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Targeting adenoviral transgene expression to neurons
Molecular and Cellular Neuroscience 39 (2008) 411–417
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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m c n e
Targeting adenoviral transgene expression to neurons Karen Sims, Zubair Ahmed, Ana Maria Gonzalez, Martin L. Read, Lisa Cooper-Charles, Martin Berry, Ann Logan ⁎ Molecular Neuroscience Group, School of Clinical and Experimental Medicine, Institute of Biomedical Research (West), University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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Article history: Received 14 March 2008 Revised 14 July 2008 Accepted 16 July 2008 Available online 3 August 2008 Keywords: CNS neurons CNS injury Adenovirus Synapsin-1 promoter Woodchuck post-transcriptional regulatory element
a b s t r a c t Adenovirus (Ad) is an efficient and safe vector for CNS gene delivery since it infects non-replicating neurons and does not cause insertional mutagenesis of host cell genomes. However, the promiscuous Ad CAR receptor targets cells non-specifically and activates a host immune response. Using Ad5 containing an expression cassette encoding the gene for green fluorescent protein, gfp, regulated by the neuron specific promoter synapsin-1 and the woodchuck post-transcriptional regulatory element (WPRE), we demonstrate efficient, prolonged and promoter-restricted gfp expression in neurons of mixed primary adult rat dorsal root ganglion (DRG) and retinal cell cultures. We also demonstrate restricted gfp expression in DRG neurons after direct injections of Ad5 containing the synapsin-1gfp/WPRE construct into L4 DRG in vivo, while Ad5 CMVgfp transfected both DRG glia and neurons. Moreover, since the effective titres of delivered Ad5 are reduced with this neuron specific promoter/WPRE expression cassette, the viral immune challenge should be attenuated when used in vivo. © 2008 Elsevier Inc. All rights reserved.
Introduction Many axotomised neurons die and are not replaced after central nervous system (CNS) injury, resulting in permanent loss of function. Moreover, the severed axons of surviving neurons do not regenerate because of the limited availability of stimulatory neurotrophic factors (NTF) and the expression of axon growth inhibitory molecules in both the wound and degenerating projections (Berry et al., 1999, 2008). However, neuron survival and axon regeneration are promoted by delivery of either NTF, or their genes (ntf) (Berry et al., 2001a; Logan et al., 2006; Lykissas et al., 2007). Targeting the expression of therapeutic genes to neurons is an essential requirement for treatments aimed at promoting CNS axon regeneration and has added value in reducing: (1), titres of delivered infective particles; (2), bystander effects of transfected non-neuronal cells (NNC — glia, fibroblasts, endothelial cells, etc); and (3), immune responses, providing safer and prolonged transgene expression (Berry et al., 2001b). Previous attempts at gene therapy in the two systems have been disappointing (Verhaagen et al., 1995–1996; Berry et al., 2001a,b), and our present experimental aim is to design and test an improved reporter gene construct that utilises gene regulatory elements to specifically target transgene expression to neurons for eventual use in gene therapy protocols. Importantly, in this study, we have used Ad5 to transfect mixed primary cultures containing adult retinal ganglion ⁎ Corresponding author. Fax: +44 121 414 8867. E-mail address: [email protected] (A. Logan). URL: http://www.medsciences.bham.ac.uk/staff/medicine/logana.htm (A. Logan). 1044-7431/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.07.020
cells (RGC) and adult dorsal root ganglion neurons (DRGN) isolated from adult rat retina and dorsal root ganglia (DRG), respectively. Furthermore we have evaluated neuronal targeting of transgenes after direct Ad5 injection into DRG in vivo. Most neuron specific promoters are either too large, and/or induce relatively weak gene expression (Glover et al., 2002; Boulos et al., 2006). Nonetheless, exclusive neuronal gene expression is conferred by the Synapsin-1 (495 bp Syn-1) promoter in vitro and in vivo and, because of its relatively small size, is suitable for incorporation into small vector systems. Syn-1 is a phosphoprotein that regulates synaptic vesicle formation (Schoch et al., 1996), and has a high level of early transcription in primary hippocampal neurons (Kügler et al., 2001) through a sequence containing a neuron restrictive silencer element (NRSE) region (Kügler et al., 2001). NNC contain a neuron restrictive silencer factor (NRSF, also termed REST) that binds to the NRSE present in many neuron-specific genes and blocks inappropriate gene expression (Millecamps et al., 1999; Lietz et al., 2003). The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) has multiple post-transcriptional roles, including extension of the RNA poly-A tail (Glover et al., 2002). Incorporating WPRE into an Adenoviral (Ad) expression cassette, combined with either the universal cytomegalovirus promoter (CMV), or the Syn-1 promoter, increases the intensity and duration of gfp expression by up to 3-fold in hippocampal cultures (Glover et al., 2002). Furthermore, the addition of the WPRE to the Syn-1 expression cassette maintains specificity of expression to hippocampal neurons in mixed primary cultures (Glover et al., 2002), and drives transcription for up to 9 m after injection into the dentate gyrus of the hippocampus (Glover et al., 2003).
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This is the first study to show targeted gene expression in both adult DRGN and RGC using gene regulatory elements; two important neuronal targets for NTF therapies after spinal cord and optic nerve injuries, respectively. Specifically, we have compared neuron specific Ad-mediated gfp expression regulated by the Syn-1 promoter (Ad5Syn-1.gfp), with that of the CMV promoter (Ad5CMV.gfp) in mixed primary DRG and retinal cultures and evaluated increased, prolonged and restricted transgene expression in DRGN and RGC after inclusion of WPRE into the expression construct (Ad5Syn-1.gfp. WPRE). We have also evaluated the neuronal targeting of gfp expression after direct injection of Ad5Syn-1.gfp.WPRE and Ad5CMV. gfp.WPRE into L4 DRG in vivo.
In DRG cultures infected with Ad5Syn-1.gfp.WPRE, DRGN expression of GFP increased from 63 ± 18% at 5 d, to 93 ± 14% at 20 d (Figs. 4A, B), but GFP was not expressed in NNC (Figs. 4A, B). As well as enhancing the duration of transgene expression in DRGN, a significantly higher frequency of DRGN expressed GFP when infected
Results Syn-1 and CMV promoter-mediated neuronal specificity of transgene expression in PC12 cells and in mixed DRG and retinal cultures To evaluate promoter-mediated neuronal restriction of Ad5 transgene expression, we examined gfp expression 5 d after delivery of Ad5Syn-1.gfp and Ad5CMV.gfp to both primary DRG and retinal cell cultures and to differentiated PC12 cells (a neuron-like rat pheochromocytoma cell line that stops dividing and undergoes terminal differentiation when treated with nerve growth factor, NGF). The efficacy of Syn-1 promoter-regulated GFP expression was first tested in NGF-differentiated PC12 cells (PC12D) that express the Syn-1 promoter (Romano et al., 1987; Stefanis et al., 2001). Five days after infection of PC12D cells with Ad5Syn-1.gfp, significantly more cells expressed GFP (p b 0.001) than after Ad5CMV.gfp infection, indicating high Syn-1 promoter activity in this neuron-like cell line (Fig. 1A). Morphologically defined RGC in primary retinal cultures (Fig. 2A) infected with Ad5Syn-1.gfp exclusively expressed GFP (Figs. 1B, 2B) but, after infection with Ad5CMV.gfp, non-RGC neurons (NRN) and non-neuron cells (NNC) also expressed GFP (Figs. 1B, 2C). The similar frequencies of GFP-expressing RGC using the Ad5Syn-1.gfp and Ad5CMV.gfp. cassettes suggested comparable transfection efficiencies and strengths of Syn-1 and CMV promoter drive of gfp in this neuron type. Morphologically defined DRGN in primary DRG cultures (Fig. 3A) infected with Ad5Syn-1.gfp exclusively expressed GFP after 5 d (Figs. 1C, 3B) but, after Ad5CMV.gfp transfection, NNC also expressed GFP (Figs. 1C, 3C), showing that Ad5Syn-1.gfp restricts transgene expression specifically to DRGN in mixed DRG cultures. Compared to Ad5CMV.gfp, DRG cultures infected with Ad5Syn-1.gfp had a significantly lower frequency (p b 0.001) of GFP-expressing DRGN (Fig. 1C), illustrating that the efficiency of Syn-1 in promoting gfp expression in DRGN is less than that of CMV. Effect of the WPRE on frequency and duration of GFP expression in DRGN We next counted the numbers of DRGN and NNC expressing GFP at 5 d and 20 d after the incorporation of WPRE into the Ad5 cassette. The expression frequencies of Ad5Syn-1.gfp and Ad5CMV.gfp either with, or without WPRE were compared in mixed DRG cultures. At 5 d, Ad5Syn-1.gfp expressed GFP exclusively in DRGN at a frequency of 46 ± 18% (Fig. 4A), a frequency which remained unchanged at 20 d (41 ± 35% — Fig. 4B). Thus, the frequency of DRGN expressing GFP was maintained over time using Ad5Syn-1.gfp. After transfecting cultures with Ad5CMV.gfp, 90 ± 16% DRGN and 100% NNC expressed GFP after 5 d (Fig. 4A) but, by 20 d, DRGN and NNC frequencies were reduced to 71 ± 33% and 60 ± 32%, respectively (Fig. 4B). Compared with Ad5Syn-1.gfp, the significantly high frequency (p b 0.001) of gfp expression in DRGN obtained with Ad5CMV.gfp at 5 d persisted until 20 d (Fig. 4A).
