In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.
In gene therapy, it is advantageous to use selective targeting strategies to control the expression of therapeutic genes. Transgene expression driven by tissue-specific promoters may help reduce the effects of off-target expression, and improve the efficiency and safety of lentiviral vectors for gene therapy. Lentivirus-derived vectors are efficient delivery vehicles for genes as they can target, and permanently integrate into hematopoietic stem cells and non-dividing cells, for example, macrophages, with high efficiency. Using gene therapy with a tissue-specific promoter, macrophages (or other cell types) can be targeted to either overexpress or silence a specific protein. The use of tissue-specific promoters can limit both genotoxicity (for example, by limitation of enhancer activity in non-target cells, which leads to reduced activation of oncogenes near the vector integration site) and cytotoxicity (ectopic transgene expression in non-target cells).1
Macrophages are key players in several common diseases, for example, infections,2 arthritis,3 atherosclerosis4 and cancer.5, 6 Macrophage-specific promoters (MSPs) can be used for gene delivery approaches as valuable tools to study the function of the macrophage per se, or to treat diseases in a macrophage-dependent manner. Several different MSPs have earlier been published in different settings.7, 8, 9, 10, 11, 12, 13, 14, 15, 16 Among these, previous studies indicate that the CD68 promoter7, 8 and a synthetic promoter (SP) (generated by randomly ligating myeloid-specific cis promoter elements for several different transcription factors and inserting them upstream from the p47phox basal promoter)16 result in the highest transgene expression levels and macrophage specificity. However, these promoters exist in different lengths and orientations, and further characterization is required to assess which promoter is superior.
In this study we sought to find an MSP with high specificity resulting in high protein expression in macrophages and low leakage in non-target cells by lentiviral delivery. To achieve an appropriate comparison between promoters, we constructed lentiviral vectors containing promoters fused with green fluorescent protein (GFP). We compared three variants of the CD68 promoter (full length, the 343-bp proximal region and the 150-bp proximal region) and two variants of the SP (in forward and reverse orientation) in vitro and in vivo. We show that MSPs can efficiently be used for lentiviral gene delivery, and that the 150-bp proximal region of the CD68 promoter results in significant protein expression and is currently the best available promoter for macrophage-specific expression of transgenes.
We generated lentiviral constructs with expression cassettes containing promoters, GFP and the woodchuck post-transcriptional regulatory element (WPRE). The following promoters were used: (i) the spleen focus-forming virus long terminal repeat promoter (SFFV), used as positive control; (ii) the full-length human CD68 promoter in reverse orientation (CD68FL(r)); (iii) the proximal 343 bp of the CD68 promoter (CD68/343(f)); (iv) the proximal 150 bp of the CD68 promoter (CD68/150(r)); (v) the SP in the forward orientation (SP(f)); and (vi) the SP in the reverse orientation (SP(r)). All constructs are shown in Figure 1.
Expression of GFP in cell line and primary macrophages
To determine whether lentiviral particles encoding GFP driven by MSPs could transduce macrophages and lead to protein expression, we transduced various cell lines and primary cells. Fluorescence-activated cell sorting (FACS) analysis was performed to determine GFP expression in different cell populations, and typical gating is shown in Figure 2. To compare the MSP-driven GFP expression in macrophages, a mouse macrophage cell line (RAW 264.7) was initially used. We found that all MSPs except CD68FL(r) were functional and led to GFP expression in a concentration-dependent manner, both in terms of number of cells (Figure 3a and Supplementary Table S1) and mean fluorescence intensity (Supplementary Figure S1), although to a lower extent compared with SFFV. CD68FL(r) was excluded from further experiments because of low GFP expression levels. Activation of macrophages by addition of lipopolysaccharide after transduction only marginally increased the number of GFP-positive cells (Figure 3b). To verify GFP expression in primary cells, we also transduced peritoneal macrophages with lentiviral particles encoding GFP driven by the MSPs. All the MSPs led to significant expression of GFP in primary macrophages (Figure 3c, Supplementary Figure S1 and Supplementary Table S1). Taken together, these data show that CD68/343(f) and CD68/150(r) resulted in the highest expression in cell line and primary macrophages. Because SP(f) and SP(r) expressed GFP to the same extent, we excluded SP(r) from further experiments.
