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Symposium Report |
1 Institute of Bioengineering and Nanotechnology, The Nanos #04–01, Singapore 2 Department of Biological Sciences, National University of Singapore, Singapore
Abstract
Recombinant baculoviruses have been employed as gene delivery vectors for mammalian cells, including neurones, during recent years. The aim of the current study was to develop a new recombinant baculovirus vector capable of enhancing gene expression in neurones. A hybrid promoter constructed by fusing the enhancer of human cytomegalovirus (CMV) immediately early promoter to the human platelet-derived growth factor (PDGF) ß-chain promoter was placed into a baculovirus expression cassette. In cultured neurones, baculovirus vectors containing the hybrid promoter augmented transgene expression up to 100-fold greater than that mediated by titre-matched baculovirus vectors with the PDGF promoter alone. Double immunostaining of tissue sections collected from the striatum and the retina injected with the new baculovirus vector demonstrated its specificity in driving gene expression almost exclusively in neurones, confirming the feasibility of using a tissue-specific promoter in the context of baculovirus vectors to provide cell type-specific transgene expression. The attributes of the new baculovirus vector might have practical implications for gene therapy in the nervous system.
(Received 22 September 2004;
accepted after revision 11 November 2004; first published online 12 November 2004)
Corresponding author S. Wang: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04–01, Singapore 138669. Email: swang{at}ibn.a-star.edu.sg
The baculovirus Autographa californica multiple nuclear polyhedrosis virus with the very strong polyhedrin promoter has been extensively used in insect cell-based recombinant protein production systems. After accommodating a promoter active in mammalian cells, the baculoviruses are able to drive exogenous gene expression in a broad spectrum of mammalian cells, both proliferating and non-proliferating, quiescent cells (Boyce & Bucher, 1996; Ghosh et al. 2002; Kost & Condreay, 2002). In vivo studies have demonstrated the baculovirus-mediated gene expression in the liver (Hofmann et al. 1995; Sandig et al. 1996), skeletal muscle (Pieroni et al. 2001) and pancreas (Ma et al. 2000), as well as the brain (Sarkis et al. 2000; Lehtolainen et al. 2002). As a gene transfer vector, the baculovirus possesses inherited advantages, including no apparent signs of cytopathic effects in mammalian cells, large gene insert capacity, and a simple production procedure (Ghosh et al. 2002; Kost & Condreay, 2002). Being non-mammalian viruses, unmodified wild-type baculoviruses are incapable of replication and expression of viral proteins in mammalian cells (Doller et al. 1983; Hartig et al. 1992), thus significantly reducing the chance of pre-existing humoral and cellular immunity in mammals.
Although the brain is on the list of successfully transfected organs by recombinant baculovirus vectors, usually accommodating a viral promoter, the transfected cell types are mostly non-neuronal cells (Sarkis et al. 2000; Lehtolainen et al. 2002). This is a disappointment in terms of gene therapy for neurodegenerative disease where transduction of neurones is required. Thus far, no research has ever been done using a neurone-specific promoter in baculovirus vectors to achieve targeted expression in neurones. In the current study we report a new recombinant baculovirus vector, namely BV-CMV E/PDGF-luc, constructed by accommodating a hybrid neurone-specific promoter, CMV E/PDGF, to drive the reporter gene expression. CMV E/PDGF is a hybrid neurone-specific promoter constructed recently in our laboratory by fusing a 380-bp fragment of the CMV enhancer 5' to the PDGF-ß promoter (Liu et al. 2004). Its expression efficiency and neuronal specificity in the context of baculovirus vectors were tested in cultured neurones and in the CNS in vivo in this study.
Methods
Recombinant baculovirus vectors were constructed according to the manual of Bac-To-Bac Baculovirus Expression System (Gibco BRL, Life Technologies, USA). Luciferase cDNA under the control of a PDGF promoter, a CMV enhancer, a hybrid CMV enhancer and PDGF promoter, or the CMV enhancer/promoter was inserted into the transfer plasmid pFastBac1 for virus particle propagation in Sf9 insect cells and the corresponding viruses were named as BV-PDGF-luc, BV-CMV E-luc, BV-CMV E/PDGF-luc and BV-CMV E/P-luc, respectively.