Fig. 1. Comparison of frequencies of cells expressing GFP after transfection with AdSyn1.gfp and AdCMV.gfp 5 d previously. Significantly higher frequency of cells expressed GFP with Ad5Syn-1.gfp compared with the Ad5CMV.gfp, in NGF-differentiated PC12 cells (A). Significantly higher frequency of cells expressed GFP with Ad5Syn-1.gfp in RGC compared to NRN/NNC (B) and in DRGN compared with NNC (C). (⁎⁎p b 0.01; ⁎⁎⁎p b 0.001; n = 9 for infected cultures; n = 6 for non-infected controls).
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Fig. 2. Brightfield, fluorescent and merged images of 5 d retinal cultures to show the cellular localisation of GFP expression after infection with Ad5Syn-1.gfp (B) and Ad5CMV.gfp (C). RGC were identified morphologically under brightfield by size and the presence of neurites (A). (Arrowheads = NRN/NNC expressing GFP; thin arrows = RGC expressing GFP; thick arrows = RGC not expressing GFP; Scale bar = 20 μm).
with Ad5Syn-1.gfp.WPRE compared to Ad5Syn-1.gfp at 20 d (p b 0.001) (Fig. 4B). By contrast, in DRG cultures infected with Ad5CMV.gfp.WPRE, the frequencies of DRGN and NNC expressing GFP were relatively stable over time (Figs. 4A, B). There was a significantly higher (p b 0.01) number of DRGN expressing GFP after infection with Ad5CMV.gfp.WPRE (92 ±16%) than with Ad5Syn-1.gfp.WPRE (63±18%) at 5 d (Fig. 4A) but, by 20 d, expression profiles were similar (Ad5Syn-1. gfp.WPRE — 93±14%; Ad5CMV.gfp.WPRE — 94 ±12%). With Ad5CMV. gfp.WPRE, GFP expression was maintained in almost all NNC, so that the frequency was 100 ±0% at 5 d and 97 ±7% at 20 d. Neuron specificity of GFP expression was maintained throughout the experimental period with the Ad5Syn-1.gfp.WPRE construct. Neuronal targeting of gfp expression after in vivo injection of Ad5Syn-1.gfp.WPRE into L4 DRG Immunohistochemistry for GFP in sections of DRG directly injected with Ad5Syn-1.gfp.WPRE revealed that GFP expression was restricted to DRGN (Fig. 5A) while no immunoreactivity was observed in contralateral control sections of the same DRG (Fig. 5B). High power magnification confirmed neuronal specificity (arrows) with Ad5Syn-1. gfp.WPRE and demonstrated little or no immunoreactivity in DRG glia (arrowheads) (Fig. 5C). However, in sections of DRG injected with Ad5CMV.gfp.WPRE, GFP expression was observed in both neurons (arrows) and DRG glia (arrowheads) (Fig. 5D) while no immunoreactivity was observed in contralateral controls (Fig. 5E). High power
magnification confirmed that immunoreactivity was present in both neurons (arrows) and DRG glia (arrowheads) (Fig. 5F). It was noted that the large diameter DRGN were more refractory to transfection with both Syn-1 and CMV constructs compared to small diameter DRGN. Discussion Vectors encoding therapeutic ntf are commonly delivered to sites of injury in CNS projection tracts. The local expression of NTF entraps growing axons, thereby preventing directed axon extension (Berry et al., 2008) and thus specific targeting of therapeutic ntf to the somata of damaged neurons is mandatory for successful target reinnervation (Berry et al., 2001b). This can be achieved by uptake from lesion sites of vectors encoding therapeutic ntf under neuron-specific promoter control, followed by their retrograde transport to distal neuronal somata where ntf expression occurs to prime neurons for growth and mobilise axon regeneration (Berry et al., 2001a,b). Ad vectors are retrogradely transported in axons (Hermans et al., 1997; Yamashito et al., 2001; Hibbert et al., 2006; Kelkar et al., 2006) and retrograde axonal delivery to axotomised neuronal somata ensures neurontargeted transgene expression. The transgene expression achieved from CMV driven expression cassettes is strong and efficient, but is not cell specific. By contrast, although gene expression regulated by the Syn-1 promoter is relatively weak in some neurons (Glover et al., 2002), it is neuron
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specific, since the NRSE blocks gene expression in NNC (Kügler et al., 2001). Here, we have shown that the Syn-1 promoter drives targeted transgene expression at high frequency in adult RGC and DRGN in mixed primary cultures. Furthermore, incorporation of the WPRE into the vector construct both enhances and sustains strong neuronal transgene expression. Neuron specificity of the Syn-1 promoter and efficiency in driving gfp expression Glover et al. (2002) developed the Ad5Syn-1.gfp construct using 495 bp of the Syn-1 promoter in place of the CMV promoter. They showed that the Ad5Syn-1.gfp vector conferred neuron specific expression both in vitro and in vivo compared to Ad5CMV.gfp, which drove GFP expression in all cell types. We have confirmed the results of Thiel et al. (1991) who showed that PC12D cells express GFP when infected with Ad5Syn-1.gfp, as would be expected of neurons. Here, the 495 bp Syn-1 promoter sequence drove GFP expression in N95% of PC12D cells i.e., at frequencies significantly higher than those achieved with the CMV promoter, suggesting that the former is the stronger promoter for this neuron-like cell line. Both adult DRG and retinal cultures demonstrated DRGN and RGC specificity of reporter gene expression with the Ad5Syn-1.gfp vector. The lack of GFP expression in NNC after Ad5Syn-1.gfp transfection of DRG and retinal cultures is presumably explained by the relatively low strength of the promoter in the presence of NRSE which suppresses expression by interacting with the NRSE region of the Syn-1 promoter
(Kügler et al., 2001; Millecamps et al., 1999). By contrast, over 5 d, the Ad5CMV.gfp vector promoted significantly higher frequency of GFP expression than did Ad5Syn-1.gfp in both NNC and neurons in both mixed cultures. Kügler et al. (2003), showed that Ad5CMV.gfp vector transgene expression was confined almost exclusively to glia after cerebral injection, while the Ad5Syn-1.gfp targeted GFP expression exclusively to neurons. Similarly, Boulos et al. (2006) showed that the CMV promoter led to very strong transgene expression in astrocytes in mixed cortical cell cultures. An important observation of these studies is that the strength of the promoters used to regulate transgene expression varies with neuronal type. Accordingly, RGC and DRGN responded differently to the same constructs, with Syn-1 being a stronger promoter in RGC than in DRGN. Enhanced frequency and duration of transgene expression with the WPRE Since fewer DRGN expressed GFP at 5 d using Ad5.Syn-1.gfp compared to Ad5.CMV.gfp constructs, we tested the ability of the WPRE to enhance and prolong GFP expression. Accordingly, we measured the frequency of cells expressing GFP at 5 d and 20 d after Ad5 infection. Importantly, in DRGN, inclusion of the WPRE into the expression cassettes sustained reporter transgene expression. Both Ad5Syn-1.gfp.WPRE and Ad5CMV.gfp.WPRE vectors promoted an increased frequency of GFP expression in DRG cultures at 20 d compared to those without WPRE by 52% and 24%, respectively. Glover et al. (2002) infected dentate gyrus neurons with Ad5Syn-1.gfp.WPRE and also
Fig. 3. Brightfield, fluorescent and merged images of 5 d DRG cultures to show the cellular localisation of GFP expression after infection with Ad5Syn-1.gfp (B) and Ad5CMV.gfp (C). DRGN were identified morphologically under brightfield by size and the presence of neurites (A). (Arrowheads = NNC expressing GFP; thin arrows = DRGN expressing GFP; thick arrows = DRGN not expressing GFP; Scale bar = 20 μm).
