Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons

1 Dual promoter lentiviral vectors for constitutive and regulated gene expression in neurons Sergio Gascon1, Juan A. Paez-Gomez2, Margarita Diaz-Guerra1, Peter Scheiffele3, Francisco G. Scholl2. 1 Instituto de Investigaciones Biomedicas “Alberto Sols”, CSIC-UAM, Arturo Duperier, 4, 28029-Madrid, Spain. 2 Departamento de Fisiologia Medica y Biofisica, Facultad de Medicina, Universidad de Sevilla, Avda. Sanchez Pizjuan, 4, 41009 Sevilla, Spain. 3 Department of Physiology and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, 630 West 168th Street, P&S 11-511, New York, New York 10032, USA. Correspondence should be addressed to: Francisco G. Scholl, Departamento de Fisiologia Medica y Biofisica, Facultad de Medicina, Universidad de Sevilla, Avda. Sanchez Pizjuan, 4, 41009 Sevilla, Spain. Phone: +34-95 4559867. Fax: +34-95 4551769. E-mail: [email protected] Number of pages (excluding figures): 27 2 Number of figures: 5 plus 1 Supplementary Figure. 3 Abstract Gene transfer methods for efficient co-expression of exogenous proteins in neurons are crucial tools towards the understanding of the molecular basis of the central nervous system. Lentiviruses are retroviral vectors that can transduce a wide variety of cells including differentiated neurons. In this work, we have generated lentiviral vectors containing dual promoters that allow efficient co-expression of exogenous proteins in neurons. We show that insertion of two copies of a human synapsin promoter/WPRE cassette in a single lentiviral vector directs robust co-expression of cDNAs in cultured neurons, while excluding expression in the surrounding glial cells. Furthermore, insertion of the tetracycline-inducible system (Tet-off) controlled by the synapsin promoter results in tightly regulated expression of EGFP when used as a transgene in cultured neurons. Transduction of primary neurons with this inducible system leads to a 100-fold increase in EGFP mRNA levels in the absence of doxycycline. In transduced cultures, EGFP transcription is inhibited within 24 hours upon addition of doxycycline. The viral systems we developed here provide neuron-specific and regulated expression mediated by single lentiviral vectors and will prove valuable tools for the study of neuronal function. Keywords: lentivirus, synapsin promoter, inducible expression, primary neurons. 4 1. Introduction Expression of exogenous proteins in cultured neurons represents an important approach towards the understanding of the molecular basis of neuronal function in normal and pathological states. In addition to the expression of single genes, efficient expression of two cDNAs in the same neuron is often desired in several experimental approaches. For example, many proteins, such as ion channels require co-expression of different subunits for normal function, or often a reporter fluorescent protein needs to be co-expressed to monitorize neuronal morphology or function when assaying the role of an exogenous cDNA. A similar problem arises when systems for regulated expression are used, as most regulated expression systems require the co-delivery in the same cell of the target gene and a transcriptional transactivator or repressor (Yamamoto et al., 2000). Therefore, the generation of molecular tools that allow for the simultaneous delivery of cDNAs into neurons is of critical importance. Viral gene delivery systems are powerful tools for the expression of exogenous cDNAs especially in neuronal cells, as they overcome problems with low efficiency of gene transfer. However, a general challenge for both, constitutive and regulated gene expression from viral vectors is the co-delivery of multiple cDNAs into neurons. In recent years, lentiviral vectors have become a widely used system for the expression of exogenous cDNAs in different cell types in 5 vivo and in vitro, (Lois et al., 2002; Pfeifer et al., 2002; Delenda, 2004). Compared to other gene transfer systems, lentiviral vectors provide several advantages. As retroviral vectors, the viral genome is integrated into the chromosome of the host cell after retrotranscription, allowing for long-term, stable expression of the transgene (Lois et al., 2002; Pfeifer et al., 2002). In contrast to other retroviruses, lentivirus can integrate into the genome of both, dividing and non-dividing cells (Naldini et al., 1996). These properties make lentiviral vectors an excellent vehicle to deliver exogenous genes in postmitotic neurons (Blomer et al., 1997; Wong et al., 2006). Most commonly, lentiviral vectors are pseudotyped with the Vesicular Stomatitis Virus