In vitro transduction experiments were performed in differentiated C17.2 cells, a multipotent neural stem cell line generated by retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells, and in primary rat cerebellar granule neurones established from the cerebellum of 8-day-old Wistar rats. Cells were infected with baculoviruses at a multiplicity of infection (m.o.i.; ratio of infectious virus particles to cells or number of virus per cell) of 50 in Opti-MEM (Invitrogen, Netherlands) at 37°C for 3 h, after which the virus-containing medium was removed and the cells were fed with fresh medium and maintained at 37°C for 24 h before luciferase activity measurement. Cells were then collected and homogenized by sonication for 10 s on ice in PBS. Samples were then centrifuged at 28 000 g for 1 h at 4°C. Ten µl of the supernatant was used to assay luciferase activity employing a kit from Promega. Measurements were made in a single-well luminometer (Berthold Lumat LB 9501) for 10 s.
For in vivo viral delivery, adult male Wistar rats (weighing 250–320 g) were used. Five µl of 5 x 106 plaque-forming unit (pfu) virus particles was injected stereotaxically into the striatum (anterior to the interaural line, +0.5 mm; lateral to the midsagittal sinus, +3 mm; ventral from the surface of the skull, –5.0 mm). For intravitreous body injection, a syringe with a 30-gauge needle was introduced through the sclera into the vitreous body posterior to the ora seratta, 10 µl of the vitreous body was slowly sucked out, and then 10 µl of the virus particles (107 pfu) was slowly injected back into the posterior chamber. Two days after injections, rats were killed to collect tissues. Frozen coronal sections of tissue samples were cut at 30 µm thickness and used for immunostaining with polyclonal anti-luciferase antibodies (Promega; dilution 1 : 150) or monoclonal antibodies against neurone specific nuclear protein (NeuN) (Chemicon International, USA; dilution 1 : 500). Sections were examined with a Carl Zeiss LSM510 confocal laser scanning microscope.
Results
We first evaluated the efficiency of transduction of BV-CMV E/PDGF-luc in cultured cells, including neuronally differentiated mouse C17.2 neurones and primary rat cerebellar granule neurones. Baculovirus vectors carrying the hybrid CMV E/PDGF promoter mediated significantly higher levels of luciferase expression in C17.2 cells than the control vectors BV-CMV E-luc with the CMV enhancer alone and BV-PDGF-luc, providing 10- to 100-fold enhancement at both day 1 and day 2 (Fig. 1). Improvement in gene expression by BV-CMV E/PDGF-luc was also noticeable in primary rat cerebellar granule neurones (Fig. 2). In particular, 6 days after transduction in these neurones, the gene expression from BV-CMV E/PDGF-luc was even higher than that provided by BV-CMV E/P-luc (Fig. 2), indicating the possibility of using the hybrid promoter to maintain expression of a transgene in neurones.
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Major functional cells in the nervous system are neurones, the type of cells that carries out the fundamental tasks of the system in receiving, conducting and transmitting signals. Therapeutic protection of these cells is one of the main goals of molecular therapy of neurological disorders. Examples include protection of dopaminergic nigrostriatal neurones in Parkinson's disease (Duvoisin, 1992), cholinergic neurones in Alzheimer's disease (Gomez-Isla et al. 1996), and spinal motoneurones in amyotrophic lateral sclerosis. With this consideration in mind, many efforts have been made to develop gene delivery vectors that are effective in mediating neuronal transduction.
The possibility of using baculovirus vectors for gene transduction in the nervous system was investigated previously in at least two studies. (1) An initial report described an efficient transduction of neural cells in vitro and in vivo by baculovirus vectors containing the human cytomegalovirus (CMV) immediately early gene promoter and enhancer (Sarkis et al. 2000). In primary cell cultures of human embryonic brains, neuro epithelial, neuroblastic and glial cells could be infected. In vivo studies using adult nude mice demonstrated that mainly astrocytes, and only a few neurones, were transduced in baculovirus vector-injected brains. (2) Another study, again using the CMV promoter and enhancer, aimed to examine the cell type-specificity of baculovirus-mediated gene expression in the brain and identified cuboidal epithelial cells of the choroids plexus as the main target cells, with modest gene expression being detected in endothelial cells but very limited or no expression found in other types of brain cells, including neurones and astrocytes (Lehtolainen et al. 2002).