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satellite glia. By contrast, when Ad5.CMV.gfp.WPRE was injected into DRG, gfp expression was apparent in both DRGN and glia. Importantly, all types of DRGN expressed the reporter gene after injection of Ad5.syn1.gfp.WPRE, although the relative levels of gfp expression were lower in the larger neurons. By contrast, the large DRGN seemed completely refractory to transfection with the more promiscuous construct, Ad5. CMV.gfp.WPRE. These observations suggest the applicability of this neuronal targeting strategy. Conclusion In this study, we have evaluated the utility of gene regulatory elements for targeting and enhancing transgene expression in two populations of neurons in primary culture. We demonstrate for the first time that the Ad5Syn-1.gfp construct restricts transgene expression to DRGN and RGC in primary adult DRG and retinal cultures, respectively. Neuron-restricted transgene expression is maintained at a higher frequency for more sustained periods if the gene cassette also incorporates the WPRE regulatory sequence. The in vivo demonstration of targeted reporter gene expression in high numbers of all types of DRGN after direct DRG injection underlines the potential benefits of employing gene regulatory elements for neuronal targeting of transgene constructs. The Ad5Syn-1.gfp.WPRE vector thus represents a good candidate for the delivery of therapeutic NTF to injured CNS neurons. Experimental methods Cell culture
Fig. 4. Comparison of the frequencies of cells expressing GFP in 5 d (A) and 20 d DRG cultures (B) after transfection with Ad5 containing gfp regulated by the Syn-1 and CMV promoters with and without WPRE. After 5 d, more cells expressed GFP after Ad5CMV. gfp.WPRE compared to Ad5Syn-1.gfp.WPRE transfection of DRG mixed cultures (A). After 20 d, the frequency of cells expressing GFP was significantly higher with Ad5Syn-1. gfp.WPRE compared to that with Ad5Syn-1.gfp (B). (⁎⁎p b 0.01; n = 9 for infected cultures; n = 6 for non-infected controls).
showed both neuronal specificity and a high frequency of GFP expression for up to 6 w. These observations have important implications for the development of gene therapy vectors for chronic neuro-degenerating conditions requiring long term ntf therapy. The addition of the WPRE will reduce the requirement for multiple vector injections by sustaining expression for longer periods. Although we have shown proof-of-concept for the use of regulatory sequences like WPRE to promote and prolong transgene expression in neurons, concerns have been raised about the potential oncogenicity of WPRE in relation to the presence of X protein-coding sequences (Kingsman et al., 2005). Thus, WPRE may require modification to eliminate the oncogenic potential of these factors.
DRG and retinae were isolated from 150 g adult male Sprague Dawley rats. L3–L6 DRG were removed bilaterally and dissociated in Neurobasal-A medium (Invitrogen, Paisley, UK) containing 0.1% collagenase (Sigma-Aldrich, Poole, UK) for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. Cells were transferred to NeurobasalA medium supplemented with 0.02% B27 (Invitrogen), 0.0025% 200 mM L-glutamine (Invitrogen) and 0.005% Gentimicin (Invitrogen) and triturated several times. After centrifugation at 120 ×g for 8 min through a 15% BSA gradient, cell pellets were re-suspended in 100 μl of supplemented Neurobasal-A medium and plated at a density of 1500 cells/well on glass coverslips coated with 100 μg/ml of poly-D-lysine (Sigma-Aldrich) and 100 μg/ml of laminin-1 (Sigma-Aldrich) in supplemented Neurobasal-A medium. As well as DRGN, DRG mixed cultures contained satellite glia, Schwann cells, fibroblasts and endothelial cells, collectively referred to as non-neuronal cells (NNC) throughout this study. DRGN were identified by size (N25 μm) and the presence of neurites (Rambourg et al., 1983; Tandrup, 2004). Retinae were dissociated according to the method of Lorber et al. (2002) using the Papain Dissociation System (Worthington Biochemical's, New Jersey, USA). Retinal cells were plated at a density of 106 cells/well (Huettner and Baughman, 1986) on glass coverslips coated with 100 μg/ml of poly-D-lysine and 100 μg/ml of laminin-1 in PBS (Invitrogen). As well as RGC, mixed retinal cultures also contained amacrine cells, interneurons and photoreceptors, collectively termed non-RGC neurons (NRN), and glia, endothelial cells and fibroblasts collectively termed non-neuronal retinal cells (NNRC). RGC were identified by size (N10 μm) (Danias et al., 2002; Kashiwagi et al., 2001) and the presence of neurites as RGC are the only retinal neurons to grow neurites (Marc and Jones, 2002). Cell lines
Targeting transgene expression to DRGN in vivo with Ad5.Syn-1gfp.WPRE The observations made in DRG cultures with the Ad5 constructs were reflected in vivo. Direct injection of DRG with Ad5.syn-1.gfp.WPRE led to rapid targeted reporter gene expression in neurons that was clearly detected at 5 d, with little or no expression seen in the surrounding
PC12 cells (ATCC, Middlesex, UK) were grown at 37 °C in 25 ml RPMI 1640 (Gibco) supplemented with 10% horse serum (Gibco) and 5% FCS (Gibco). The cells were plated at 50,000 cells/ml on 200 μg/ml collagen type 1 (Upstate, Dundee, UK) and 20 μg/ml poly-D-lysine. PC12 cells were differentiated (PC12D) with daily feeds of 100 ng/ml of NGF (Caltech
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Fig. 5. Immunohistochemistry to localise GFP in sections of DRG 5 d after injection of Ad5Syn-1.gfp.WPRE (A–C) and Ad5CMV.gfp.WPRE (D–F) in vivo. (A) GFP immunoreactivity was only observed in neurons, (B) no immunoreactivity was present in contralateral controls and (C) high power confirms that GFP immunoreactivity is absent in DRG glia but present in neurons in Ad5Syn-1.gfp.WPRE injected DRG. In contrast, injection of Ad5CMV.gfp.WPRE into DRG showed (D) GFP immunoreactivity in both DRG neurons and glia, (E) no immunoreactivity was present in contralateral controls and (F) high power confirms that GFP immunoreactivity was present in both DRG neurons and glia. Scale bar in A–F = 20 μm. (Arrows - DRGN; arrowheads - glia).
