Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo
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Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.
We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC1D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. In vivo studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC1D promoters.
This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.
KeywordsPC12 Cell Glial Fibrillary Acidic Protein 1321N1 Cell Bidirectional Promoter Gene Expression Cassette
The widespread phenotype diversity within the central nervous system underscores the importance of restricting transgene expression to a specified target cell type [1, 2, 3, 4]. Failure to do so results in gene expression in non target cells that confounds data interpretation and may lead to undesirable side effects. Restricting gene expression to a specified cell population within the brain by using cell-selective promoters remains an attractive approach [5, 6]. In addition, cell-type-specific promoters are advantageous since they are less likely to activate host cell defense machinery and are less sensitive to cytokine-induced promoter inactivation than viral promoters . As such, improved stability and longevity of gene expression can be expected.
The recent demonstration of synthetic bidirectional promoters that mediate coordinate transcription of two mRNAs  prompted us to test whether this design is applicable to TAS. In synthetic bidirectional promoters a minimal core promoter is joined upstream to an efficient promoter positioned in the opposite orientation . The rationale of the design was that upstream elements in the efficient promoter, when closely flanked by minimal promoters on both sides, drive transcriptional activity in both directions [18, 19, 20, 21, 22]. Earlier, Baron et al. (1995) constructed tetracycline-inducible bidirectional promoters by duplicating a minimal promoter on both sides of a series of Tet operator repeats to obtain exogenously regulated expression of two transgenes in a correlated, dose-dependent manner . Here we applied bidirectional promoter design in combination with TAS in vitro and in vivo. We tested two cell-specific promoters, SYN and GfaABC1D promoters. The properties of these two promoters were described earlier.
Results and discussion
Five self-inactivated HIV-derived lentiviral vectors (Figure 1B) were constructed for this study containing: (1) the EGFP reporter gene under the control of the SYN promoter alone (LV-1 × SYN-EGFP), (2) the EGFP reporter gene under the control of the GfaABC1D promoter alone (LV-1 × GfaABC1D-EGFP), (3) SYN-based bidirectional promoter driving the synthesis of the transcriptional activator GAL4p65 (for details about GAL4p65, refer to ) and the reporter gene EGFP (LV-mCMV/SYN-EGFP), (4) GfaABC1D-based bidirectional promoter driving the synthesis of the transcriptional activator GAL4p65 and the reporter gene EGFP (LV-mCMV/GfaABC1D-EGFP). LV-1 × SYN-EGFP and LV-1 × GfaABC1D-EGFP served as controls lacking the transcriptional activator GAL4p65. In LV-mCMV/SYN-EGFP and LV-mCMV/GfaABC1D-EGFP, a minimal CMV core promoter (mCMV, 65 bp) derived from pTRE-Tight-DsRed2 (Clontech) was joined in the opposite orientation to either the SYN or GfaABC1D promoter to form bidirectional promoters mCMV/SYN and mCMV/GfaABC1D. Two gene expression cassettes flanked the bidirectional promoters. The 5' cassette transcribed the strong GAL4p65 transactivator. The 3' cassette drove the synthesis of the reporter gene with 5 tandem GAL4 binding sequences at the 5' end of the specific promoter. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE [24, 25, 26]) was included in all of the four constructs to further enhance the expression level of the reporter gene. If the bidirectional promoters mCMV/SYN and mCMV/GfaABC1D are active in both directions, upstream product GAL4p65 would bind to GAL4 binding sequences introduced 5' of SYN or GfaABC1D promoter. This we anticipated would then lead to boosted expression of EGFP.
We next evaluated the performance of the new vectors in vivo in the rat brain. LVV were stereotaxically injected into the hypoglossal motor nucleus. To allow for direct comparison, we set the dose for each virus for one rat as 106 infectious units and transgene expression was scored one week postinjection. As shown in Figure 3A, significantly stronger EGFP expression was observed from LV-mCMV/SYN-EGFP and LV-mCMV/GfaABC1D-EGFP than that from LV-1 × SYN-EGFP and LV-1 × GfaABC1D-EGFP. NIH ImageJ was used to quantitatively compare the relative EGFP fluorescence levels. We observed a ~4-fold increase in the level of fluorescence in tissues transduced by LV-mCMV/SYN-EGFP than by LV-1 × SYN-EGFP [Figure 3B(1)] and ~9-fold increase by LV-mCMV/GfaABC1D-EGFP than by LV-1 × GfaABC1D-EGFP [Figure 3B(2)].
Although we used two heterogeneous core promoters other investigators reported that a unidirectional promoter may be bidirectionalized by fusing either a homogeneous or heterogenous minimal core promoter at its 5' end in the opposite orientation [17, 27, 28, 29]. Apart from cell-specific promoters, which can be made bidirectional as demonstrated in the current study, constitutive and inducible promoters can also be bidirectionalized [17, 23, 28]. Thus, we believe that the ability to confer bidirectional expression to a promoter is not a special feature of just a few selected promoters. Future studies would benefit from applying the bidirectional TAS as described in this study to create potent phenotype specific-viral gene expression systems.