Glycoprotein (VSVG) and therefore infect a broad range of cell types. This low cell type preference represents a challenge when cell type specific expression is desired. For example, in studies on neuron-specific signaling mechanisms it is often essential to avoid infection of surrounding astroglia in tissue or in cell culture. A strategy for circumventing such problems is to insert regulatory elements into the viral vectors that direct cell type specific or inducible gene expression. This is possible due to the relatively large packaging capacity of lentiviral particles, which can accommodate the incorporation of several transcriptional-regulatory units into the viral genome (Azzouz et al., 2002). In addition, the promoter activity within the viral long terminal repeat (LTR) is irreversibly disabled in self-inactivating (SIN) vectors, allowing expression of the transgene to be controlled by the activity of exogenous promoters (Lois et al., 2002). Among the different neuronal 6 promoters analyzed, the human synapsin (hSYN) promoter is very well suited for neuron-specific expression of transgenes in vitro and in vivo due to its small size and high cell type specificity (Kugler et al., 2001; Glover et al., 2002; Nakagawa et al., 2006; Hioki et al., 2007). In this work, we have developed and tested new dual promoter lentiviral vectors for constitutive and regulated neuron-specific expression. First, we show that the insertion of two hSYN promoter/WPRE cassettes into a SIN-lentiviral vector efficiently drives simultaneous expression of two cDNAs in a neuron-specific manner. In a second system, we use expression of the tetracycline-dependent transactivator, tTA, under control of the hSYN promoter to drive inducibleexpression of a transgene delivered on the same lentiviral vector. These dual promoter vectors should facilitate the analysis of neuronal morphology and function in normal and pathological conditions. 2. Materials and methods 2.1. Generation of lentiviral vectors. All dual-gene lentiviral vectors are based on the LenLox3.7 plasmid (generously provided by L. Van Parijs) (Rubinson et al., 2003). LenLox3.7 vector carries a U6 promoter followed by a CMV-WPRE cassette that controls EGFP expression 7 (Rubinson et al., 2003). To insert two copies of the hSYN promoter, two XbaI- HpaI and NsiI-NheI DNA fragments containing a 480 bp region from the distal most 3’ end of the human synapsin promoter were amplified by polymerase chain reaction (PCR) from the vector pMH4-I-hSYN (generously provided by M. Bahr, University of Göttingen, Germany) (Kugler et al., 2001). The amplification fragments were ligated into the LenLox3.7 plasmid from which the U6 and CMV promoters had been removed by XbaI-HpaI and NsiI-NheI digestions, respectively. From this vector, the WPRE sequence was amplified by PCR with NotI-NsiI sites and cloned downstream of the first synapsin promoter to generate the SYN-SYN-GFP vector. The plasmid SYN-DsRed-SYN-GFP (Fig. 1A) was generated by inserting the DsRed sequence obtained by PCR with NotI-XhoI sites from the vector pDsRed-N1 (Clontech) under the control of the first SYNWPRE cassette. One-single synapsin promoter vectors coding for EGFP (SYNGFP) or DsRed proteins (SYN-DsRed) were generated from SYN-DsRed-SYN- GFP by removing the SYN-DsRed or SYN-GFP cassettes using NotI-EcoRI and XbaI-NotI restriction sites, respectively. Inducible lentiviral vectors were originated in several cloning steps from the SYN- DsRed-SYN-GFP vector. First, the TRE inducible promoter from the pTRE vector (Clontech) was amplified by PCR with NsiI-AscI ends and cloned in the SYNSYN-GFP to replace the second hSYN promoter, after digestion with NsiI-AscI. Next, a 1008-bp fragment obtained by PCR from the pTet-Off plasmid (Clontech) 8 containing the tTA coding sequence was cloned in BamHI-NotI sites downstream the hSYN promoter to generate SYN-Tetoff-GFP (Fig. 1B). To construct the SV40-Tetoff-GFP vector, a 441-bp fragment containing the SV40 promoter was inserted in SYN-Tetoff-GFP vector after removal of the hSYN promoter by XbaI digestion. All constructs were verified by DNA sequencing. 2.2 Cell culture Primary culture of embryonic cortical neurons was performed as described (Brewer et al., 1993) with some modifications. Plates were treated with poly-L- lysine (100 µg/ml) and laminin (4 µg/ml) overnight at 37°C before seeding. Cerebral cortices from 18-days old rat embryos (Wistar) were dissected and mechanically dissociated in culture medium (Eagle minimum medium supplemented with 22.2 mM glucose, 0.1 mM glutamine, 5% FBS, 5% donor horse serum and penicillin/streptomycin). Cells were seeded at a density of 50.000 cells/cm2 in the same media and maintained at 37 °C and 5% CO2. 