Biosystems, CA, USA) per well for 10 d to prevent apoptosis and promote neurite outgrowth (Thiel et al., 1991; Okamoto et al., 1999). Viruses Glover et al. (2002) developed composite expression cassettes comprising 495 bp of the Syn-1 promoter with and without 800 bp of the WPRE. The cassette contained the gfp reporter gene inserted into the multiple cloning site (MCS). The Ad5 constructs comprised either the CMV, or the Syn-1 promoter, the gfp reporter gene (Ad5CMV.gfp and Ad5Syn-1. gfp., respectively) and WPRE (Ad5CMV.gfp WPRE and Ad5Syn-1.gfp. WPRE, respectively) (vectors were donated by Dr James Uney, University of Bristol, UK). Cell infection Cells were infected with Ad5 at a concentration of 500 MOI (multiplicities of infection), and studied after 5 d and 20 d. Cells in 200 μl of DMEM (Gibco) with 2% Foetal Bovine Serum (FBS) had Ad5 added and were incubated at 37 °C. After 2 h, a further 500 μl of DMEM with 2% FBS were added and the cells incubated for 5 d and 20 d. Control wells contained no virus, but had the same number of media changes. The controls were used to set fluorescent background level at zero. Injection of DRG in vivo Adult Sprague Dawley rats (200–250 g) were anaesthetised and the L4 DRG was exposed. 2 μl of either Ad5Syn-1.gfp.WPRE, or Ad5CMV. gfp.WPRE was injected into the L4 DRG and animals were returned to their cages. After 5 d, rats were killed and intracardially perfused using 4% paraformaldehyde. Tissues were removed, cryoprotected in a graded series of sucrose and blocked up in OCT embedding medium (Raymond A. Lamb, East Sussex, UK). 10 μm thick sections were cut on a cryostat (Bright Instrument Company, Huntingdon, UK) and collected onto glass slides prior to immunohistochemistry for GFP. Immunohistochemistry for gfp DRG sections were thawed, post-fixed in 100% methanol for 1 min and washed in phosphate buffered saline (PBS). Sections were then permeabilised in Triton X-100 for 10 min, blocked in 4% normal goat
serum and incubated with 1:500 diluted rabbit polyclonal anti-GFP (Abcam, Cambridge, UK) overnight in a humidified chamber at 4 °C. Sections were then washed in several changes of PBS and incubated with a biotin-labelled anti-rabbit antibody and GFP immunoreactivity was visualised using the Vectastain ABC kit (Vector Labs, Peterborough, UK) following the manufacturer's instructions. Acknowledgments We would like to thank Dr James Uney, University of Bristol, for providing the adenoviral vectors used in this study and Dr. Barbara Lorber, University of Birmingham, for the technical assistance with GFP immunohistochemistry. We would also like to thank the BBSRC (grant number BB/C50466X/1) for funding this project. References Berry, M., Butt, A., Logan, A., 1999. Cellular responses to penetrating CNS injury. In: Berry, M., Logan, A. (Eds.), CNS Injury: Cellular Responses and Pharmacological Strategies. CRC Press LLC, pp. 1–18. Berry, M., Gonzalez, A.M., Clarke, W., Greenlees, L., Barrett, L., Tsang, W., Seymour, L., Bonadio, J., Logan, A., Baird, A., 2001a. Sustained effects of gene-activated matrices after CNS injury. Mol. Cell Neurosci. 17, 706–716. Berry, M., Barrett, L., Seymour, L., Baird, A., Logan, A., 2001b. Gene therapy for central nervous system repair. Curr. Op. Mol. Ther. 3, 338–349. Berry, M, Ahmed, A, Lorber, B, Logan, A., 2008. Regeneration of axons in the visual system. Restor. Neurol. Neurosci. 28, 1–28. Boulos, S., Meloni, B.P., Arthur, P.G., Bojarski, C., Knuckey, N.W., 2006. Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures. Brain Res. 1102, 27–38. Danias, J., Shen, F., Goldblum, D., Chen, B., Ramos-Esteban, J., Podos, S.M., Mittag, T., 2002. Cytoarchitecture of the retinal ganglion cells in the rat. Invest. Opthalmol. Vis. Sci. 43, 587–594. Glover, C., Bienemann, A., Heywood, D., Cosgrave, A., Uney, J., 2002. Adenoviral mediated, high level, cell specific transgene expression: a Syn-1.WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol. Ther. 5, 509–516. Glover, C., Bienemann, A., Hopton, M., Harding, T.C., Kew, J.N., Uney, J., 2003. Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used. J. Gene Med. 5, 554–559. Hermans, W.T.J.M.C., Giger, R.J., Holtmaat, A.J.G.D., Dijkhuizen, P.A., Houweling, D.A., Verhaagen, J., 1997. Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS. J. Neuro. Meth. 71, 85–98. Hibbert, A.P., Kramer, B.M., Miller, F.D., Kaplan, D.R., 2006. The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons. Mol. and Cell. Neurosci. 32, 387–402.
K. Sims et al. / Molecular and Cellular Neuroscience 39 (2008) 411–417 Kashiwagi, K., Iizuka, Y., Araie, M., Suzuki, Y., Tsukahara, S., 2001. Effects of retinal glial cells on isolated rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 42, 2686–2694. Kelkar, S., De, B.P., Gao, G., Wilson, J.M., Crystal, R.G., Leopold, P.L., 2006. A common mechanism for cytoplasmic dyenin-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes. J. Virol. 80, 7781–7785. Kingsman, S.M., Mitrophanous, K., Olsen, J.C., 2005. Potential oncogene activity of the Woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther. 12, 3–4. Kügler, S., Meyn, L., Holzmller, H., Gerhardt, E., Isenmann, S., Schulz, J., Bähr, M., 2001. Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors. Mol. Cell. Neurosci. 17, 78–96. Kügler, S., Killic, E., Bähr, M., 2003. Human synapsin-1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347. Lietz, M., Hohl, M., Thiel, G., 2003. RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-specific DNA binding site. Eur. J. Biochem. 270, 2–9. Logan, A., Ahmed, Z., Baird, A., Gonzalez, A.M., Berry, M., 2006. Neurotrophic factor synergy is required for neuronal survival and disinhibited axon regeneration after CNS injury. Brain 129, 490–502. Lorber, B., Berry, M., Logan, A., Tonge, D., 2002. Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro. Mol. Cell. Neurosci. 21, 301–311. Lykissas, M.G., Batistatou, A.K., Charalabopoulous, K.A., Beris, A.E., 2007. The role of neurotrophins in axonal growth, guidance and regeneration. Curr. Neurovasc. Res. 4, 143–151. Marc, R.E., Jones, B.W., 2002. Molecular phenotyping of retinal ganglion cells. J. Neurosci. 15, 413–427.
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Millecamps, S., Kiefer, H., Navarro, V., Geoffroy, M.-C., Robert, J.-J., Finiels, F., Mallet, J., Barkats, M., 1999. Neuron-restrictive silencer elements mediate neuron specificity of adenoviral gene expression. Nature Biotech 17, 865–869. Okamoto, S.-I., Sherman, K., Lipton, S., 1999. Absence of binding activity of neuron restrictive silencer factor is necessary, but not sufficient for transcription of NMDA receptor subunit type I in neuronal cells. Mol. Brain Res. 74, 44–54. Rambourg, A., Clermont, Y., Beaudet, A., 1983. Ultrastructural features of six types of neurons in rat dorsal root ganglia. J. Neurocytol. 12, 47–66. Romano, C., Nichols, R.A., Greengard, P., Greene, L.A., 1987. Synapsin I in PC12 cells: I. Characterization of the phosphoprotein and effect of chronic NGF treatment. J. Neurosci. 7, 1294–1299. Schoch, S., Cibelli, G., Thiel, G., 1996. Neuron-specific gene expression of synapsin I. J. Biol. Chem. 271, 3317–3323. Stefanis, L., Kholodilov, N., Rideout, H.J., Burke, R.E., Greene, L.A., 2001. Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells. J. Neurochem. 76, 1165–1176. Tandrup, T., 2004. Estimates of number and size of rat dorsal root ganglion cells in studies of structure and cell survival. J. Neurocytol. 33, 173–192. Thiel, G., Greengard, P., Südhof, T., 1991. Characterisation of tissue-specific transcription by the human synapsin I gene promoter. PNAS 88, 3431–3435. Verhaagen, J., Hermens, W.T., Dijkhuizen, P.A., Holtmaat, A.J., Gispen, W.H., 19951996. Use of viral vectors to promote neuroregeneration. Clin Neurosci. 3, 275–283. Yamashito, S., Mita, S., Arima, T., Maeda, Y., Kimura, E., Nisheda, Y., Murakami, T., Okado, M., Uchino, M., 2001. Bcl-2 expression b retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice. Gene Ther. 8, 977–986.
