus resulted in a similar pattern of neuronal and glial-specific transgene expression and these regulatable Ad-vector systems have been used in functional studies (Harding et al. 2001; Noel et al. 1999; Ralph et al. 2001; Xia et al. 2001).
Thomas et al. (2000) have shown that the inflammatory response elicited against Ad vectors is dramatically increased when a titre of greater than 107 plaque-forming units (pfu) is injected into the brain. However, this study also showed that as little as 10–100 pfu of Ad vector could mediate transgene expression in the CNS (with no immune response being elicited) if powerful promoters are also used. In our studies adenoviral vectors have been used to deliver the transactivator and TRE elements of the Tet-system (described in Fig. 1) and this could result in: (i) the total viral titre delivered to the cell being increased to mediate effective transgene expression and (ii) a significant inflammatory response being induced. Therefore, to develop a system that could be used at low titres while mediating strong tightly regulatable gene expression in the CNS we incorporated the WPRE into a neurone-specific Tet-regulatable Ad system (Ralph et al. 2000). This system was injected at low titres (104 pfu) to transduce hippocampal neurones in vivo, and transgene expression with no loss of specificity or ability to regulate expression was obtained (Lee et al. 2004). Furthermore, Fig. 2 shows that at these titres the dual vector Tet system containing the WPRE (Ad.Syn1-tTA-WPRE + Ad.TRE-EGFP-WPRE) mediated strong transgene expression but did not elicit a substantial immune response. However, when control vectors not containing the WPRE (Ad.Syn1-tTA + Ad.TRE.EGFP and Ad0) were used at higher viral titres (7 x 107 pfu) transgene expression was reduced and a substantial immune response was generated. It is also important to note that these in vivo experiments were conducted with the Ad.Syn1-tTA + Ad.TRE-EGFP (and Ad.Syn1-tTA-WPRE + Ad.TRE-EGFP-WPRE) vectors at a ratio of 10 : 1 because initial experiments conducted using a 1 : 1 ratio did not mediate regulatable transgene expression. These results demonstrate the ability to manipulate independently the expression levels of the Tet transactivator and TRE allows optimal gene induction and reduced leakiness in the off state to be obtained.
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Acknowledgements
We would like to thank the Wellcome Trust, BBSRC and MRC for grant support.
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