Specificity of MSPs in vitro
To study the specificity of MSPs in non-macrophage cells in vitro, we used a B-cell line (A20), a T-cell line (HDBR1), an endothelial cell line (H5V) and a fibroblast cell line (NIH/3T3). In B cells, transduction with CD68/343(f) resulted in significant expression of GFP (5% positive cells) (Figure 4a and Supplementary Table S2). In T cells, CD68/343(f) and CD68/150(r) resulted in 40% and 47% GFP positive cells, respectively (Figure 4b and Supplementary Table S2). In endothelial cells, all MSPs except CD68/150(r) led to significant expression of GFP (CD68/343(f) and SP(f) resulted in 10% and 6% GFP-positive cells, respectively) (Figure 4c and Supplementary Table S2). In fibroblasts (which express fairly high levels of CD68(ref. 17)), transduction with CD68/343(f) and CD68/150(r) resulted in significant expression of GFP (35% and 31%, respectively) (Figure 4d and Supplementary Table S2). Thus, transduction of non-macrophage cell lines in vitro shows that all MSPs tested were to some extent promiscuous. However, CD68/150(r) and SP(f) showed a more specific expression pattern than CD68/343(f).
Expression and specificity of MSPs in vivo
To study the expression and specificity of the MSPs in different cell populations in vivo, hematopoietic stem cells were transduced with CD68/343(f), CD68/150(r) or SP(f) and transplanted into C57Bl6 recipient mice. We found that all the selected MSPs were well integrated both in bone marrow and in peripheral cells 8 weeks after transplantation (Table 1). FACS analysis was performed to determine GFP expression in different cell populations in the bone marrow, peritoneal lavage and spleen (Figure 2). Using CD68/150(r), 25–40% of the macrophages (F4/80+ cells) expressed GFP in the different organs (Figures 5a–c). When we combined data from the bone marrow, peritoneal lavage and spleen, the average GFP positivity for macrophages was 31% (P<0.001). On the other hand, dendritic cells (DCs) (CD11c+), neutrophils (Gr-1+), T cells (CD3+) and B cells (CD19+) did not express significant levels of GFP (Supplementary Table S3), suggesting a highly macrophage-specific expression pattern. By contrast, SP(f) showed a more nonspecific pattern, as both macrophages (44%, P<0.05) as well as DCs (42%, P<0.05), neutrophils (37%, P<0.05) and B cells (44%, P<0.05) expressed GFP (Figures 5a–c and Supplementary Table S3). Transduction with CD68/343(f) showed that surprisingly few cells expressed GFP and in a nonspecific pattern (Figures 5a–c). Analyses of mean fluorescence intensity also showed that CD68/150(r) led to macrophage-specific expression of GFP (Figure S2). In addition, when markers were analyzed in all GFP-expressing cells, CD68/150(r) resulted in 90–100% cells that were positive for the macrophage marker F4/80, whereas expression of T-cell, B-cell, DC and neutrophil markers was as low as 10–50% (Figure 6). Our results clearly demonstrate that CD68/150(r) provides primarily macrophage-specific expression of GFP and is an excellent MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.
In this study, we used a lentiviral vector system to compare different MSPs with the aim of finding the promoter with the highest macrophage specificity that leads to stable high protein expression. Our data show that the 150-bp proximal part of the CD68 promoter is superior to the other promoters tested and results in the highest macrophage specificity and significant protein expression.