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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m c n e
Targeting adenoviral transgene expression to neurons Karen Sims, Zubair Ahmed, Ana Maria Gonzalez, Martin L. Read, Lisa Cooper-Charles, Martin Berry, Ann Logan ⁎ Molecular Neuroscience Group, School of Clinical and Experimental Medicine, Institute of Biomedical Research (West), University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
a r t i c l e
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Article history: Received 14 March 2008 Revised 14 July 2008 Accepted 16 July 2008 Available online 3 August 2008 Keywords: CNS neurons CNS injury Adenovirus Synapsin-1 promoter Woodchuck post-transcriptional regulatory element
a b s t r a c t Adenovirus (Ad) is an efficient and safe vector for CNS gene delivery since it infects non-replicating neurons and does not cause insertional mutagenesis of host cell genomes. However, the promiscuous Ad CAR receptor targets cells non-specifically and activates a host immune response. Using Ad5 containing an expression cassette encoding the gene for green fluorescent protein, gfp, regulated by the neuron specific promoter synapsin-1 and the woodchuck post-transcriptional regulatory element (WPRE), we demonstrate efficient, prolonged and promoter-restricted gfp expression in neurons of mixed primary adult rat dorsal root ganglion (DRG) and retinal cell cultures. We also demonstrate restricted gfp expression in DRG neurons after direct injections of Ad5 containing the synapsin-1gfp/WPRE construct into L4 DRG in vivo, while Ad5 CMVgfp transfected both DRG glia and neurons. Moreover, since the effective titres of delivered Ad5 are reduced with this neuron specific promoter/WPRE expression cassette, the viral immune challenge should be attenuated when used in vivo. © 2008 Elsevier Inc. All rights reserved.
Introduction Many axotomised neurons die and are not replaced after central nervous system (CNS) injury, resulting in permanent loss of function. Moreover, the severed axons of surviving neurons do not regenerate because of the limited availability of stimulatory neurotrophic factors (NTF) and the expression of axon growth inhibitory molecules in both the wound and degenerating projections (Berry et al., 1999, 2008). However, neuron survival and axon regeneration are promoted by delivery of either NTF, or their genes (ntf) (Berry et al., 2001a; Logan et al., 2006; Lykissas et al., 2007). Targeting the expression of therapeutic genes to neurons is an essential requirement for treatments aimed at promoting CNS axon regeneration and has added value in reducing: (1), titres of delivered infective particles; (2), bystander effects of transfected non-neuronal cells (NNC — glia, fibroblasts, endothelial cells, etc); and (3), immune responses, providing safer and prolonged transgene expression (Berry et al., 2001b). Previous attempts at gene therapy in the two systems have been disappointing (Verhaagen et al., 1995–1996; Berry et al., 2001a,b), and our present experimental aim is to design and test an improved reporter gene construct that utilises gene regulatory elements to specifically target transgene expression to neurons for eventual use in gene therapy protocols. Importantly, in this study, we have used Ad5 to transfect mixed primary cultures containing adult retinal ganglion ⁎ Corresponding author. Fax: +44 121 414 8867. E-mail address: [email protected] (A. Logan). URL: http://www.medsciences.bham.ac.uk/staff/medicine/logana.htm (A. Logan). 1044-7431/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.07.020
cells (RGC) and adult dorsal root ganglion neurons (DRGN) isolated from adult rat retina and dorsal root ganglia (DRG), respectively. Furthermore we have evaluated neuronal targeting of transgenes after direct Ad5 injection into DRG in vivo. Most neuron specific promoters are either too large, and/or induce relatively weak gene expression (Glover et al., 2002; Boulos et al., 2006). Nonetheless, exclusive neuronal gene expression is conferred by the Synapsin-1 (495 bp Syn-1) promoter in vitro and in vivo and, because of its relatively small size, is suitable for incorporation into small vector systems. Syn-1 is a phosphoprotein that regulates synaptic vesicle formation (Schoch et al., 1996), and has a high level of early transcription in primary hippocampal neurons (Kügler et al., 2001) through a sequence containing a neuron restrictive silencer element (NRSE) region (Kügler et al., 2001). NNC contain a neuron restrictive silencer factor (NRSF, also termed REST) that binds to the NRSE present in many neuron-specific genes and blocks inappropriate gene expression (Millecamps et al., 1999; Lietz et al., 2003). The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) has multiple post-transcriptional roles, including extension of the RNA poly-A tail (Glover et al., 2002). Incorporating WPRE into an Adenoviral (Ad) expression cassette, combined with either the universal cytomegalovirus promoter (CMV), or the Syn-1 promoter, increases the intensity and duration of gfp expression by up to 3-fold in hippocampal cultures (Glover et al., 2002). Furthermore, the addition of the WPRE to the Syn-1 expression cassette maintains specificity of expression to hippocampal neurons in mixed primary cultures (Glover et al., 2002), and drives transcription for up to 9 m after injection into the dentate gyrus of the hippocampus (Glover et al., 2003).
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This is the first study to show targeted gene expression in both adult DRGN and RGC using gene regulatory elements; two important neuronal targets for NTF therapies after spinal cord and optic nerve injuries, respectively. Specifically, we have compared neuron specific Ad-mediated gfp expression regulated by the Syn-1 promoter (Ad5Syn-1.gfp), with that of the CMV promoter (Ad5CMV.gfp) in mixed primary DRG and retinal cultures and evaluated increased, prolonged and restricted transgene expression in DRGN and RGC after inclusion of WPRE into the expression construct (Ad5Syn-1.gfp. WPRE). We have also evaluated the neuronal targeting of gfp expression after direct injection of Ad5Syn-1.gfp.WPRE and Ad5CMV. gfp.WPRE into L4 DRG in vivo.
In DRG cultures infected with Ad5Syn-1.gfp.WPRE, DRGN expression of GFP increased from 63 ± 18% at 5 d, to 93 ± 14% at 20 d (Figs. 4A, B), but GFP was not expressed in NNC (Figs. 4A, B). As well as enhancing the duration of transgene expression in DRGN, a significantly higher frequency of DRGN expressed GFP when infected
Results Syn-1 and CMV promoter-mediated neuronal specificity of transgene expression in PC12 cells and in mixed DRG and retinal cultures To evaluate promoter-mediated neuronal restriction of Ad5 transgene expression, we examined gfp expression 5 d after delivery of Ad5Syn-1.gfp and Ad5CMV.gfp to both primary DRG and retinal cell cultures and to differentiated PC12 cells (a neuron-like rat pheochromocytoma cell line that stops dividing and undergoes terminal differentiation when treated with nerve growth factor, NGF). The efficacy of Syn-1 promoter-regulated GFP expression was first tested in NGF-differentiated PC12 cells (PC12D) that express the Syn-1 promoter (Romano et al., 1987; Stefanis et al., 2001). Five days after infection of PC12D cells with Ad5Syn-1.gfp, significantly more cells expressed GFP (p b 0.001) than after Ad5CMV.gfp infection, indicating high Syn-1 promoter activity in this neuron-like cell line (Fig. 1A). Morphologically defined RGC in primary retinal cultures (Fig. 2A) infected with Ad5Syn-1.gfp exclusively expressed GFP (Figs. 1B, 2B) but, after infection with Ad5CMV.gfp, non-RGC neurons (NRN) and non-neuron cells (NNC) also expressed GFP (Figs. 1B, 2C). The similar frequencies of GFP-expressing RGC using the Ad5Syn-1.gfp and Ad5CMV.gfp. cassettes suggested comparable transfection efficiencies and strengths of Syn-1 and CMV promoter drive of gfp in this neuron type. Morphologically defined DRGN in primary DRG cultures (Fig. 3A) infected with Ad5Syn-1.gfp exclusively expressed GFP after 5 d (Figs. 1C, 3B) but, after Ad5CMV.gfp transfection, NNC also expressed GFP (Figs. 1C, 3C), showing that Ad5Syn-1.gfp restricts transgene expression specifically to DRGN in mixed DRG cultures. Compared to Ad5CMV.gfp, DRG cultures infected with Ad5Syn-1.gfp had a significantly lower frequency (p b 0.001) of GFP-expressing DRGN (Fig. 1C), illustrating that the efficiency of Syn-1 in promoting gfp expression in DRGN is less than that of CMV. Effect of the WPRE on frequency and duration of GFP expression in DRGN We next counted the numbers of DRGN and NNC expressing GFP at 5 d and 20 d after the incorporation of WPRE into the Ad5 cassette. The expression frequencies of Ad5Syn-1.gfp and Ad5CMV.gfp either with, or without WPRE were compared in mixed DRG cultures. At 5 d, Ad5Syn-1.gfp expressed GFP exclusively in DRGN at a frequency of 46 ± 18% (Fig. 4A), a frequency which remained unchanged at 20 d (41 ± 35% — Fig. 4B). Thus, the frequency of DRGN expressing GFP was maintained over time using Ad5Syn-1.gfp. After transfecting cultures with Ad5CMV.gfp, 90 ± 16% DRGN and 100% NNC expressed GFP after 5 d (Fig. 4A) but, by 20 d, DRGN and NNC frequencies were reduced to 71 ± 33% and 60 ± 32%, respectively (Fig. 4B). Compared with Ad5Syn-1.gfp, the significantly high frequency (p b 0.001) of gfp expression in DRGN obtained with Ad5CMV.gfp at 5 d persisted until 20 d (Fig. 4A).
