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Dual Transgene Expression by Foamy Virus Vectors Carrying an Endogenous Bidirectional Promoter

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Dual Transgene Expression by Foamy Virus Vectors Carrying an Endogenous Bidirectional Promoter Gene Therapy (2010) 17, 380–388 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt ORIGINAL ARTICLE Dual transgene expression by foamy virus vectors carrying an endogenous bidirectional promoter A Andrianaki1,4, EK Siapati1,4, RK Hirata2, DW Russell2 and G Vassilopoulos1,3 1Cell and Gene Therapy Laboratory, Center for Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece; 2Division of Haematology, Department of Medicine, University of Washington, Seattle, WA, USA and 3Division of Haematology, University of Thessaly Medical School, Larissa, Greece Several gene therapy applications require the transfer and (range from 76 to 18%). Comparison of EGFP and DNGFR simultaneous expression of multiple genes in the same cell. levels revealed that the side of the promoter that drives the In this study, we analyzed the potential for coordinated expression of the HNRNPH2 gene in the genome was expression of an endogenous bidirectional promoter located stronger and in accordance to its in situ activity. When tested on chromosome X, which controls the expression of the with CD34+ cells, transgene coexpression reached 35.3% heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) of all positive cells in progenitor assays and 16.8% of all and alpha-galactosidase (GLA) genes. The promoter was positive cells after transplantation in NOD/severe combined cloned in both transcriptional orientations in a foamy virus immunodeficient mice. In summary, we show that the (FV) vector backbone, whereas the enhanced green fluor- endogenous promoter used in this study holds bidirectional escent protein (EGFP) and low-affinity nerve growth factor activity in the context of FV vectors and can be used in gene receptor (DLNGFR) reporter genes were cloned in the 50–30 therapy applications requiring synchronized expression of two and 30–50 transcriptional orientations, respectively. In all the genes. cell lines tested, both vectors showed high levels of trans- Gene Therapy (2010) 17, 380–388; doi:10.1038/gt.2009.147; gene coexpression that reached 76% of total positive cells published online 12 November 2009 Keywords: FV; bidirectional promoter; gene transfer; hematopoietic stem cells Introduction expression of the transgene downstream of the IRES element and cell type-dependent efficiency of balanced The expression of multiple genes in gene transfer expression.6–8 Another method has been the construction applications is a desirable property of viral vectors. of viral vectors with two transcription units cloned in Coexpression of a marker gene or an antibiotic resistance tandem but this design has been characterized by gene could be used to enrich gene-corrected cells before transcriptional interference.9,10 This strategy has also their in vivo administration. In applications in which been exploited in lentiviral vectors, but the published regulated gene expression is required, co-delivery of a reports present conflicting data on the problem of transcriptional activator or repressor to the same target promoter interference.11,12 Expression of multiple genes cell could also be a useful feature of any vector design. by a single vector can also be accomplished with artificial In vivo selection of transduced cells can also be carried genes encoding multiple proteins linked by the 2A out by concomitant expression of the MGMT gene.1–5 sequences of the foot-and-mouth disease virus in which Finally, the phenomenon of vector-mediated insertional cleavage of the expressed polyproteins is mediated by mutagenesis could be tackled by simultaneous expres- the 2A peptides.13–16 However, addition of the 2A sion of conditionally cytotoxic genes. peptide requires engineering of the proteins, although A number of approaches have been used to generate the expression of the cDNAs has been reported to be bicistronic vectors capable of simultaneous transgene dependent on the nature of the expressed genes and the expression. A widely used method is the inclusion of cloning order in the construct.3 Coordinated expression viral internal ribosome entry site (IRES) elements that has also been achieved with the construction of a permit cap-independent mRNA translation. However, synthetic bidirectional promoter using elements from this technology has been characterized by reduced the viral CMV, the UBI-C and the PGK promoter sequences.17 Recently, a ubiquitously acting chromatin Correspondence: Dr G Vassilopoulos, Laboratory of Cell and Gene opening element (A2UCOE), located between the Therapy, Centre of Basic Research II, Biomedical Research Founda- divergently transcribed heterogeneous nuclear ribonu- tion of the Academy of