Previous studies using MSPs have shown that the CD68 promoter7, 8 and an SP generated by fusing transcription factors to the p47phox minipromoter16 result in high transgene expression levels and macrophage specificity. However, these promoters have never been compared and moreover they exist in different lengths and orientations. Further evaluation of these promoters is consequently required to assess which promoter is superior. We therefore compared three different CD68 promoters of different length and orientation (CD68FL(r), CD68/343(f) and CD68/150(r)), and two constructs of a macrophage-specific SP of different orientation (SP(f) and SP(r)). The rationale for using different orientations is that the full-length CD68 contains the 83-bp CD68 IVS1, which on the one hand mediates enhancer activity when inserted downstream from the promoter,18 but on the other hand introns contained within the insert may be removed during vector replication.19 Indeed, previous attempts to use the CD68 full-length promoter in forward orientation showed that it does not result in any GFP expression at all (I Gjertsson, unpublished observation) and the full-length CD68 promoter is therefore used in reverse orientation (CD68FL(r)). However, CD68/343(f) has previously been shown to be functional in forward orientation.20 Construction of CD68/150(r), which lacks the intron, was cloned in the same vector backbone as CD68FL(r); thus this construct is also in reverse orientation. The constructs of the SP are tested in forward SP(f) orientation as well as reverse (SP(r)) orientation.
Our data show that all tested MSPs, except CD68FL(r), are functional in vitro and in vivo. Surprisingly, the shortest version of the CD68 promoter, CD68/150(r), gives the highest macrophage specificity in vitro as well as in vivo. This promoter has been characterized previously,8 but has to our knowledge not been used for any functional studies. Previous characterization of the CD68 promoter has shown that sequences between −150 and +2 of the CD68 gene give maximal promoter activity,8 most likely owing to the fact that multiple transcription factors bind to this sequence. Ets transcription factors (for example, PU.1) are the most important regulatory elements for macrophage specificity.21, 22 At −110 bp of the CD68 promoter, there is an Ets cis-element that binds to PU.1, which is crucial for promoter activity.8 Furthermore, at −145 bp of the CD68 promoter there is an AP-1 transcription factor cis-element, which has been shown to act synergistically together with PU.1 to activate the mouse CD68 promoter. These previous notions together with our results suggest that the proximal −150 bp of the CD68 promoter is sufficient for maximal promoter activity specifically in macrophages, and it is possible that inclusion of larger parts of the promoter sequence actually decreases specificity.
The SP has been suggested to be highly specific for macrophages,16 but in vivo characterization of promoter specificity in other types of bone marrow-derived cells has so far been limited. Our results suggest that the SP is specific for all cells of hematopoietic lineage.
Our data show that the macrophage specificity for CD68/150(r) as well as CD68/343(f) is higher in vivo than in vitro. One reason for this discrepancy may be that the cells analyzed in vivo are fully differentiated and are immunologically defined. Cell lines do not show the same cell-specific expression patterns as cells in vivo.
Lentiviral vectors have become popular delivery vehicles for gene therapy. In particular, the capacity of lentiviral vectors to integrate into non-dividing cells and provide long-term and stable gene expression in vivo is an attractive attribute for gene therapy approaches. As a consequence, clinical trials for treatment of HIV (reviewed by Lemiale and Korokhov23) and β-thalassemia24 have been performed using lentiviral vector delivery systems with general viral promoters driving ubiquitous expression in all cell types. However, it is advantageous to use a selective targeting strategy to control the expression of therapeutic genes. Transgene expression driven by tissue-specific promoters may help reduce the effects of off-target expression, and improve the efficiency and safety of lentiviral vectors for gene therapy. Gene therapy with a macrophage-specific promoter could, for instance, be an option in some lysosomal storage diseases such as Morbus Gaucher, where the genetic defect is confined to macrophages.25
In conclusion, we found that CD68/150(r) is the MSP with highest macrophage-specific activity among the compared promoters, and that it is currently the best available promoter for lentiviral delivery of transgenes in vitro and in vivo.