Fig. 1. Comparison of frequencies of cells expressing GFP after transfection with AdSyn1.gfp and AdCMV.gfp 5 d previously. Significantly higher frequency of cells expressed GFP with Ad5Syn-1.gfp compared with the Ad5CMV.gfp, in NGF-differentiated PC12 cells (A). Significantly higher frequency of cells expressed GFP with Ad5Syn-1.gfp in RGC compared to NRN/NNC (B) and in DRGN compared with NNC (C). (⁎⁎p b 0.01; ⁎⁎⁎p b 0.001; n = 9 for infected cultures; n = 6 for non-infected controls).
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Fig. 2. Brightfield, fluorescent and merged images of 5 d retinal cultures to show the cellular localisation of GFP expression after infection with Ad5Syn-1.gfp (B) and Ad5CMV.gfp (C). RGC were identified morphologically under brightfield by size and the presence of neurites (A). (Arrowheads = NRN/NNC expressing GFP; thin arrows = RGC expressing GFP; thick arrows = RGC not expressing GFP; Scale bar = 20 μm).
with Ad5Syn-1.gfp.WPRE compared to Ad5Syn-1.gfp at 20 d (p b 0.001) (Fig. 4B). By contrast, in DRG cultures infected with Ad5CMV.gfp.WPRE, the frequencies of DRGN and NNC expressing GFP were relatively stable over time (Figs. 4A, B). There was a significantly higher (p b 0.01) number of DRGN expressing GFP after infection with Ad5CMV.gfp.WPRE (92 ±16%) than with Ad5Syn-1.gfp.WPRE (63±18%) at 5 d (Fig. 4A) but, by 20 d, expression profiles were similar (Ad5Syn-1. gfp.WPRE — 93±14%; Ad5CMV.gfp.WPRE — 94 ±12%). With Ad5CMV. gfp.WPRE, GFP expression was maintained in almost all NNC, so that the frequency was 100 ±0% at 5 d and 97 ±7% at 20 d. Neuron specificity of GFP expression was maintained throughout the experimental period with the Ad5Syn-1.gfp.WPRE construct. Neuronal targeting of gfp expression after in vivo injection of Ad5Syn-1.gfp.WPRE into L4 DRG Immunohistochemistry for GFP in sections of DRG directly injected with Ad5Syn-1.gfp.WPRE revealed that GFP expression was restricted to DRGN (Fig. 5A) while no immunoreactivity was observed in contralateral control sections of the same DRG (Fig. 5B). High power magnification confirmed neuronal specificity (arrows) with Ad5Syn-1. gfp.WPRE and demonstrated little or no immunoreactivity in DRG glia (arrowheads) (Fig. 5C). However, in sections of DRG injected with Ad5CMV.gfp.WPRE, GFP expression was observed in both neurons (arrows) and DRG glia (arrowheads) (Fig. 5D) while no immunoreactivity was observed in contralateral controls (Fig. 5E). High power
magnification confirmed that immunoreactivity was present in both neurons (arrows) and DRG glia (arrowheads) (Fig. 5F). It was noted that the large diameter DRGN were more refractory to transfection with both Syn-1 and CMV constructs compared to small diameter DRGN. Discussion Vectors encoding therapeutic ntf are commonly delivered to sites of injury in CNS projection tracts. The local expression of NTF entraps growing axons, thereby preventing directed axon extension (Berry et al., 2008) and thus specific targeting of therapeutic ntf to the somata of damaged neurons is mandatory for successful target reinnervation (Berry et al., 2001b). This can be achieved by uptake from lesion sites of vectors encoding therapeutic ntf under neuron-specific promoter control, followed by their retrograde transport to distal neuronal somata where ntf expression occurs to prime neurons for growth and mobilise axon regeneration (Berry et al., 2001a,b). Ad vectors are retrogradely transported in axons (Hermans et al., 1997; Yamashito et al., 2001; Hibbert et al., 2006; Kelkar et al., 2006) and retrograde axonal delivery to axotomised neuronal somata ensures neurontargeted transgene expression. The transgene expression achieved from CMV driven expression cassettes is strong and efficient, but is not cell specific. By contrast, although gene expression regulated by the Syn-1 promoter is relatively weak in some neurons (Glover et al., 2002), it is neuron
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specific, since the NRSE blocks gene expression in NNC (Kügler et al., 2001). Here, we have shown that the Syn-1 promoter drives targeted transgene expression at high frequency in adult RGC and DRGN in mixed primary cultures. Furthermore, incorporation of the WPRE into the vector construct both enhances and sustains strong neuronal transgene expression. Neuron specificity of the Syn-1 promoter and efficiency in driving gfp expression Glover et al. (2002) developed the Ad5Syn-1.gfp construct using 495 bp of the Syn-1 promoter in place of the CMV promoter. They showed that the Ad5Syn-1.gfp vector conferred neuron specific expression both in vitro and in vivo compared to Ad5CMV.gfp, which drove GFP expression in all cell types. We have confirmed the results of Thiel et al. (1991) who showed that PC12D cells express GFP when infected with Ad5Syn-1.gfp, as would be expected of neurons. Here, the 495 bp Syn-1 promoter sequence drove GFP expression in N95% of PC12D cells i.e., at frequencies significantly higher than those achieved with the CMV promoter, suggesting that the former is the stronger promoter for this neuron-like cell line. Both adult DRG and retinal cultures demonstrated DRGN and RGC specificity of reporter gene expression with the Ad5Syn-1.gfp vector. The lack of GFP expression in NNC after Ad5Syn-1.gfp transfection of DRG and retinal cultures is presumably explained by the relatively low strength of the promoter in the presence of NRSE which suppresses expression by interacting with the NRSE region of the Syn-1 promoter
(Kügler et al., 2001; Millecamps et al., 1999). By contrast, over 5 d, the Ad5CMV.gfp vector promoted significantly higher frequency of GFP expression than did Ad5Syn-1.gfp in both NNC and neurons in both mixed cultures. Kügler et al. (2003), showed that Ad5CMV.gfp vector transgene expression was confined almost exclusively to glia after cerebral injection, while the Ad5Syn-1.gfp targeted GFP expression exclusively to neurons. Similarly, Boulos et al. (2006) showed that the CMV promoter led to very strong transgene expression in astrocytes in mixed cortical cell cultures. An important observation of these studies is that the strength of the promoters used to regulate transgene expression varies with neuronal type. Accordingly, RGC and DRGN responded differently to the same constructs, with Syn-1 being a stronger promoter in RGC than in DRGN. Enhanced frequency and duration of transgene expression with the WPRE Since fewer DRGN expressed GFP at 5 d using Ad5.Syn-1.gfp compared to Ad5.CMV.gfp constructs, we tested the ability of the WPRE to enhance and prolong GFP expression. Accordingly, we measured the frequency of cells expressing GFP at 5 d and 20 d after Ad5 infection. Importantly, in DRGN, inclusion of the WPRE into the expression cassettes sustained reporter transgene expression. Both Ad5Syn-1.gfp.WPRE and Ad5CMV.gfp.WPRE vectors promoted an increased frequency of GFP expression in DRG cultures at 20 d compared to those without WPRE by 52% and 24%, respectively. Glover et al. (2002) infected dentate gyrus neurons with Ad5Syn-1.gfp.WPRE and also
Fig. 3. Brightfield, fluorescent and merged images of 5 d DRG cultures to show the cellular localisation of GFP expression after infection with Ad5Syn-1.gfp (B) and Ad5CMV.gfp (C). DRGN were identified morphologically under brightfield by size and the presence of neurites (A). (Arrowheads = NNC expressing GFP; thin arrows = DRGN expressing GFP; thick arrows = DRGN not expressing GFP; Scale bar = 20 μm).