2.3. Lentivirus production and infection of primary cell cultures Recombinant lentiviral particles were produced as previously described (Lois et al., 2002). Briefly, HEK293T cells were triple transfected using the calcium phosphate method with the lentiviral expression vector and two packaging 9 vectors: pCAG-VSVg, a plasmid expressing the VSV-G envelope gene, and pCMVDR8.91, a plasmid expressing the gag/pol genes. The supernatants containing the viral particles were collected 48-60 hours after transfection and concentrated using filtration columns (Centricon Plus-20, molecular weight cutoff 100 kDa; Millipore) as described (Reiser, 2000). Viral titers were determined by serial dilution on PC12 pheochromocytoma cells by FACScan analysis as the synapsin promoter is active in this cell line (Thiel et al., 1991). Primary cell cultures were transduced at the indicated DIV by adding concentrated lentivirus at a multiplicity of infection (m.o.i.) of 0.5 to the growing media. When indicated, doxycycline (Calbiochem) was added at 2 µg/ml to the culture media. 2.4. Fluorescence activated cell sorting analysis PC-12 cells were seeded at a density of 50.000 cells per well in 12-well plates. Twenty-four hours later the cultures were transduced with LV-hSYN-DsRed, LV- hSYN-GFP or LV-hSYN-DsRed-hSYN-GFP at m.o.i. 0.5. Seventy-two hours later the cells were resuspended in 500 µl of PBS solution and analyzed by FACScan (B-9320 Becton Dickinson). Filter sets of 515-545 and 564-606 nm wavelength band pass were used for the detection of EGFP and DsRed proteins, respectively. The 488 nm wavelength line of a mercury lamp was used to excite both fluorescent proteins. BD CellQuest Pro software was used for quantitation analysis. The threshold for gating was determined as the fluorescence value 16 transgene controlled by the TRE inducible promoter. Transcriptional activation is negatively regulated by the presence of tetracycline or its analog, doxycycline (DOX). The presence of DOX blocks the binding of tTA to the TRE promoter, thereby inhibiting transgene expression. Therefore, regulated expression with the Tet-off system requires the delivery in the same target cell of both the tTA transactivator and the transgene. To direct inducible transgene expression in cultured neurons with a single vector, we combined in a dual promoter lentiviral vector the neuronal-specificity of the hSYN promoter and the inducibility of the Tet-off system (Fig. 1B). In this vector, the hSYN promoter mediates the expression of the tTA transactivator, whereas the TRE promoter drives expression of the cDNA of interest, e.g. EGFP in the SYN-Tetoff-GFP vector (Fig. 1B). Therefore, hSYN-mediated expression of tTA would in turn regulate EGFP expression in neurons in a DOX-dependent manner. We also generated a second lentiviral vector, SV40-Tetoff-GFP, where the ubiquitous SV40 promoter controls tTA expression (Fig. 1B). 3.5. Doxycycline-regulated expression of EGFP in mixed neuronal cultures To study inducible transgene expression, dissociated cortical cultures were transduced with LV-SYN-Tetoff-GFP at 1 DIV and EGFP fluorescence was analyzed 7-8 days later. As shown in Figure 4, EGFP expression was clearly detected in cultured neurons grown in the absence of DOX (“on” state). Staining 17 of the transduced cultures with antibodies against the neuronal marker bIII- tubulin, showed that over 90% of EGFP-expressing cells were identified as neurons, whereas only a 5.5 % were co-stained with the glial marker GFAP (Fig. 4C). The opposite situation was found for vector LV-SV40-Tetoff-GFP, where transgene expression was mainly detected in glial cells and in only few neurons (92% glia, 8% neurons). These data confirm that the VSVG-pseudotyped lentiviral particles efficiently infect both, neuronal and non-neuronal cells but that the hSYN promoter restricts expression to neurons (Fig 4A and B). When transduced parallel cultures were grown in the presence of DOX for 7 days (“off” state), EGFP expression was inhibited irrespectively of whether the cultures were incubated with LV-SYN-Tetoff-GFP or LV-SV40-Tetoff-GFP viruses (Figs. 4 and 5). Addition of DOX does not alter neuronal density as compared with untreated controls, suggesting that neuronal viability is not affected by the presence of the drug (Fig. 4A). These results indicate that the hSYN promoter directs inducible gene expression in cultured neurons mediated by a dual promoter vector containing the Tet-off system. 