Materials and methods
Generation of lentiviral constructs
The following constructs were used in this study and are summarized in Figure 1. The SFFV promoter construct (previously referred to as pHRSIN-cPPT-SEW) that is used as a positive control has been described previously.26 To generate the construct containing the full-length human CD68 promoter (2940 bp) in reverse orientation (the CD68FL(r)), its expression cassette was put together stepwise in the cloning plasmid pTZ18R Amersham Biosciences, GE Healthcare (Chalfont St Giles, UK) using standard cloning procedures. Briefly, pTZ18R was digested with HindIII/EcoRI and a multiple cloning site (MCS) linker, SalI–AscI–HindIII–XbaI–PacI–PmeI–ClaI–XhoI–NheI–MfeI, was inserted with overhang that destroys the original HindIII/EcoRI sites. A 3-bp HindIII/XbaI fragment containing the CD68 promoter was excised from pcDNA3(ref. 7) and inserted into the corresponding sites in the pTZ18R MCS. The WPRE27 was excised from SFFV using ClaI and inserted into the corresponding site in the MCS. The bovine growth hormone poly adenylation signal (bGHpA) was amplified by a PCR from pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) to generate a 248-bp fragment with suitable sites (forward primer: 5′-IndexTermGTATGTCGACACTGTGCCTTCTAGTTGG-3′; reverse primer: 5′-IndexTermGTATTCTAGAAGCCATAGAGCCCAC-3′). The PCR product was subcloned into pCR2.1 TOPO (Invitrogen) and verified by sequencing. The bGHpA fragment was then excised with SalI/XbaI and inserted into the XhoI/NheI sites in the MCS. The EGFP was excised from pEGFP1 (Clontech, Mountain View, CA, USA) with BamHI/NotI and inserted into the corresponding sites in another pTZ18R plasmid with its MCS exchanged to PacI–SalI–BamHI–SnaBI–NotI–PmeI. The EGFP was then excised with PacI/PmeI and inserted into the corresponding sites in the MCS in the first pTZ18R plasmid. Finally, the whole expression cassette was excised from pTZ18R with MfeI/SalI and inserted, in reverse orientation, into SFFV using the EcoRI/XhoI site and hence exchanging the SFFV-GFP-WPRE expression cassette. Generation of a promoter consisting of the proximal 343 bp of the CD68 promoter (CD68/343(f)) has been described previously.20 To generate a promoter consisting of the proximal 150 bp of the CD68 promoter (CD68/150(r)), the 150-bp CD68 proximal promoter was PCR-amplified from pBSCD68, generating a 187-bp fragment with suitable sites (forward primer: 5′-IndexTermGATAGGCGCGCCAAGCTTCTGAGGCCCCTGAGTCAG-3′; reverse primer: 5′-IndexTermGATAGTCGACTTAATTAATGGCTGAACCGCCTCAC-3′). The PCR product was subcloned into pCR2.1 TOPO and verified by sequencing. The promoter fragment was then excised with AscI/SalI and inserted into the corresponding sites in the CD68FL(r), exchanging the 3097-bp CD68 promoter fragment. To generate the SP in the forward orientation (SP(f)), a 1720-bp EcoRI/SacII fragment containing the SP, GFP and part of the WPRE was excised from SP-GFP16 and inserted into the corresponding sites of SFFV, exchanging the SFFV-GFP-WPRE expression cassette. To generate the SP in the reverse orientation (SP(r)), the CD68FL(r) was digested with AscI/SalI, removing the full-length CD68 promoter, and an MCS linker, AscI–EcoRI–XmaI–SalI, was inserted. The SP promoter was then excised from SP-GFP16 with EcoRI/AgeI and inserted into the EcoRI/XmaI sites in the MCS linker.
