Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible ‘all-in-one’ lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16–24 h) and dose-dependent (>0.04 μg ml−1 Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34+ hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34+-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.
Self-inactivating lentiviral vectors (SIN-LVs) have evolved as a powerful tool for gene delivery applications and the technology is used in basic biology and preclinical research, as well as in clinical gene therapy studies. SIN-LVs have the ability to transduce a variety of dividing and nondividing mammalian cells, allowing for robust and long-term transgene (over)expression.1, 2, 3, 4 As a rule, transgene expression is accomplished by constitutive active promoter elements; however, this can be problematic in experimental scenarios requiring rapid on/off regulation of the investigated transgene or in situations where the therapeutic transgene is toxic to critical off-target cells.5, 6 Here, regulated transgene expression systems appear warranted. Although various approaches towards this aim have been described, the tetracycline (Tet)-regulated system introduced by Gossen and Bujard7 in 1992 has remained one of the most powerful and commonly applied strategies. The Tet system is based on the coexpression of two important elements in the respective target cell: (i) the tetracycline response element containing repeats of the Tet-operator sequences (TetO) fused to a minimal promoter and connected to a gene of interest and (ii) the transcriptional transactivator (tTA), a fusion protein of the Tet-repressor (TetR) and the transactivation domain of the herpes simplex virus-derived VP16 protein. Whereas in the originally described version, transgene expression was active in the absence of tetracycline or its potent analogue doxycycline (Dox; referred to as Tet-OFF system), modification of four amino acids within the transactivator protein resulted in a reverse tTA (rtTA), which only binds to TetO in the presence of Dox (Tet-ON system).8
Meanwhile, the Tet system has been successfully incorporated into viral constructs including lentiviral vectors to direct regulated transgene expression in a large number of different tissues and cells.9, 10, 11, 12 In this context, the previous ‘two-vector’ approach expressing the transactivator and the Tet- regulated transgene from two different constructs, more recently, has been replaced by the more advanced ‘all-in-one’ systems expressing the transactivator and Tet-regulated transgene from a single vector construct.13, 14, 15, 16, 17, 18, 19, 20, 21, 22 However, the combination of high inducible transgene expression levels and minimal background activity in the non-induced state has remained as a problem with these constructs. Thus, various other modifications have been introduced to refine the technology. For the transactivator, this included replacement of the original VP16 domain by minimal activation domains, removal of potential splice-donor and splice-acceptor sites and codon optimization, resulting in the improved transactivator variant rtTA2S-M2 with higher sensitivity to Dox and lower baseline activity.23 Furthermore, new Tet-responsive promoter elements have been generated, including modification in the TetO with 36-nucleotide spacing from neighbouring operators to enhance regulation.24 More recently, further refinements generated Tet-responsive promoter elements with low basal activity and an improved dynamic range of expression.25, 26 This includes the pTet-T11 (short: TII) variant displaying a high dynamic range and minimal background activity in the context of γ-retroviral-mediated gene transfer into murine hematopoietic cells.25
An application particularly suited for inducible transgene expression systems is myeloprotective gene therapy. This refers to the transgenic expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem and progenitor cells to prevent or reduce the toxicity of anticancer chemotherapeutic agents.27, 28, 29, 30, 31 To achieve myeloprotection, however, expression of the therapeutic transgene is required only during the relatively short periods of actual chemotherapy application. In this context, we and others have shown that the CTX-R gene cytidine deaminase (CDD) confers protection against nucleoside analogue-type drugs such as cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C).32, 33, 34 Myeloprotective properties of CDD gene transfer in the context of Ara-C application have been established in vitro for murine and human clonogenic hematopoietic progenitor cells as well as in murine in vivo CDD gene transfer models.32, 35, 36, 37, 38 In one of these studies, however, substantial lymphopenia was noted with constitutive CDD overexpression.38
In the current study, we, therefore, use SIN lentivirus with an improved inducible ‘all-in-one’ Tet-system based on the rtTA2S-M2 transactivator variant and the TII promoter element for regulated transgene expression in human hematopoietic cells. We here demonstrate rapid and reliable induction of transgene expression with minimal background activity, and in case of CDD gene transfer, this results in significant protection from Ara-C toxicity in K562 cells as well as in primary hematopoietic cells.
