Bidirectional promoter interference between two widely used ... - Nature

Gene Therapy (2008) 15, 384–390 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt

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Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct JA Curtin1,2, AP Dane1, A Swanson1, IE Alexander1,2 and SL Ginn1 1

Gene Therapy Research Unit, The Children’s Hospital at Westmead and Children’s Medical Research Institute, Westmead, New South Wales, Australia and 2Discipline of Paediatrics and Child Health, The University of Sydney, Westmead, New South Wales, Australia

Gene transfer vectors encoding two or more genes are potentially powerful research tools and are poised to play an increasingly important role in gene therapy applications. Common strategies employed to express more than one transgene per vector include the use of multiple promoters, internal ribosome entry site (IRES) elements, splicing signals and fusion proteins. Of these, the IRES elements and multiple promoters have been most widely used. The use of multiple promoters, however, may be compromised by interference between promoters, promoter silencing and vector rearrangements or deletions. In this study, we demonstrate promoter interference between two internal

heterologous promoters in the context of a late-generation lentiviral vector. The interference, involving the human cytomegalovirus-immediate-early promoter and human elongation-factor-1a promoter, occurred bidirectionally with both promoters markedly impairing expression of the adjacent transcription unit. The data presented not only highlight the potential for interference between these widelyused promoters, but also the value of a sequential approach to vector construction that allows such effects to be recognized. Gene Therapy (2008) 15, 384–390; doi:10.1038/sj.gt.3303105; published online 24 January 2008

Keywords: promoter interference; lentiviral vectors; gene expression

Gene transfer vectors encoding two or more genes are potentially powerful research tools and are poised to play an increasingly important role in gene therapy applications. The challenge, however, is to achieve consistent high-level gene expression from each transcriptional unit. In practise, transgene expression is unpredictable when multiple cistrons are expressed from a single gene transfer vector. Additional open reading frames can be exploited to confer selectable phenotypes, both positive and negative, on target cell populations, and to modify physiological and pathophysiological processes that demand multiple therapeutic genes to be introduced.1–3 Strategies to express more than one transgene per vector include the use of multiple promoters, internal ribosome entry site (IRES) elements, alternate splicing signals and fusion genes,4,5 with the first two strategies being most frequently employed. IRES elements have been used successfully to generate polycistronic vectors, with the advantage of ensuring co-expression of the adjacent genes.4,6–8 When expression levels of the genes upstream and downstream of the IRES element are compared, however, the downstream gene is typically expressed at lower levels,9 and the extent of this effect Correspondence: Dr IE Alexander, Gene Therapy Research Unit, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, New South Wales 2145, Australia. E-mail: [email protected] Received 23 September 2007; revised 12 December 2007; accepted 15 December 2007; published online 24 January 2008

varies unpredictably with target cell type and genes used. The use of splicing signals, to generate separate mRNA transcripts from a single promoter, has been limited due to difficulty in controlling the splicing mechanism.5 The expression of one cistron is frequently at the expense of the other and the ratio of spliced to unspliced mRNA is highly dependent on the vector and cellular context.10 The use of fusion proteins is limited to the subset of situations where fusion does not interfere with the function of each subunit.11 Another alternative is the use of self-processing polyproteins where ciscleavage is mediated by sequences such as the 2A region from the foot-and-mouth disease virus.12,13 Promoter or transcriptional interference is defined as the perturbation of one transcription unit by another.14 The concept of promoter interference has long been recognized in the context of gammaretroviral vectors. Cullen et al.15 demonstrated that the downstream (30 ) long terminal repeat of an avian retroviral provirus is unable to act as an efficient promoter of transcription when a transcriptionally active upstream (50 ) long terminal repeat is present. Emerman and Temin16 found, while analysing cell clones transduced with a single integrated provirus containing two promoters and two selectable genes, that usually only one of the adjacent cistrons was active, with the other gene being suppressed. This suppression was epigenetic, reversible, cis-acting and was influenced by the strength and position of the different promoters. Furthermore, the gene suppression effect was shown to occur either upstream or downstream to the adjacent active gene.