Production of lentiviral particles
A vesicular stomatitis virus-G pseudotyped lentivirus was produced by transient transfection of 293FT cells with three plasmids, one of the self-inactivating transfer vector plasmids (SFFV, CD68FL(r), CD68/343(f), CD68/150(r), SP(f), SP(r)); the multi-deleted packaging plasmid pCMVÄR8.74; and the vesicular stomatitis virus-G envelope pMD.G2, by calcium phosphate co-precipitation (ProFection Mammalian Transfection System Promega Biotech AB, Stockholm, Sweden). Because this method did not lead to a sufficiently high titer of lentiviral particles encoding CD68/150(r), this particular transfer vector plasmid was co-precipitated with pCMVΔR8.74 and pMD.G2 using polyethylenimine (Sigma-Aldrich, St Louis, MO, USA). At 72 h after transfection, the medium was harvested and concentrated by ultracentrifugation at 90 000 g. The pellets were resuspended in phosphate-buffered saline (PBS) containing 2% fetal calf serum and stored at −80 °C. The concentration of lentiviral particles was determined by transducing a defined number of HeLa cells with serially diluted lentiviral particles, and measuring the expression of GFP using FACSCalibur (BD Biosciences, San Jose, CA, USA) and analyzing by using the FlowJo Software (Tree Star Inc., Ashland, OR, USA).
Transduction of peritoneal macrophages and cell lines
Peritoneal macrophages were isolated by flushing the peritoneum of wild-type C57Bl6/J mice with PBS. Cells were collected and cultured for 2 h at 37 °C in serum-free RPMI-1640 media supplemented with sodium-pyruvate (2 mM), non-essential amino acids, sodium bicarbonate (1.5 g l−1), penicillin (100 U ml−1) and streptomycin (100 mg l−1). The cells were washed three times with PBS and cultured at 37 °C in RPMI-1640 media containing 10% fetal calf serum supplemented as described above. The following mouse cell lines were used: the B-cell line A20, the T-cell line HDBR1, the macrophage cell line RAW 267.4, the endothelial cell line H5V and the fibroblast cell line NIH/3T3. All cells were cultured in a humidified incubator with 5% CO2 at 37 °C. Cells were transduced at the different multiplicities of infection (MOIs) indicated. The cells were harvested after 5 days (peritoneal macrophages, RAW 267.4, NIH/3T3 and H5V) or 7 days (A20 and HDBR1), and GFP expression was analyzed by flow cytometry.
To detect GFP expression in different cell populations, a single-cell preparation of 1 × 106 cells from cell culture, spleen, bone marrow and peritoneal cells was placed in 96-well plates and pelleted (3 min, 300 g, 4 °C). To avoid nonspecific binding by means of Fc–receptor interactions, cells were incubated with Fc-block (2.4G2; BD Biosciences) for 10 min at room temperature. The antibodies used were anti-CD19 (ID3), anti-CD11c (HL3), anti-Gr1 (RB68C), anti-CD3 (145-2C11) purchased from (BD Bioscience) and anti-F4/80 (BM8) purchased from (eBioscience, San Diego, CA, USA). All antibodies were diluted in FACS buffer (PBS containing 1% fetal calf serum and 0.5 mM EDTA). The antibodies were directly conjugated with fluorescein isothiocyanate, phycoerythrin, allophycocyanin (APC) and APC-H7. Cells were stained as described previously and gating of cells was performed using fluorochrome minus one settings,28 and detected using FACSCanto II (BD Biosciences). Analyses with respect to the number of cells and MFI (geometric mean) were performed using the FlowJo Software (Tree Star Inc.). Typical gating is shown in Figure 2.
Transduction of hematopoietic stem cells
Bone marrow was harvested from femur from 3-month-old C57Bl6/J donor mice and hematopoietic stem cells were isolated by negative selection using the EasySep Mouse Hematopoietic Progenitor Cell Enrichment kit (Stemcell Technologies, Manchester, UK). After isolation, hematopoietic stem cells were resuspended in StemSpan with 1% penicillin/streptomycin and the cytokines mouse stem cell factor (100 ng ml−1), Flt-3L (100 ng ml−1), interleukin-11 (100 ng ml−1) and interleukin-3 (20 ng ml−1), and cultured in 12-well plates at a concentration of 1 × 106 cells ml−1. The cells were transduced with the lentiviral constructs CD68/343(f), CD68/150(r) and SP(f) at MOI 30, and incubated at 37 °C overnight. The next morning cells were washed with PBS twice, counted and resuspended at a concentration of 2 × 105 cells/100 μl.