Few endogenous bidirectional promoters have been described until recently. Surprisingly, the human genome survey disclosed a prevalence of bidirectional gene pairs, representing more than 10% of the genes in the genome, whose transcription sites are separated by less than 1000 base pairs [30, 31, 32]. The significance of divergent gene organization is uncertain. Takai and Jones hypothesized that divergent gene organization might stem from the evolution of the human genome from a more compact genome . Alternatively, divergently transcribed gene pairs and their bidirectional promoters may act as unique constructs to coordinate gene expression. Although the structural and functional implications of the widespread occurrence of bidirectional promoters in the human genome are not fully understood, transcription of these clusters of closely spaced genes may contribute to enhance communication and interplay between promoter elements and transcriptional factors . Therefore, the synthetic bidirectional promoter design validated in this study may mimic a well-represented and evolutionarily conserved feature of eukaryotic transcription, providing a structural architecture for their robust performance.
Our study presents an updated TAS with improved suitability for viral vector-based expression systems. This strategy should be useful for constructing powerful gene expression systems based on other weak cell-specific promoters of larger sizes. We have also constructed AVV based on similar expression cassettes and confirmed their improved performance, although these results are not presented in this communication. The LVV based on TAS and the bidirectional promoters as constructed in this study will be of value for the exploration of in vivo gene function and future gene therapy applications.
Lentiviral shuttle vectors used in the current study.
pTYF-1 × SYN-EGFP
SYN with GAL4 binding sites
bidirectional promoter mCMV/SYN
GAL4p65 and EGFP
pTYF-1 × GfaABC1D-EGFP
GfaABC1D with GAL4 binding sites
bidirectional promoter mCMV/GfaABC1D
GAL4p65 and EGFP
Production of lentiviral vectors
The LVV system used in this study is derived from HIV-1 and pseudotyped with the vesicular stomatitis virus coat glycoprotein. Stocks were produced by transient cotransfection of the shuttle plasmids, the packaging vector pNHP, and the envelope plasmid pHEF-VSVG in HEK293FT cells (Invitrogen, Carlsbad, CA, USA). Viral concentration and titration were carried out as described earlier .
Cell culture and in vitro lentiviral vector transduction
The in vitro transduction experiments were carried out in neurone-derived rat pheochromocytoma PC12 cell line (ATCC, No. CRL-1721) and 1321N1 glial cell line from human brain astrocytoma (ECACC, No. 86030402). PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS and 5% horse serum. 1321N1 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. Cells were split one day prior to transfection and plated in 24-well plates at a cell density of 5 × 104 per well. After overnight incubation, cells were transduced with lentiviral vectors in the presence of polybrene (8 ug/ml). Cells were then washed in PBS and were cultured in DMEM for a further 48 hrs.
In vivo lentiviral vector transduction into the rat hypoglossal motor nucleus
Lentiviral vectors were stereotaxically injected into the hypoglossal motor nucleus of male Wistar rats (250–300 g). All procedures were carried out according to the Home Office animals Scientific Procedures Act 1986, UK. Animals were deeply anaesthetized with an intramuscular injection of ketamine (60 mg/kg) and medetomidine (250 μg/kg). They were placed in a stereotaxic head holder and the caudal dorsal medulla was exposed through a midline incision in the dorsal neck. A total of six microinjections of viral vector were made bilaterally at the level of the calamus scriptorius and 400 μm rostral and caudal to it, 300–500 μm from the midline and 450–550 μm ventral to the dorsal surface of the medulla as described previously . The injection rate was 0.5 μl/min and the needle was allowed to remain in situ for 5 min before being slowly retracted at the end of each injection. To allow for direct comparison, we set the dose for each virus for one rat as 106 infectious units. At 7 days after injection, rat brain stems were collected. Three rats were used for each virus. Frozen coronal sections of each brain were cut at 40 um thickness and used for imaging.
Frozen coronal transverse sections were cut at 40 μm thickness and free-floating sections were washed 3 times for 20 min in 0.1 M PBS at pH 7.4 containing 0.2% Triton X-100, then blocked with 5% normal horse serum (NHS) in PBS for 1 h. Sections were then incubated overnight with monoclonal antibody against NeuN or GFAP (both from Chemicon International, USA; dilution 1:500). This was followed by 2 hrs incubations in biotinylated donkey-anti mouse F(ab)2 fragments (1: 500, Jackson Immunolabs, PA, USA) and 2% NHS in PBS, then ExtrAvidin-Cy3 in PBS (1: 1000, Sigma). They were collected on gelatincoated slides with non-quenching mounting medium Vectashield (Vector labs, CA, USA). Images were captured using an Inverted Leica Confocal Imaging Spectrophotometer System (TCS-SP2) at 1–2 μm intervals through the thickness of the section. The two channels (EGFP and Cy3) were scanned separately to avoid "bleed" of fluorescence between channels and merged using Leica software.
The work was supported by British Heart Foundation.
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