Rapid and robust transgene induction and shut-off kinetics in human hematopoietic K562 cells
First, we evaluated the improved inducible ‘all-in-one’ LV design in human erythroleukemic K562 cells using the Lv.TII.GFP and Lv.TII.CDD construct, expressing either GFP (green fluorescent protein) alone or a CDD-IRES-GFP (CDD-internal ribosome entry site-GFP) cassette (Figure 1a). Transduction of K562 cells showed an efficiency of 30–50% for both constructs by eGFP expression after exposure to 2 μg ml−1 Dox (Figure 1b), and purity of the eGFP-positive population, subsequently, was increased to >90% by fluorescence-activated cell sorting (data not shown). Thereafter, Dox induction kinetics were performed on sorted Lv.TII.CDD-transduced K562 cells following several days of Dox starvation. As demonstrated in Figure 2a, rapid transgene expression was observed upon administration of 2 μg ml−1 of Dox. Induction of eGFP expression already was detectable after 24 h and the plateau levels were reached within 48 h. This observation was confirmed by the analysis of hCDD mRNA (Figure 2b) as well as by protein expression (Figure 2c). The rapid induction of transgene expression by 2 μg ml−1 of Dox was further validated by time-lapse studies highlighting eGFP-positive cells already 15–17 h after Dox administration (Supplementary Movie 1).
To investigate the ‘turn-off’ of transgene expression, Lv.TII.CDD-transduced cells were cultured in the presence of 2 μg ml−1 of Dox for >2 weeks before Dox withdrawal experiments were performed. Rapid shut-off of transgene expression was demonstrated resulting in a reduction of eGFP fluorescence intensity within 24 h and almost non-detectable levels 96–120 h after Dox withdrawal (Figure 2d and Supplementary Movie 2). Similarly, analysis of hCDD expression from the Lv.TII.CDD construct demonstrated a 70% reduction of hCDD mRNA 24 h after Dox withdrawal and almost undetectable levels after 120 h (Figure 2e) and this pattern was confirmed by hCDD protein analysis (Figure 2f).
Minimal background activity of TII-containing lentiviral vectors in K562 cells
To assess eGFP and hCDD background expression in the absence of Dox, Lv.TII.GFP- and Lv.TII.CDD-transduced cells were cultured in the presence or absence of 2 μg ml−1 of Dox. In the absence of Dox, mean fluorescence intensity (MFI) of eGFP in Lv.TII.GFP and Lv.TII.CDD gene-modified cells was comparable to non-transduced K562 cells (Figure 2g). Upon application of Dox, eGFP expression increased 230-fold in Lv.TII.GFP-transduced cells, whereas only a 58-fold induction of eGFP reporter gene was detected in Lv.TII.CDD-transduced cells, a difference that may be explained by reduced transgene expression from the 3′ position relative to the IRES.39 In addition, low to absent background hCDD transgene expression was demonstrated in Lv.TII.CDD-transduced cells by quantitative reverse transcriptase-PCR (qRT-PCR) in the absence of Dox, with a 110-fold increase in expression upon administration of 2 μg ml−1 Dox, further highlighting the robustness of the TII promoter element (Figure 2h).
Dose-dependent transgene expression in K562 cells
To investigate the dose dependency of Dox-induced transgene expression, Lv.TII.CDD-transduced K562 cells were exposed to Dox concentrations ranging from 0 to 2.0 mg ml−1 for various time intervals. As depicted in Figure 3a, eGFP fluorescence was detected already at a Dox concentration of 0.04 μg ml−1 (plateaus of MFI values 180) and reached its maximum at concentrations from 0.2 μg ml−1 onwards (plateau of MFI values 650). For all concentrations, transgene expression was detected as early as 24 h after Dox application, reaching plateau levels after 48 h. Again, the results were confirmed by time-lapse video analysis (Supplementary Video 1). Mean fluorescence intensity levels measured in the presence of 0.002 and 0.008 μg ml−1 of Dox were indistinguishable from non-transduced or non-Dox-treated K562 cells reiterating the stringent regulation of transgene expression from the Ptet-11 promoter also in the off-state (Figures 3a and b).