Promoter interference in lentiviral vectors JA Curtin et al

Historically, vectors with two internal promoters have been widely investigated.5 In the case of gammaretroviruses, the use of multiple promoters may be compromised by promoter interference, promoter suppression and rearrangements.15–18 In vectors containing two genes each with its own promoter, suppression of an individual gene may also occur through epigenetic silencing or by promoter deletion.16,17 Epigenetic silencing may occur when transcription of one cistron alters the chromatin structure of surrounding DNA such that transcription from the second promoter is inhibited.16 Despite these limitations, gammaretroviral vectors have been used extensively in gene therapy applications.19 Internal promoters have also been widely used in other viral vectors, where some of the disadvantages seen in gammaretroviruses have not been observed.5,20 Lentiviral vectors, a class of complex retroviruses, offer a number of potential advantages over gammaretroviruses including the ability to transduce post-mitotic and non-dividing cells.21–24 In addition, differences in integration site patterns between lentiviral and gammaretroviral vectors25–27 may alter susceptibility to promoter interference. Lentiviral vectors, harbouring two or more independent transcription units have been described without promoter interference being a recognized problem.20,28,29 We, however, have previously reported promoter interference in the context of an earlygeneration lentiviral vector between the U3 region in the long terminal repeat and an internal heterologous

cytomegalovirus immediate early (CMV/IE) promoter.30 This effect was shown to significantly influence transgene expression in a cell-type and species-specific manner. In late-generation lentiviral vectors the U3 region, responsible for this effect, has been deleted to ablate long terminal repeat promoter activity in an effort to enhance safety and reduce the risk of insertional mutagenesis.31 Here we report substantial promoter interference between two internal heterologous promoters in the context of a series of late-generation lentiviral vectors (Figure 1). In the process of developing a polycistronic vector for liver-directed gene therapy, we observed reduced expression of both transgenes. We demonstrated that the reduced gene expression was due to promoter interference between two widely-used promoters, the human CMV/IE promoter and the human elongationfactor-1a (EF1a) promoter. The interference observed was bidirectional because each promoter affected expression of the adjacent transgene. In addition, this phenomenon was seen in both mouse and human cell lines indicating that this effect is not restricted to certain species. In the NIH3T3 murine cell line, enhanced green fluorescent protein (EGFP) expression was markedly reduced in cells transduced with both the LV-2 or LV-3 vectors (with two internal heterologous promoters) when compared to the LV-1 vector (with single promoter and reporter) as determined by direct visualization under

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Figure 1 Schematic diagram of the lentiviral vectors. The transfer vectors used in this study were derived from the third generation selfinactivating vector pRRLsin18.cPPT.CMV.EGFP.WPRE.38 The vector backbone was initially modified by converting the Cla I site to an Xba I site. A polylinker with unique restriction endonuclease sites was then inserted downstream of the EGFP cassette to create the vector designated LV-1. The 1186-bp EF1a promoter from pEF/myc/nuc (Invitrogen, Carlsbad, CA, USA) was then cloned into the Pme I site of the polylinker to create the vector designated LV-2. The heavy chain of GCSh was inserted at the Sal I site of the polylinker to create the vector designated LV-3. In the final vector, the CMV/IE promoter was removed from LV-3 by digestion with Xba I and then religated to create the vector designated LV-4. Vector stocks were produced by transient transfection as described previously.30 Transduction titres, to calculate the MOI used in each experiment, were determined on HEK293 human embryonic kidney cells39 in the presence of Polybrene (8 mg ml1; SigmaAldrich, St Louis, MO, USA) by using qPCR and previously published primer sequences (FPLV2 and RPLV2).40 Briefly, genomic DNA was extracted 4 days after transduction using the QIAamp DNA blood Mini Kit (Qiagen GmbH, Hilden, Germany) and 200 ng added to reactions containing 1 PCR reaction buffer, 5 mM MgCl2, 200 mM each dNTP, 1 mM of each primer, 0.1 SYBR Green (Molecular Probes, Eugene, OR, USA) and 1.25 units Plantinum Taq DNA polymerase (Invitrogen). The reactions were performed on a Rotor-Gene 3000 (Corbett Research, Australia). cPPT, central polypurine tract; GA, fragment of the HIV-1 gag gene; GCSh, g-glutamylcysteine synthetase; RRE, Rev responsive element; RSV, Rous sarcoma virus and HIV chimeric long terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. Gene Therapy