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satellite glia. By contrast, when Ad5.CMV.gfp.WPRE was injected into DRG, gfp expression was apparent in both DRGN and glia. Importantly, all types of DRGN expressed the reporter gene after injection of Ad5.syn1.gfp.WPRE, although the relative levels of gfp expression were lower in the larger neurons. By contrast, the large DRGN seemed completely refractory to transfection with the more promiscuous construct, Ad5. CMV.gfp.WPRE. These observations suggest the applicability of this neuronal targeting strategy. Conclusion In this study, we have evaluated the utility of gene regulatory elements for targeting and enhancing transgene expression in two populations of neurons in primary culture. We demonstrate for the first time that the Ad5Syn-1.gfp construct restricts transgene expression to DRGN and RGC in primary adult DRG and retinal cultures, respectively. Neuron-restricted transgene expression is maintained at a higher frequency for more sustained periods if the gene cassette also incorporates the WPRE regulatory sequence. The in vivo demonstration of targeted reporter gene expression in high numbers of all types of DRGN after direct DRG injection underlines the potential benefits of employing gene regulatory elements for neuronal targeting of transgene constructs. The Ad5Syn-1.gfp.WPRE vector thus represents a good candidate for the delivery of therapeutic NTF to injured CNS neurons. Experimental methods Cell culture
Fig. 4. Comparison of the frequencies of cells expressing GFP in 5 d (A) and 20 d DRG cultures (B) after transfection with Ad5 containing gfp regulated by the Syn-1 and CMV promoters with and without WPRE. After 5 d, more cells expressed GFP after Ad5CMV. gfp.WPRE compared to Ad5Syn-1.gfp.WPRE transfection of DRG mixed cultures (A). After 20 d, the frequency of cells expressing GFP was significantly higher with Ad5Syn-1. gfp.WPRE compared to that with Ad5Syn-1.gfp (B). (⁎⁎p b 0.01; n = 9 for infected cultures; n = 6 for non-infected controls).
showed both neuronal specificity and a high frequency of GFP expression for up to 6 w. These observations have important implications for the development of gene therapy vectors for chronic neuro-degenerating conditions requiring long term ntf therapy. The addition of the WPRE will reduce the requirement for multiple vector injections by sustaining expression for longer periods. Although we have shown proof-of-concept for the use of regulatory sequences like WPRE to promote and prolong transgene expression in neurons, concerns have been raised about the potential oncogenicity of WPRE in relation to the presence of X protein-coding sequences (Kingsman et al., 2005). Thus, WPRE may require modification to eliminate the oncogenic potential of these factors.
DRG and retinae were isolated from 150 g adult male Sprague Dawley rats. L3–L6 DRG were removed bilaterally and dissociated in Neurobasal-A medium (Invitrogen, Paisley, UK) containing 0.1% collagenase (Sigma-Aldrich, Poole, UK) for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. Cells were transferred to NeurobasalA medium supplemented with 0.02% B27 (Invitrogen), 0.0025% 200 mM L-glutamine (Invitrogen) and 0.005% Gentimicin (Invitrogen) and triturated several times. After centrifugation at 120 ×g for 8 min through a 15% BSA gradient, cell pellets were re-suspended in 100 μl of supplemented Neurobasal-A medium and plated at a density of 1500 cells/well on glass coverslips coated with 100 μg/ml of poly-D-lysine (Sigma-Aldrich) and 100 μg/ml of laminin-1 (Sigma-Aldrich) in supplemented Neurobasal-A medium. As well as DRGN, DRG mixed cultures contained satellite glia, Schwann cells, fibroblasts and endothelial cells, collectively referred to as non-neuronal cells (NNC) throughout this study. DRGN were identified by size (N25 μm) and the presence of neurites (Rambourg et al., 1983; Tandrup, 2004). Retinae were dissociated according to the method of Lorber et al. (2002) using the Papain Dissociation System (Worthington Biochemical's, New Jersey, USA). Retinal cells were plated at a density of 106 cells/well (Huettner and Baughman, 1986) on glass coverslips coated with 100 μg/ml of poly-D-lysine and 100 μg/ml of laminin-1 in PBS (Invitrogen). As well as RGC, mixed retinal cultures also contained amacrine cells, interneurons and photoreceptors, collectively termed non-RGC neurons (NRN), and glia, endothelial cells and fibroblasts collectively termed non-neuronal retinal cells (NNRC). RGC were identified by size (N10 μm) (Danias et al., 2002; Kashiwagi et al., 2001) and the presence of neurites as RGC are the only retinal neurons to grow neurites (Marc and Jones, 2002). Cell lines
Targeting transgene expression to DRGN in vivo with Ad5.Syn-1gfp.WPRE The observations made in DRG cultures with the Ad5 constructs were reflected in vivo. Direct injection of DRG with Ad5.syn-1.gfp.WPRE led to rapid targeted reporter gene expression in neurons that was clearly detected at 5 d, with little or no expression seen in the surrounding
PC12 cells (ATCC, Middlesex, UK) were grown at 37 °C in 25 ml RPMI 1640 (Gibco) supplemented with 10% horse serum (Gibco) and 5% FCS (Gibco). The cells were plated at 50,000 cells/ml on 200 μg/ml collagen type 1 (Upstate, Dundee, UK) and 20 μg/ml poly-D-lysine. PC12 cells were differentiated (PC12D) with daily feeds of 100 ng/ml of NGF (Caltech
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Fig. 5. Immunohistochemistry to localise GFP in sections of DRG 5 d after injection of Ad5Syn-1.gfp.WPRE (A–C) and Ad5CMV.gfp.WPRE (D–F) in vivo. (A) GFP immunoreactivity was only observed in neurons, (B) no immunoreactivity was present in contralateral controls and (C) high power confirms that GFP immunoreactivity is absent in DRG glia but present in neurons in Ad5Syn-1.gfp.WPRE injected DRG. In contrast, injection of Ad5CMV.gfp.WPRE into DRG showed (D) GFP immunoreactivity in both DRG neurons and glia, (E) no immunoreactivity was present in contralateral controls and (F) high power confirms that GFP immunoreactivity was present in both DRG neurons and glia. Scale bar in A–F = 20 μm. (Arrows - DRGN; arrowheads - glia).