Inducible CDD expression mediates robust protection from Ara-C-induced toxicity in K562 cells
Functionality of Dox-inducible hCDD expression was evaluated by exposing Lv.TII.CDD- or Lv.TII.GFP-transduced K562 cells to Ara-C following 7 days of preculture in the presence or absence of Dox. As depicted in Figure 4a, Lv.TII.CDD-transduced K562 cells cultured in the presence of 2.0 μg ml−1 Dox survived exposure to up to 2000 nm of Ara-C, whereas non-Dox-treated Lv.TII.CDD cells or Lv.TII.GFP control cells (irrespective of Dox administration) were highly susceptible to Ara-C exposure from 50 nm onwards. We have no explanation, however, for the slightly higher LD50 (lethal dose, 50%) of Lv.TII.GFP-transduced K562 cells following Dox addition as compared with the same cells without Dox exposure, nor have we observed this in the past with the same or similar vectors in other cell types. Moreover, LD50 levels of 150–260 nm Ara-C were observed in our K562 cells, whereas LD50 levels for Lv.TII.CDD-transduced cells cultured in the presence of Dox were not reached in our studies (>2000 nm). Furthermore, in Dox dose–response studies, cultivation of Lv.TII.CDD-transduced K562 cells even in the presence of 0.04 μg ml−1 Dox already resulted in a significantly increased LD50 of 1750 nm Ara-C, with a Dox concentration of 0.2–2.0 nm resulting in stable LD50 values of >2000 nm (LD50 not reached at 2000 nm Ara-C; Figure 4b). On the other hand, exposure to very low concentrations of Dox (0.002 and 0.008 μg ml−1) resulted in LD50 values of ~50 nm Ara-C similar to non-Dox-exposed Lv.TII.CDD- or Lv.TII.GFP-transduced cells (±Dox).
Protection of K562 cells from Ara-C toxicity went hand in hand with rescues from Ara-C-induced cell cycle arrest. Using the fluorescent dye e670 as an indicator for cellular proliferation (Figure 4c), we first demonstrated unperturbed proliferation of Lv.TII.CDD- and Lv.TII.GFP-transduced K562 cells in the presence of 2.0 μg ml−1 of Dox ruling out a significant influence of inducible hCDD transgene expression or the presence of Dox on the proliferative behaviour of K562 cells. Thereafter, we investigated the effect of Ara-C treatment (200 nm) on cell proliferation of non-, Lv.TII.GFP- or Lv.TII.CDD-transduced cells in the presence and absence of 2.0 μg ml−1 of Dox. Unaltered proliferation could only be observed for Lv.TII.CDD-transduced cells in the presence of Ara-C but not for control- or Lv.TII.GFP cells, demonstrating the rescue of K562 cells from Ara-C-induced cell cycle arrest by inducible hCDD expression (Figure 4d).
Tightly regulated Dox-induced transgene expression in primary hematopoietic cells
To investigate inducible transgene expression in primary hematopoietic cells, CD34+ cells were transduced with Lv.TII.GFP and exposed to 2.0 μg ml−1 Dox. Transduction efficiency was ~20%. Again transgene expression was detectable within 24 h of Dox administration with plateau levels reached at 48–72 h (Figure 5a). Furthermore, when dose–response kinetics were assessed for Dox concentrations ranging from 0 to 2.0 μg ml−1, a dose- dependent increase in eGFP expression was observed for concentrations of 0.04 μg ml−1, with plateau levels reached at 1.0–2.0 μg ml−1 of Dox (Figure 5b and Supplementary Figure 1) resembling the data in K562 cells. Upon Dox administration, an ~300-fold increase in eGFP expression was detected as shown 72 h after Dox application (Supplementary Figure 2). The dose response was confirmed by western blot analysis of Lv.TII.GFP-transduced CD34+ cells 4 days after Dox administration. Of note, low to if any background eGFP expression was observed in Lv.TII.GFP-transduced CD34+ cells exposed to 0–0.002 μg ml−1 Dox, further confirming the tight control of transgene expression by the Lv.TII vector design (Figure 5c). Similarly, high inducible transgene expression with low background activity was demonstrated for the hCDD gene by western blot analysis in primary CD34+ cells (Figure 5d).