3.6. Doxycycline-regulated switching of transgene expression To analyze the time-course kinetics of transgene expression, mixed cultures of cortical neurons were incubated with LV-SYN-Tetoff-GFP at 1 DIV and the expression of EGFP analyzed by Western blotting at different time points in 18 culture. In the absence of DOX, EGFP protein started to accumulate at 4 DIV, reaching maximal expression levels at 9-12 DIV (Figs. 5A,C). These maximal levels were maintained until the end of the culture period (18 DIV). Addition of DOX to the media at the time of transduction led to negligible EGFP levels in cultures grown for as long as 18 DIV (Fig. 5A). To study the kinetics of switching off transgene expression, we transduced cortical neurons with LV-SYN-Tetoff-GFP at 1 DIV, grew cells for 9 DIV to allow expression, and then added DOX to the media. As shown in Figure 5B, EGFP levels decreased three days after addition of the drug, reaching minimal protein levels 9 days after DOX addition (Fig. 5C). Due to the long half-life of the EGFP protein that can lead to discrepancies in the repression kinetics of protein and mRNA levels (Kafri et al., 2000), we performed quantitative real-time PCR experiments in cortical neuronal cultures transduced with LV-SYN-Tetoff-GFP. An approximately 100-fold induction in EGFP mRNA levels was obtained in cultures grown for 9-12 days in the absence of DOX. Addition of DOX to cultures grown for 9 days in the activation state (absence of DOX), drastically inhibited EGFP mRNA levels within the first 24 hours (Fig. 5D). Three days after addition of DOX, EGFP mRNA was barely above background levels, indicating efficient inhibition of transgene expression. 4. Discussion 19 Many basic research and therapeutic applications require co-expression of different cDNAs in one population of target cells. In this study, we have generated and tested dual promoter lentiviral vectors that drive constitutive, neuron-specific expression of cDNAs. We then applied this system for regulated neuron-specific expression under the control of the tetracycline-mediated transactivator. These new vectors provide rapid and tightly controlled expression and are easy to use. An alternative strategy for co-expression of exogenous proteins is the use of bicistronic mRNAs containing an internal ribosomal entry site (IRES). However, due to the significantly reduced expression of the cDNA placed downstream of the IRES, this approach often does not yield efficient co-expression of two cDNAs (Yu et al., 2003). Moreover, bicistronic constructs are less easily adapted for regulated expression without the need of introducing additional transgenes. Dual promoter viral vectors have been used previously in non-neuronal cells (Yu et al., 2003; Amendola et al., 2005; Osti et al., 2006). We integrated into viral vectors two hSYN promoters which direct neuron-specific expression of transgenes in vivo and in vitro (Kugler et al., 2001; Kugler et al., 2003a; Nakagawa et al., 2006). Bähr and colleagues used a similar approach for neuron-specific co-expression of two cDNAs in adeno-associated viral vectors (Kugler et al., 2003b). Our studies show co-expression at similar levels of two 20 cDNAs in primary neurons using a dual promoter lentiviral system. The two copies of the hSYN promoter/WPRE cassette each yield similar expression levels for the two cDNAs. Moreover, the hSYN promoters restricted expression of both cDNAs to neurons despite efficient infection of glial cells. Finally, the expression levels achieved in cultures infected at low m.o.i were sufficiently high to allow direct fluorescence detection of reporter proteins in living neurons, without the need for immunostaining. Therefore, our dual promoter lentiviral vectors should greatly facilitate imaging studies with genetically encoded reporters of neuronal function (Granseth et al., 2006). We further modified our dual promoter system for regulated gene expression. Insertion of the Tet-off system into a single lentiviral vector controlled by the hSYN promoter mediated expression of EGFP in cultured neurons. Absence of DOX from the cultures induced expression of the transgene by increasing EGFP mRNA levels up to 100-fold, whereas addition of DOX to transduced cultures efficiently inhibited transcription within 24 hours. Single lentiviral vectors that allow DOX-regulated expression of transgenes controlled by ubiquitous promoters have been previously generated and their use in the CNS reported (Kafri et al., 2000; Vogel et al., 2004; Blesch et al., 2005; Szulc et al., 2006) Our data with the SV40-Tet-off-GFP vector suggest a preference of this ubiquitous promoter for glial cells in mixed neuronal