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Figure 2 Promoter interference in murine cells. (a) NIH3T3 cells (ATCC; CRL 1658) were transduced with vector at an MOI of 30 in the presence of Polybrene (8 mg ml1). The upper panels are representative fluorescent images and the lower panels are bright field images of the same field 7 days after transduction with the LV-1, LV-2 or LV-3 vectors. (b) Percentage of NIH3T3 cells expressing EGFP determined by FACS analysis 8 days after transduction with LV-1 (open bars), LV-2 (grey bars) or LV-3 (closed bars) at MOIs of 10, 20 and 40. (c) MFI representing the mean channel number of the EGFP fluorescence, of cells determined by FACS analysis using a FACScan cytometer (BD Biosciences, San Jose, CA, USA) running CellQuest software (version 3.1f). (d) Integrated vector genome copy number per cell determined by qPCR. The mean±s.e.m. of triplicate measurements from a representative experiment is shown. EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; MFI, mean fluorescence intensity; MOI, multiplicity of infection; qPCR, real-time quantitative PCR.

fluorescent microscopy (Figure 2a). This observation was confirmed by fluorescence-activated-cell-sorting analysis of transduced cells (Figure 2b). At multiplicity of infections of 10, 20 and 40, LV-1-transduced cells had the greatest proportion of EGFP-expressing cells, LV-2-transduced cells an intermediate proportion and LV-3-transduced cells the lowest proportion. For the LV-2 and LV-3 vector constructs, which contain the EF1a promoter downstream of the CMV/IE-EGFP expression cassette, the mean fluorescence intensity of the EGFP positive cell population was also significantly reduced (Figure 2c). To ensure that the differences observed in EGFP expression levels were not the result of differences in transduction efficiency, the integrated vector genome copy number was determined. Cells transduced by the LV-2 and LV-3 vectors, which displayed reduced EGFP expression, did not have reduced numbers of integrated genome copies per cell (Figure 2d). Together these results support the conclusion that the EF1a promoter is interfering with the ability of the upstream CMV/IE promoter to drive EGFP expression. To assess whether the reduced EGFP expression observed was species restricted, experiments were repeated in the human HEK293 cell line. Consistent with the results obtained in mouse cells, EGFP expression was markedly reduced in LV-2 and LV-3-transduced cells compared to LV-1-transduced cells as determined by fluorescence microscopy (Figure 3a). Flow cytometry of Gene Therapy

the same populations revealed a modest reduction in the relative proportion of cells expressing detectable EGFP (Figure 3b), and marked reduction in the intensity of EGFP expression in individual cells as indicated by the analysis of mean fluorescence intensities (Figure 3c). Again, differences in EGFP expression were not the result of differences in transduction efficiency (Figure 3d). These results, obtained in human cells, are qualitatively similar to those obtained in mouse cells, consistent with an absence of any overt species-specific effects. At equivalent multiplicity of infections, however, both the percentage of EGFP-positive cells and intensity of EGFP expression were higher in 293 cells. This likely reflects the greater transcriptional activity of the CMV promoter in human cells and differences in relative permissiveness for lentivirus-mediated transduction. The absence of overt species-specific effects on the promoter interference effects observed contrasts with U3 region-mediated promoter interference observed in early generation lentiviral vectors by our group.30 Interestingly, the degree to which the EF1a promoter interferes with the upstream CMV/IE promoter was more pronounced when the GCSh (g-glutamylcysteine synthetase) transgene was expressed (LV-3) and may reflect differences in its state of transcriptional activity (Figures 2 and 3). To determine whether the presence of the CMV/IE promoter was interfering with the expression of GCSh from the downstream EF1a promoter, the CMV/IE