In addition to CD34+ progenitor cells, robust Dox-induced GFP (Figure 5e) or hCDD (Figure 5f) transgene expression also was detected in more mature myeloid cells differentiated by exposure to granulocyte–macrophage-colony-stimulating factor (GM-CSF) or GM-CSF/granulocyte colony-stimulating factor/interleukin-3 (GM-CSF/G-CSF/IL-3), respectively. Furthermore, in terms of functional CDD expression, marked drug resistance was observed for myeloid cells derived from Lv.TII.CDD-transduced CD34+ cells by exposure to G-CSF/IL-3 for 7 days. In the presence of 2.0 μg ml−1 of Dox, Lv.TII.CDD-transduced cells were protected from up to 300 nm Ara-C, whereas Lv.TII.GFP-transduced control cells died from concentrations of 50 nm Ara-C onwards (Figure 5g).
No differences in differentiation capabilities between Lv.TII.GFP- and Lv.TII.CDD-transduced cells were observed in these experiments. Thus, differentiation of Lv.TII.CDD- or Lv.TII.GFP-transduced CD34+ cells by GM-CSF/G-CSF/IL-3 yielded equal numbers and proportions of myeloid cells on cytospins (Table 1 and Figure 5h) with similar surface expression of CD66b, CD11b and CD33 (Figure 5i).
In the present study, we describe an optimized Dox-inducible ‘all-in-one’ SIN lentiviral vector construct allowing for robust and tightly regulated transgene expression. The construct uses the pTet-T11 (TII) promoter element to direct transgene expression25 and constitutively expresses a reverse transactivator protein (rtTA2S-M2)23 from the PGK promoter. When the vector was explored in human hematopoietic cell lines and primary cells, robust and rapid inducible transgene expression with low background expression was demonstrated, as well as the potential to direct high-level expression of the drug resistance gene CDD, thus rendering the construct suitable for potential use in myeloprotective gene therapy strategies. The data extend our previous work in the murine model with an earlier generation Tet-ON system.37
A core component of the new ‘all-in-one’ SIN-LV vector design is represented by the TII promoter element, which previously has been investigated successfully in the context of γ-retroviral and transposon-based gene transfer. This element comprises consensus sequences of the TATA box (cTATA), binding sites for the transcription factor IIB and a turnip yellow mosaic virus 5′-untranslated region and mouse mammary tumour virus promoter sequences fused to optimized TetO heptamers. When integrated in γ-retroviral vectors, the TII element allowed for highly dynamic transgene upregulation upon Dox exposure with minimal background activity in murine hematopoietic cells.25 We here confirmed and extended these results in the context of an SIN-LV vector and human hematopoietic K562, as well as primary CD34+ cells. These data are in line with results of other groups evaluating alternative γ-retroviral or lentiviral ‘all-in-one’ or two-vector systems.12, 15, 17, 37, 40, 41, 42 Transgene regulation by Dox, in our hands, was stringently dose-dependent with levels >0.04 μg ml−1 needed for efficient induction. Although response to even lower Dox concentrations has been described for certain improved pTet promoter variants,26, 43 our data of >0.04 μg ml−1 Dox as a threshold for induction are well within the range reported so far.13, 41 Certainly, the exact levels of Dox required to trigger transgene expression vary between cell sources and are influenced by parameters specific to individual cell types such as the specific activity of the TII promoter in these cells, the levels of transactivator generated by the respective constitutive active promoter (here the PGK promoter) or the uptake capacity of the cell for the effector Dox.43, 44 Based on this background, the observed differences in the minimal effective Dox concentrations between K562 and primary CD34+ hematopoietic cells in our study are not surprising.