cultures, although it has been previously shown that lentiviral vectors containing the SV40 promoter can 21 transduce neuronal cell lines (Brun et al., 2003). While the use of non-specific promoters might raise concerns when regulated expression in neurons is desired, the dual-gene lentiviral vector we have developed offers the possibility of targeting specific cell populations (neurons versus glial cells) by using appropriate tissue-specific promoters. In comparison to gene delivery systems where the transfer genome is split in two plasmids, the use of dual promoter lentiviral vectors offers several advantages. All the molecular components are contained in a unique vector, which guarantees expression of the two cDNAs in the target cells. As a direct consequence, the viral titer and the number of integrations into the host genome are decreased, which reduces the chances of insertional mutation. In addition, the use of short regulatory units, such as the hSYN promoter and WPRE, results in vectors that remain well below the packaging capacity of a recombinant lentivirus. The modular design of the dual promoter vectors developed in this study allows for an easy replacement of the transgenes. We believe that the systems described here may prove an important tool for the understanding of neuronal function in normal and pathological conditions. Acknowledgements 22 The authors wish to thank Carlos Lois and David Baltimore for pCMVDR8.91 and VSV-G protein plasmids; Luke Van Parijs for the LenLox3.7 vector and Mathias Bähr for the pMH4-I-hSYN vector. We thank Amalia Martinez-Mir and Rafael Fernandez-Chacon for critical reading of the manuscript. This work was supported by grants PI051667 from Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III and Programa Ramón y Cajal (P-2004/1209) to FGS and SAF2003-02327 and SAF2006-03064 from Ministerio de Educación y Ciencia to MD-G. SG is a recipient of a predoctoral fellowship from Comunidad de Madrid. JAP-G is a recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia. 23 References Amendola M, Venneri MA, Biffi A, Vigna E, Naldini L. Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol, 2005; 23: 108-16. Azzouz M, Martin-Rendon E, Barber RD, Mitrophanous KA, Carter EE, Rohll JB, Kingsman SM, Kingsman AJ, Mazarakis ND. Multicistronic lentiviral vectormediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease. J Neurosci, 2002; 22: 10302-12. Blesch A, Conner J, Pfeifer A, Gasmi M, Ramirez A, Britton W, Alfa R, Verma I, Tuszynski MH. Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons. Mol Ther, 2005; 11: 916-25. Blomer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol, 1997; 71: 6641-9. Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res, 1993; 35: 567-76. 24 Brun S, Faucon-Biguet N, Mallet J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Mol Ther, 2003; 7: 782-9. Delenda C. Lentiviral vectors: optimization of packaging, transduction and gene expression. J Gene Med, 2004; 6 Suppl 1: S125-38. Glover CP, Bienemann AS, Heywood DJ, Cosgrave AS, Uney JB. Adenoviralmediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol Ther, 2002; 5: 509-16. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A, 1992; 89: 5547-51. Granseth B, Odermatt B, Royle SJ, Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron, 2006; 51: 773-86. Hioki H, Kameda H, Nakamura H, Okunomiya T, Ohira K, Nakamura K, Kuroda M, Furuta T, Kaneko T. Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Ther, 2007. Kafri T, van Praag H, Gage FH, Verma IM. Lentiviral vectors: regulated gene expression. Mol Ther, 2000; 1: 516-21. Kugler S, Kilic E, Bahr M. 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, 2003a; 10: 337-47. 25 Kugler S, Lingor P, Scholl U, Zolotukhin S, Bahr M. Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units. Virology, 2003b; 311: 89-95. Kugler S, Meyn L, Holzmuller H, Gerhardt E, Isenmann S, Schulz JB, Bahr M. Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors. Mol Cell Neurosci, 2001; 17: 78-96. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 2002; 295: 868-72. Nakagawa T, Feliu-Mojer MI, Wulf P, Lois C, Sheng M, Hoogenraad CC. Generation of lentiviral transgenic rats expressing glutamate receptor interacting protein 1 (GRIP1) in brain, spinal cord and testis. J Neurosci Methods, 2006; 152: 1-9. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996; 272: 263-7. Osti D, Marras E, Ceriani I, Grassini G, Rubino T, Vigano D, Parolaro D, Perletti G. Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes. J Virol Methods, 2006; 136: 93-101. Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A, 2002; 99: 2140-5.