Bone marrow transplantation
C57Bl/6 were obtained from Charles Rivers Laboratories International Inc. (Wilmington, MA, USA) and housed in a pathogen-free barrier facility (12-h light/12-h dark cycle) and fed rodent chow. All animal studies were approved by the local Animal Ethics Committee. One week prior to and 2 weeks after the transplantation 8-week-old C57Bl6/J-recipient mice were given acidified water supplemented with neomycin (100 mg l−1) and polymyxin-B sulfate (10 mg l−1). The recipient mice were irradiated with 9 Gy and reconstituted with transduced stem cells (2 × 105) by an intravenous injection. Mice were killed after 8 weeks and tissues and cells were harvested for analyses.
Assessment of in vivo transgene integration by PCR
To detect vector integration in different cell populations, DNA was prepared from the bone marrow and spleen using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and the WPRE was amplified with the forward primer 5-IndexTermGGCACTGACAATTCCGTGGT-3′, the reverse primer 5′-IndexTermAGGGACGTAGCAGAAGGACG-3′ and the probe 5′IndexTermFAM-ACGTCCTTTCCATGGCTGCTCGC-TAMRA.29 The WPRE copy number was normalized to exon-5 of the mouse titin gene, which was amplified with forward primer 5′-IndexTermAAAACGAGCAGTGACGTGAGC-3′, the reverse primer 5′-IndexTermTTCAGTCATGCTGCTAGCGC-3′ and the probe 5′IndexTermFAM-TGCACGGAAGCGTCTCGTCTCAGTC-TAMRA-3′. Real-time PCR was performed in a mixture containing 2 μl of DNA, 1 × Taqman Universal Mastermix (Applied Biosystems, Foster City, CA, USA), 300 nM each of forward and reverse primers, and 200 nM FAM-labeled probe in a 20-μl reaction. The analysis was performed using the 7500 Real-Time PCR System (Applied Biosystems). All primers were obtained from Sigma-Aldrich and probes from Applied Biosystems.
GFP expression was compared between constructs using a linear regression model, with MOI as continuous variable and construct as fixed variable. The model included construct as a fixed term and MOI as an interaction term (MOI *construct), for example, a regression model with different slopes and intercepts for each construct. For nonlinear responses, MOI was log-transformed before analysis. Expression levels are presented as normalized expression calculated at an average MOI and are presented as mean±s.e.m. Mean expression was compared to zero expression by t-test and was adjusted for multiple tests within each experiment using Bonferroni correction and a P-value less than 0.05 was considered significant. All statistical analyses were performed using IBM SPSS Statistics 19 (IBM Corporation, Armonk, NY, USA).
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We thank Louise Henningsson, Kristina Skålén, Elin Stenfeldt and Maria Heyden (Gothenburg University) for expert technical assistance; Yan Ru Su and John Blakemore (Vanderbilt University) for stimulating discussions and technical expertise; and Rosie Perkins (Gothenburg University) for editing the manuscript. This work was supported by the Swedish Research Council; the VINNOVA Foundation; the IngaBritt and Arne Lundgren Foundation; the Swedish Foundation for Strategic Research; the Sahlgrenska University Hospital ALF research grants; and NIH Grants HL105375, HL57986, HL106845 and HL65709.
The authors declare no conflict of interest.
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Levin, M., Lidberg, U., Jirholt, P. et al. Evaluation of macrophage-specific promoters using lentiviral delivery in mice. Gene Ther 19, 1041–1047 (2012). https://doi.org/10.1038/gt.2011.195
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