For the majority of settings, a successful clinical application of regulated transgene expression systems will require a reliable high-level induction of the therapeutic transgene (On-state) with minimal background activity in the Off-state (‘leakiness’). In this context, we here demonstrate ~110-fold increased hCDD mRNA expression levels in Lv.TII.CDD and a ~230-fold induction of GFP in Lv.TII.GFP-transduced K562 cells highlighting the capacity of our Dox-mediated gene induction system. In addition, ~300-fold transgene induction was observed in our experiment with Lv.TII.GFP- transduced primary CD34+. These data recapitulate or even compare favourably with other studies using all-in-one lentiviral vectors for inducible transgene expression.13, 15, 45 Even more important, however, our study reveals low background activity in the non-induced state in both K562 and CD34+ cells, confirming the results achieved with the pTet-T11 promoter in murine hematopoietic cells in the context of γ-retroviral gene transfer25 and extending these to SIN-LV constructs and human cells. Also, in this respect, our data show improvements over other investigations in the field. Although the two groups have reported low background activity with considerable induction rates in murine and human hematopoietic cells,13, 14 other studies using the Tet system in the context of lentiviral gene transfer were compromised by considerable background activity.45, 46 The good results for the TII come to no surprise, as the intrinsic background activity of the TII promoter has been systematically reduced by stepwise modification of its predecessor, the Ptet-1 promoter including the removal of hidden binding sites for transcription factors.25, 26
Furthermore, our vector architecture positioning the Tet-expression cassette within the vector backbone represents an important safety feature. As a consequence in our construct, only a single Tet-inducible promoter integrates. In contrast, position of the Tet-inducible cassette within the long-terminal repeat results in two copies of the Tet-inducible promoter in the integrated provirus.22 Whereas the inducible promoter at the 5′ end of the vector expresses the transgene of choice, also the 3′ portion of the vector contains a transactivator binding cassette, which, although not used for Tet-dependent transgene regulation, potentially increases the risk of deregulation of adjacent genomic regions by the integrated vector.
Given that myeloprotective gene therapy approaches require expression of the therapeutic drug resistance gene in principle for the relatively short periods of cytotoxic drug application, we here tested our vector construct for regulated expression of the drug resistance gene CDD.29 Confirming previous data generated in murine hematopoietic cells with an earlier generation Tet-ON system,37 we here demonstrated Lv.TII.CDD-transduced K562 cells to be effectively protected from Ara-C-induced toxicity at Dox concentrations >0.04 μg ml−1. Furthermore, also primary human CD34+-derived myeloid cells were efficiently protected from Ara-C toxicity upon Lv.TII.CDD and Dox exposure. Of note, protection of differentiated myeloid cells was observed over a similar dose range of Ara-C as previously described for constitutive hCDD overexpression in human clonogenic progenitor cells.35
In general, the SIN-LV vector design, as used in our studies, has been shown to substantially reduce the risk of insertional mutagenesis when compared with γ-retroviral vectors47, 48 even though definite proof for this concept also in the context of Dox-regulated vectors is still missing. In addition, promoter/enhancer activity has been shown to influence markedly the risk of vector-induced oncogenesis.49, 50 Thus, the only temporary activity of the T11 promoter may offer a way to reduce genotoxicity despite the employment of strong promoter/enhancer elements as may be required for adequate expression of therapeutic transgenes. This appears to carry particular relevance for myeloprotective gene therapy approaches, as here the additional exposure of cells to potentially DNA-damaging chemotherapeutic agents may further increase the risk of genotoxicity.
A potential drawback of Tet-regulated expression systems with regard to clinical applications clearly is associated with the immunogenicity of the continuously expressed transactivator protein as studies in rodents and non-human primates have demonstrated a cellular as well as a humoral immune response to the TET activator.51, 52, 53 Given the therapeutic efficacy of Ara-C primarily in acute leukaemias, allogeneic transplant settings in this disease entity represents the most likely scenario for the clinical application of Dox-regulated hCDD expression.29 In this situation, however, immunogenicity may be less an issue, as de novo formation of the immune system might induce tolerance to components of the Tet system.54 In case of not or only partially myeloablated hosts, however, additional strategies such as immunologically inert transactivators or immunomodulatory approaches such as regulatory T cells or dendritic cells may be required.55, 56, 57, 58
We here introduced an ‘all-in-one’ lentiviral vector featuring an improved pTet promoter element with very low background activity for regulated transgene expression in human hematopoietic cells. Using this construct, we could demonstrate stringently regulated functional expression of the drug resistance gene CDD in human hematopoietic cells, rendering our improved ‘all-in-one’ SIN lentiviral construct a highly interesting choice for gene therapy approaches in which only temporary transgene expression is required such as myeloprotective gene therapy.