With the assistance of an enhanced neurone-specific promoter, we demonstrated that baculovirus vectors could be modified to mediate gene expression effectively in neurones. Combination of a viral enhancer/promoter element and a cellular promoter has been evaluated in previous studies, with several of them showing improved activity of a cellular promoter. In one of such studies using plasmid vectors, enhancers and promoters from muscle-specific genes were substituted for or combined with the CMV enhancer/promoter and one of these chimeric vectors offered an expression level up to twice that of the parental plasmid (Barnhart et al. 1998). An adeno-associated virus (AAV)-2 expression cassette that uses the CMV enhancer/promoter in combination with a 1.2-kb human skeletal actin promoter increased transgene expression in the muscle significantly, providing a therapeutic range of expression of coagulation factor IX with a 2- to 4-fold lower vector dose (Hagstrom et al. 2000). The most widely used hybrid promoter is probably the CMV enhancer/chicken 
-actin promoter (CAG), which can improve expression in several tissues (Niwa et al. 1991; Xu et al. 2001). The CMV enhancer also stimulated the elongation factor (EF)-1 promoter and the ubiquitin promoter for increased levels of transgene expression (Kobayashi et al. 1997; Yew et al. 2001). The improved neuronal transfection activity of the hybrid CMV E/PDGF promoter has been verified in plasmid DNA vectors in our previous study (Liu et al. 2004). The current study further confirmed the feasibility of using the CMV enhancer to positively stimulate a neurone-specific promoter in the context of a baculovirus expression cassette for improved expression of a reporter gene in the cultured neurones and in the brain. The mechanism underlying the enhancement is not clearly understood yet. We can only speculate that transcriptional factors and auxiliary proteins attracted by the CMV enhancer may interact with those attracted by the PDGF promoter to generate a synergistic action that favours gene expression (Khachigian et al. 1995; Rafty & Khachigian, 1998).
The hybrid approach adopted in the current study has enhanced the transcriptional activity of the PDGF promoter. But it might not be universally applicable to other promoters. We have tried and failed to improve the transcriptional activity of the neuron-specific enolase (NSE) promoter with this method (authors' unpublished observations). In one of the previous efforts that tested 19 different gene regulatory elements produced by combination of a muscle-specific promoter with the CMV enhancer/promoter, only one offered a significant increase in transgene expression compared to a control (Barnhart et al. 1998). The placement of the CMV enhancer sequence relative to a cellular promoter may vary in location, orientation and/or number, leading to hybrid promoters with varying degrees of activities. Rational design to enhance the transcriptional activity of a specific promoter by this hybrid approach requires a clear view of molecular mechanisms underlying the aforementioned successful case studies. Analysis of the recognition of transcriptional regulation elements by RNA polymerase, transcription factors and auxiliary proteins in our hybrid promoter may provide a clue for developing such rational design strategies.
The wide anatomical distribution of affected neurones in certain neurological disorders and the relatively inaccessible neuronal location in others make direct local delivery of therapeutic agents problematic and present obstacles to effective gene therapy of the disorders. Several virus vectors seem able to override the problems by taking advantage of the natural process of axonal transport. This process starts from endocytosis of extracellular substances. In the case of axonal endocytosis, axon terminals are used as ports of entry to take up the substances. The incorporated substances are then transported in axoplasm to the cell body. The viruses capable of doing so include adenovirus (Soudais et al. 2001; Peltekian et al. 2002), adeno-associated virus (Kaspar et al. 2002) and herpes virus (Breakefield & DeLuca, 1991; Jin et al. 1996). However, the application of these viruses as gene delivery vectors is hampered by certain inherited disadvantages, including the small insert size of AAV (Jooss & Chirmule, 2003) and immune and inflammatory responses elicited by the herpes simplex virus (HSV) and adenovirus (McMenamin et al. 1998; Lawrence et al. 1999). We have recently demonstrated that baculoviruses are able to migrate by axonal transport to cell bodies, resulting in transgene expression in projection neurones (Li et al. 2004). We are currently investigating whether baculovirus-derived vectors accommodating the enhanced neurone-specific promoter reported in this study may prolong transgene expression in the brain.
In conclusion, we have developed a baculovirus expression cassette harbouring a hybrid CMV E/PDGF promoter and confirmed its specificity in neurones. The hybrid promoter was obviously superior to its original promoter element in terms of driving gene expression in neurones, offering a means to achieve meaningful transgene expression required for gene therapy of neurodegenerative disorders and functional studies of neurones.
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Acknowledgements
This work was supported by the Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), Singapore. The authors thank Ms X.Y. Ma and Dr X. Wang for their technical assistance.
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