Athens, 4, Soranou Efesiou Street, Athens cleoprotein H2 (HNRPA2B1) and CBX3 genes, has 11527, Greece. shown more stable and position-independent expression E-mail: [email protected] 18 4These authors contributed equally to this work. compared with viral promoters. The A2UCOE element Received 11 March 2009; revised 29 September 2009; accepted 2 is a methylation-free CpG island that is present October 2009; published online 12 November 2009 in divergently transcribed housekeeping genes, but it Foamy viral vectors with a bidirectional promoter A Andrianaki et al 381 has not been tested for its bidirectional promoter activity in vitro. Computational studies have identified a large number Cla I Age I of divergently transcribed gene pairs, representing about 10% of all human genes, located on opposite strands with transcriptional start sites o1000 bp apart.19–21 The GLA HNRNPH2 transcripts of many bidirectional gene pairs were simultaneously coexpressed, indicating that their shared Age I Cla I cis-regulatory elements could initiate transcription at both directions.21 This bidirectional regulation system ΔΦΔΦ.HG.F HG GFP has been observed in the mammalian genome and it may reflect the requirement for tightly coordinated expression Cla I Age I in certain cellular pathways, such as cell cycle,22 histone 23 24 gene expression and heat shock responses. Such dual ΔΦΔΦ.GH.F GH GFP promoters are theoretically less likely to activate trans- gene silencing as they are often driving the expression of housekeeping genes.21 In this study, we have used such a ΔΔΦΦ.HG.F ΔΔΦΦ.GH.F putative bidirectional promoter which was identified in the course of a gene targeting experiment (DW Russell, unpublished data) and is located on chromosome Xq22.1. The promoter is driving the expression of the HNRNPH2 and the alpha-galactosidase (GLA) human genes, which are expressed in many tissues, including the hemato- 100 101 102 103 104 100 101 102 103 104 poietic system (HNRNPH2 (http://genome.ucsc.edu/ GFP GFP cgi-bin/hgTracks?db=hg18&position=chrX:100549847- 100555773&hgsid=146016699&knownGene=full); GLA 50 ΔΦ (http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg18& N.HG.F ΔΦ position=chrX:100539435-100549657&hgsid=146016699& 40 N.GH.F IRES2GFP knownGene=full)). We show that when cloned in a 30 foamy virus (FV) vector backbone, the promoter holds GFP bidirectional activity and can transcribe two reporter 20 MFI genes in a coordinated manner, recapitulating its endogenous activity. 10 0 HeLa HT1080 Results Figure 1 Construction and functional assays of foamy virus (FV) Both promoter orientations are active in diverse vectors with an endogenous bidirectional promoter. (a) Genomic organization of the bidirectional promoter located on chromosome cellular environments Xq22 in the intragenic region between the alpha-galactosidase The putative bidirectional promoter, located at chromo- (GLA) and the heterogeneous nuclear ribonucleoprotein H2 some position Xq22.1, lies in the intragenic region (HNRNPH2) genes. A 402-bp region (dotted line) was PCR between the GLA (NM_000169.2) and the HNRNPH2 amplified with primers carrying ClaI and AgeI restriction sites and genes (NM_001032393.1) (Figure 1a). The sequence was subsequently cloned in the deleted foamy viral vector backbone DF. (b) Illustration of the FV viral constructs generated after insertion of 402 bp long and included the region immediately up- the putative promoter in either orientation. HG denotes the stream of the translation start site of the GLA gene and transcription towards the GLA gene, whereas GH denotes tran- part of the HNRPH2 50-untranslated region up to scription towards the HNRNPH2 gene. An enhanced green position +103 of the mRNA. The promoter was PCR fluorescent protein (EGFP) reporter gene was placed downstream isolated from human genomic DNA and cloned in an of the promoter in sense orientation relative to viral transcription. intermediate vector (see Materials and methods) from (c) Transduction of HT1080 cells with DFN.HG.F and DFN.GH.F vectors carrying the EGFP expression cassette. Flow cytometry which it was further amplified with primers carrying analysis of EGFP expression in comparison with cells transduced ClaIorAgeI restriction enzyme sites, fitted to permit with FV vectors carrying a standard PGK promoter (filled directional cloning into a deleted FV vector with an histogram). (d) GFP mean fluorescence intensity (MFI) levels from enhanced green fluorescent protein (EGFP) reporter gene an IRES2GFP vector were compared with the MFI levels generated (Figure 1b). The constructs were designated DF.GH.F from the bicistronic DF.N.HG.F and DF.N.GH.F vectors. Results and DF.HG.F to mark the sequence orientation towards from at least three independent experiments with s.d. are shown. the HNRNPH2 or the GLA gene respectively, followed by the EGFP gene (F). The mean titer for
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