Materials and methods
Human peripheral blood or umbilical cord blood samples were collected after written informed consent of the donors by either Hannover Medical School (Hannover, Germany) or Vinzenz Hospital (Hannover, Germany) as approved by the Institutional Ethic Committees.
Lentiviral constructs were based on third-generation SIN lentiviral vectors modified with a woodchuck hepatitis virus-derived posttranscriptional regulatory element.59 The RRL.PPT.TII.eGFP.pre (kindly provided by Niels Heinz, Langen, Germany), referred to as Lv.TII.GFP, served as a control vector. To generate RRL.PPT.TII.CDD.IRES.eGFP.pre, referred to as Lv.TII.CDD, cDNA of hCDD (Open Biosystems, IHS1380-OB-97652440, Epsom, UK) and IRES.eGFP expression cassette were excised with AgeI and BsrGI restriction enzymes from the lentiviral construct RRL.PPT.SFFV.CDD.IRES.eGFP.pre. The resulting hCDD and IRES.eGFP fragments were cloned in a two-step process into the AgeI/BsrGI-digested Lv.TII.GFP control vector substituting eGFP with hCDD.IRES.eGFP expression cassette. All products were verified by restriction enzyme digestion and sequencing.
Production of viral vector and titration
Viral supernatants were generated by transient transfection of 293 T cells as described previously.60 In brief, 7 × 106 293 T cells cultured in high-glucose Dulbecco’s modified Eagle’s Medium (PAA, Pasching, Germany) supplemented with 10% foetal calf serum, 100 Uml−1 penicillin/streptomycin and 2 mmol l−1 glutamine (all PAA) were seeded 24 h before transfection in 10 cm dishes. Cells were co-transfected with 5 μg lentiviral vector and third-generation packaging constructs (8 μg pcDNA3.GP.4xCTE (expressing HIV-1 gag/pol; 5 μg pRSV-Rev; 2 μg pMD.G (encoding the vesicular stomatitis glycoprotein)) using calcium phosphate precipitation in the presence of 20 mmol l−1 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PAA) and 25 mmol l−1 chloroquine (Sigma-Aldrich, Darmstadt, Germany). Supernatants were harvested 44 and 68 h after transfection and concentrated using ultracentrifugation (Beckman Coulter, Krefeld, Germany) for 3 h at 30 000 g and 4 °C. Viral titres were determined by transduction of murine SC1 fibroblasts with serial dilution of viral supernatant in the presence of 2 μg ml−1 Dox-Hyclate (Dox) (Sigma-Aldrich). The percentage of transduced cells was determined 72 h after transduction by flow cytometric analysis (FACSCalibur; Becton Dickinson, Heidelberg, Germany) of eGFP-expressing cells.
Isolation of human CD34+ cells, cultivation and myeloid differentiation
Peripheral blood mononuclear cells were isolated from human umbilical cord blood samples or G-CSF-mobilized peripheral blood using density gradient centrifugation. Subsequently, CD34+ cells were positively selected using magnetic separation according to the manufacturer’s instructions (Miltenyi, Bergisch Gladbach, Germany). Following purification, cells were seeded in 24-well cell culture plates and cultured in StemSpan (StemCell Technologies, Grenoble, France) supplemented with 100 ng ml−1 murine kit ligand/stem cell factor, 50 ng ml−1 murine thrombopoietin and 100 ng ml−1 human FMS-like tyrosine kinase 3-ligand (STF media). For myeloid differentiation, CD34+ were cultivated for ~10 days in RPMI supplemented with 2 μg ml−1 Dox, 10% foetal calf serum, 100 U ml−1 penicillin/streptomycin and 2 mmol l−1 glutamine (Life Technologies, Ober-Olm, Germany), 50 ng ml−1, hG-CSF, 50 ng ml−1, hGM-CSF and 25 ng ml−1 hIL-3. Differentiation into granulocytes was performed by the addition of 100 ng ml−1 hG-CSF and 50 ng ml−1 hIL-3 and for granulocyte–macrophage generation, 100 ng ml−1 hGM-CSF and 50 ng ml−1 hIL-3 were used (all cytokines from Peprotech, Hamburg, Germany).