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Figure 3 Promoter interference in human cells. (a) HEK293 cells 7 days after transduction with LV-1, LV-2 or LV-3 vectors at an MOI of 30. The upper panels are representative fluorescent images and the lower panels are bright field images of the same field. (b) Percentage of HEK293 cells expressing EGFP determined by FACS analysis 8 days after transduction with LV-1 (open bars), LV-2 (grey bars) or LV-3 (closed bars) at MOIs of 10, 20 and 40; (c) MFI of cells determined by FACS analysis and (d) integrated vector genome copy number per cell determined by qPCR. The mean±s.e.m. of triplicate measurements from a representative experiment is shown. EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; MFI, mean fluorescence intensity; MOI, multiplicity of infection; qPCR, real-time quantitative PCR.

promoter was removed to create the vector designated LV-4 (Figure 1). Glutamylcysteine synthetase is the first and rate-limiting enzyme in glutathione synthesis. Expression was therefore determined by the measurement of glutathione concentration, which was higher in both NIH3T3 and HEK293 cells transduced with the LV-4 vector than in cells transduced with the LV-3 vector, containing the CMV/IE promoter (Figures 4a and b). Glutathione has a negative feedback effect on glutamylcysteine synthetase that limits the amount of glutathione at steady state. Western blot analyses were therefore performed to determine the quantity of protein present in transduced cells. Cells transduced with LV-4 exhibited substantially higher GCSh protein production than the cells transduced with LV-3 (Figures 4c and d), a result consistent with the negative feedback of glutathione on glutamylcysteine synthetase activity. Differences in transduction efficiencies of LV-3 and LV-4 were excluded as a contributing factor by the analysis of proviral copy number in transduced mouse and human cells (data not shown). Taken together, these results confirm that the presence of the CMV/IE promoter in the LV-3 construct significantly reduced expression of GCSh from the downstream EF1a promoter. A number of mechanisms have been proposed for transcriptional interference. There may be read-through

transcription from an upstream gene into the regulatory region of another gene, which disrupts the binding of transcription factors and other DNA-binding proteins. This is often referred to as promoter occlusion and can be blocked by an intervening transcriptional terminator.32 In the context of our study, this would explain interference with the EF1a promoter by the upstream CMV/IE promoter, but not the converse, which was also observed. A second proposed mechanism that could explain the bidirectional interference observed is the competition between promoters for cis- or trans-acting factors including transcription factors, enhancers or their binding proteins. Steric or topological changes induced by transcription, such as negative supercoiling upstream of promoters and positive supercoiling ahead of polymerases, also have the potential to prevent transcription complexes from being efficiently formed on an adjacent promoter. Promoter interference-like effects could also occur through post-transcriptional mechanisms, such as competition between the different vector-encoded transcripts for ribosomal association or altered transcript stability. The first possibility would require limiting ribosomal activity as might occur when one or more vector-encoded transcripts are present at very high levels. In the current study, there was no apparent correlation with transgene expression levels as Gene Therapy


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Figure 4 Promoter interference is bidirectional. Glutathione concentration ([GSH]) in (a) NIH3T3 or (b) HEK293 cells exposed to LV-1 (open bars), LV-3 (closed bars) or LV-4 (hatched bars) at MOIs of 10, 20 and 40. The cells to be analysed were harvested by trypsinization and resuspended in 300 ml of 10 mM Tris-HCl (pH 7.6). Subsequent steps were carried out on ice. The cells were lysed by sonciation (three 10 s pulses, Branson Sonifier S-250A, Danbury, CT, USA) and the lysate cleared by centrifugation (3000 g for 10 min at 4 1C). A 200 ml aliquot of the supernatant was analysed for GSH levels. GSHwas measured using the Bioxytech GSH-400 kit (Oxis International, Portland, OR, USA) according to the manufacturer’s instructions. The mean±s.e.m. of triplicate measurements from a representative experiment is shown. Western blot analysis on protein extracts from (c) NIH3T3 or (d) HEK293 cells exposed to LV-1 (lane 1, endogenous protein expression), LV-3 (lane 2) or LV-4 (lane 3) at an MOI of 40. Protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Western blot analysis was performed on 250 mg of protein from transduced cells as described previously.41 After transfer to nitrocellulose (Bio-Rad), blots were probed with primary rabbit polyclonal antibody to GCSh (1:200 dilution; NeoMarkers, Fremont, CA, USA). A secondary goat anti-rabbit-HRP conjugate (1:25 000 dilution; Bio-Rad) in conjunction with SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL, USA) was used for detection. To confirm equal loading, the membrane was stripped using 0.2 M NaOH and re-probed with rabbit anti-actin antibody (1:250 dilution; Sigma-Aldrich). GCSh, g-glutamylcysteine synthetase; GSH, glutathione; MOI, multiplicity of infection; qPCR, real-time quantitative PCR.