Biosystems, CA, USA) per well for 10 d to prevent apoptosis and promote neurite outgrowth (Thiel et al., 1991; Okamoto et al., 1999). Viruses Glover et al. (2002) developed composite expression cassettes comprising 495 bp of the Syn-1 promoter with and without 800 bp of the WPRE. The cassette contained the gfp reporter gene inserted into the multiple cloning site (MCS). The Ad5 constructs comprised either the CMV, or the Syn-1 promoter, the gfp reporter gene (Ad5CMV.gfp and Ad5Syn-1. gfp., respectively) and WPRE (Ad5CMV.gfp WPRE and Ad5Syn-1.gfp. WPRE, respectively) (vectors were donated by Dr James Uney, University of Bristol, UK). Cell infection Cells were infected with Ad5 at a concentration of 500 MOI (multiplicities of infection), and studied after 5 d and 20 d. Cells in 200 μl of DMEM (Gibco) with 2% Foetal Bovine Serum (FBS) had Ad5 added and were incubated at 37 °C. After 2 h, a further 500 μl of DMEM with 2% FBS were added and the cells incubated for 5 d and 20 d. Control wells contained no virus, but had the same number of media changes. The controls were used to set fluorescent background level at zero. Injection of DRG in vivo Adult Sprague Dawley rats (200–250 g) were anaesthetised and the L4 DRG was exposed. 2 μl of either Ad5Syn-1.gfp.WPRE, or Ad5CMV. gfp.WPRE was injected into the L4 DRG and animals were returned to their cages. After 5 d, rats were killed and intracardially perfused using 4% paraformaldehyde. Tissues were removed, cryoprotected in a graded series of sucrose and blocked up in OCT embedding medium (Raymond A. Lamb, East Sussex, UK). 10 μm thick sections were cut on a cryostat (Bright Instrument Company, Huntingdon, UK) and collected onto glass slides prior to immunohistochemistry for GFP. Immunohistochemistry for gfp DRG sections were thawed, post-fixed in 100% methanol for 1 min and washed in phosphate buffered saline (PBS). Sections were then permeabilised in Triton X-100 for 10 min, blocked in 4% normal goat
serum and incubated with 1:500 diluted rabbit polyclonal anti-GFP (Abcam, Cambridge, UK) overnight in a humidified chamber at 4 °C. Sections were then washed in several changes of PBS and incubated with a biotin-labelled anti-rabbit antibody and GFP immunoreactivity was visualised using the Vectastain ABC kit (Vector Labs, Peterborough, UK) following the manufacturer's instructions. Acknowledgments We would like to thank Dr James Uney, University of Bristol, for providing the adenoviral vectors used in this study and Dr. Barbara Lorber, University of Birmingham, for the technical assistance with GFP immunohistochemistry. We would also like to thank the BBSRC (grant number BB/C50466X/1) for funding this project. References Berry, M., Butt, A., Logan, A., 1999. Cellular responses to penetrating CNS injury. In: Berry, M., Logan, A. (Eds.), CNS Injury: Cellular Responses and Pharmacological Strategies. CRC Press LLC, pp. 1–18. Berry, M., Gonzalez, A.M., Clarke, W., Greenlees, L., Barrett, L., Tsang, W., Seymour, L., Bonadio, J., Logan, A., Baird, A., 2001a. Sustained effects of gene-activated matrices after CNS injury. Mol. Cell Neurosci. 17, 706–716. Berry, M., Barrett, L., Seymour, L., Baird, A., Logan, A., 2001b. Gene therapy for central nervous system repair. Curr. Op. Mol. Ther. 3, 338–349. Berry, M, Ahmed, A, Lorber, B, Logan, A., 2008. Regeneration of axons in the visual system. Restor. Neurol. Neurosci. 28, 1–28. Boulos, S., Meloni, B.P., Arthur, P.G., Bojarski, C., Knuckey, N.W., 2006. Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures. Brain Res. 1102, 27–38. Danias, J., Shen, F., Goldblum, D., Chen, B., Ramos-Esteban, J., Podos, S.M., Mittag, T., 2002. Cytoarchitecture of the retinal ganglion cells in the rat. Invest. Opthalmol. Vis. Sci. 43, 587–594. Glover, C., Bienemann, A., Heywood, D., Cosgrave, A., Uney, J., 2002. Adenoviral mediated, high level, cell specific transgene expression: a Syn-1.WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol. Ther. 5, 509–516. Glover, C., Bienemann, A., Hopton, M., Harding, T.C., Kew, J.N., Uney, J., 2003. Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used. J. Gene Med. 5, 554–559. Hermans, W.T.J.M.C., Giger, R.J., Holtmaat, A.J.G.D., Dijkhuizen, P.A., Houweling, D.A., Verhaagen, J., 1997. Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS. J. Neuro. Meth. 71, 85–98. Hibbert, A.P., Kramer, B.M., Miller, F.D., Kaplan, D.R., 2006. The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons. Mol. and Cell. Neurosci. 32, 387–402.
K. Sims et al. / Molecular and Cellular Neuroscience 39 (2008) 411–417 Kashiwagi, K., Iizuka, Y., Araie, M., Suzuki, Y., Tsukahara, S., 2001. Effects of retinal glial cells on isolated rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 42, 2686–2694. Kelkar, S., De, B.P., Gao, G., Wilson, J.M., Crystal, R.G., Leopold, P.L., 2006. A common mechanism for cytoplasmic dyenin-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes. J. Virol. 80, 7781–7785. Kingsman, S.M., Mitrophanous, K., Olsen, J.C., 2005. Potential oncogene activity of the Woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther. 12, 3–4. Kügler, S., Meyn, L., Holzmller, H., Gerhardt, E., Isenmann, S., Schulz, J., Bähr, M., 2001. Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors. Mol. Cell. Neurosci. 17, 78–96. Kügler, S., Killic, E., Bähr, M., 2003. Human synapsin-1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347. Lietz, M., Hohl, M., Thiel, G., 2003. RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-specific DNA binding site. Eur. J. Biochem. 270, 2–9. Logan, A., Ahmed, Z., Baird, A., Gonzalez, A.M., Berry, M., 2006. Neurotrophic factor synergy is required for neuronal survival and disinhibited axon regeneration after CNS injury. Brain 129, 490–502. Lorber, B., Berry, M., Logan, A., Tonge, D., 2002. Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro. Mol. Cell. Neurosci. 21, 301–311. Lykissas, M.G., Batistatou, A.K., Charalabopoulous, K.A., Beris, A.E., 2007. The role of neurotrophins in axonal growth, guidance and regeneration. Curr. Neurovasc. Res. 4, 143–151. Marc, R.E., Jones, B.W., 2002. Molecular phenotyping of retinal ganglion cells. J. Neurosci. 15, 413–427.
417
Millecamps, S., Kiefer, H., Navarro, V., Geoffroy, M.-C., Robert, J.-J., Finiels, F., Mallet, J., Barkats, M., 1999. Neuron-restrictive silencer elements mediate neuron specificity of adenoviral gene expression. Nature Biotech 17, 865–869. Okamoto, S.-I., Sherman, K., Lipton, S., 1999. Absence of binding activity of neuron restrictive silencer factor is necessary, but not sufficient for transcription of NMDA receptor subunit type I in neuronal cells. Mol. Brain Res. 74, 44–54. Rambourg, A., Clermont, Y., Beaudet, A., 1983. Ultrastructural features of six types of neurons in rat dorsal root ganglia. J. Neurocytol. 12, 47–66. Romano, C., Nichols, R.A., Greengard, P., Greene, L.A., 1987. Synapsin I in PC12 cells: I. Characterization of the phosphoprotein and effect of chronic NGF treatment. J. Neurosci. 7, 1294–1299. Schoch, S., Cibelli, G., Thiel, G., 1996. Neuron-specific gene expression of synapsin I. J. Biol. Chem. 271, 3317–3323. Stefanis, L., Kholodilov, N., Rideout, H.J., Burke, R.E., Greene, L.A., 2001. Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells. J. Neurochem. 76, 1165–1176. Tandrup, T., 2004. Estimates of number and size of rat dorsal root ganglion cells in studies of structure and cell survival. J. Neurocytol. 33, 173–192. Thiel, G., Greengard, P., Südhof, T., 1991. Characterisation of tissue-specific transcription by the human synapsin I gene promoter. PNAS 88, 3431–3435. Verhaagen, J., Hermens, W.T., Dijkhuizen, P.A., Holtmaat, A.J., Gispen, W.H., 19951996. Use of viral vectors to promote neuroregeneration. Clin Neurosci. 3, 275–283. Yamashito, S., Mita, S., Arima, T., Maeda, Y., Kimura, E., Nisheda, Y., Murakami, T., Okado, M., Uchino, M., 2001. Bcl-2 expression b retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice. Gene Ther. 8, 977–986.