interference was equally apparent across a range of vector multiplicity of infections, and in mouse and human cells where the promoters tested differ in transcriptional strength. Differing stability of CMVdriven transcripts caused by the presence or absence of the downstream promoter sequence in the 30 untranslated region is possible, but fails to explain the bidirectional effects observed. Finally, epigenetic factors affecting chromatin structure, including methylation or histone modification, may influence surrounding gene transcription. To date, many explanations for promoter interference have been suggested, however, the interactions between cis-acting elements are complex and a clear mechanism for this phenomenon has not been identified. Therefore, irrespective of the cause, promoter interference cannot always be anticipated and should be assessed empirically when constructing gene transfer vectors containing more than one promoter. The strong CMV/IE promoter contains many transcription factor binding sites, including sites for the Sp1 family of transcription factors,33 and it is interesting to speculate that this may play a role in the promoter interference described in this report. Indeed, binding sites for this family of transcription factors are also present on the EF1a promoter.34 The use of these two promoters in combination in lentiviral vectors has been reported previously;28,35 however, the possibility of promoter interference was not examined in these studies. Therefore, while gene expression was seen from both promoters, as is the case in our study, promoter Gene Therapy

interference effects may have gone undetected. In such cases, reduced transgene expression may result in the loss of therapeutic benefit or, alternatively, require the use of a promoter containing unnecessarily strong promoter/enhancer sequences. Promoter interference also has the potential to occur between vector-borne promoters and endogenous promoters following vector integration. The resulting reduction in the expression of an endogenous gene would constitute a form of insertional mutagenesis, although would be less likely to cause pathology than aberrant gene activation. The reduction of these genotoxic risks associated with integration are theoretically achievable by optimized expression cassettes including the avoidance of strong promoter/enhancer sequences, promoters containing high core activity and inclusion of enhancer-blocking insulator elements, such as the 50 -HS4 chicken b-globin locus.36 Such elements have the potential to be exploited to create independent domains of transcriptional activity,37 both within polycistronic vectors, between vector-borne promoters and endogenous promoters. In summary, we have observed marked promoter interference between the widely-used CMV/IE and EF1a promoters in the context of a late-generation SIN lentiviral vector. Interestingly, this phenomenon occurred bidirectionally with the upstream promoter impairing expression from the downstream promoter and vice versa. In addition, the promoter interference observed was not species restricted. The data presented highlight


not only the potential for interference between these widely-used promoters, but also the value of a sequential approach to vector construction that allows such effects to be recognized. We conclude that, to a variable degree, interference between internal heterologous promoters may be relatively common and should be considered when constructing polycistronic lentiviral vectors.

Acknowledgements We thank Professor Inder Verma (Salk Institute, San Diego, USA) for providing the lentiviral vector reagents used in this study. This work was supported by an ASTRA fellowship from the Royal Australasian Collage of Physicians and by the Haemophilia Foundation of Australia. Julie A Curtin was the recipient of a NHMRC post-graduate research scholarship and a CHW New Investigator Grant. We also thank ACCO Australia, for funding support. Samantha L Ginn is the recipient of a fellowship honouring the memory of Noel Dowling.

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