Transduction of human hematopoietic cells
The human hematopoietic cell line K562 was cultured in RPMI-1640 supplemented with 10% foetal calf serum, 100 U ml−1 penicillin/streptomycin and 2 mmol l−1 glutamine. For transduction, 1 × 105 cells were seeded in 24-well cell culture plates in the presence of 10 μg ml−1 protamine sulphate (Sigma- Aldrich) and concentrated lentivirus was added. Twenty-four hours after transduction, cells were exposed to 2 μg ml−1 of Dox for 48 h and subsequently sorted for eGFP expression (FACS AriaIIu; Becton Dickinson, Franklin Lakes, NJ, USA) to establish transgenic K562 cells of purity >97%.
Primary CD34+ cells were prestimulated for 24 h in complete medium (as mentioned above) and transduced with Lv.TII.CDD or Lv.TII.GFP on retronection-coated dishes (10 μg cm−2; Takara, Kyoto, Japan) as recommended by the manufacturer. Twenty-four hours after transduction, cells were exposed to 2 μg ml−1 of Dox to induce transgene expression and non-sorted cells were used for further experiments.
For surface molecule expression of CD34+-derived myeloid cells, cells were stained for 45 min with conjugated antibodies as recommended by the manufacturer using CD11b PE, CD66b APC, CD33 PE-Cy5 and CD34 e450 (eBioscience, Frankfurt, Germany). Analysis was carried out by pregating on GFP+ cells using the FlowJo software (TreeStar, Ashland, OR, USA).
Transgene expression kinetics
Gene-modified K562 cells were cultured in the presence of 2 μg ml−1 of Dox and transgene expression (hCDD and GFP) was analysed by flow cytometry and qRT-PCR analysis at different time points (Dox-ON kinetic). For Dox-OFF kinetic studies, transgenic K562 cells were prestimulated for 2 weeks with 2 μg ml−1 of Dox. Subsequently, cells were washed with phosphate-buffered saline (GE Healthcare, Munich, Germany) and cultured without Dox. Again, transgene expression was analysed at different time points by flow cytometry and qRT-PCR. Furthermore, induction of transgene expression was determined by flow cytometry in the presence of different concentrations of Dox for transgenic K562 cells and primary CD34+ cells, respectively. The rate of induction was calculated as a ratio between MFI of GFP+ cells and MFI of non-induced cells (background MFI).
In vitro protection assay
For in vitro protection as well as LD50 analysis, transgenic K652 cells were cultured for 3 days in the presence of different concentrations of Ara-C (Neocorp AG, Weilheim, Germany) and without or with different concentrations of Dox. Cell survival was assessed by flow cytometric analysis.
Gene-modified CD34+ cells, presorted for GFP expression and further differentiated into granulocytes as described above, were cultured in the presence of different concentrations of Ara-C for 4 days, as well as with 2.0 μg ml−1 of Dox. Cell survival was assessed by flow cytometry analysis.
Quantitative reverse transcriptase-PCR
For qRT-PCR, total RNA was isolated using the GeneElute Mammalian Total RNA Kit (Sigma-Aldrich) followed by DNaseI digestion (Thermo Scientific, Erlangen, Germany) according to the manufacturer’s instructions. RNA was reverse transcribed using RevertAid reverse transcriptase and oligo(dT) primers (Thermo Scientific) and 30 ng cDNA was used for subsequent quantification by qRT-PCR. TaqMan-based qRT-PCR was performed using TaqMan Universal Master Mix II (Applied Biosystems, Darmstadt, Germany) with the following predesigned assays: hCDD (HS00156401_m1) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Hs99999905_m1). qRT-PCR was performed on an Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems).
Protein isolation was performed by cell lysis using RIPA buffer (Sigma-Aldrich) in the presence of protease inhibitors (Roche, Mannheim, Germany). Proteins were resolved by electrophoresis on 12% sodium dodecyl sulphate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, Madison, WI, USA). After blocking in TBST containing 5% dried skimmed milk powder for 1 h, membranes were incubated with the following primary antibodies at 4 °C overnight: anti-vinculin (V9131; Sigma-Aldrich), anti-GFP (A11122; Life Technologies, Darmstadt, Germany) and anti-hCDD (ab56053; Abcam, Cambridge, UK). Subsequently, membranes were rinsed with TBST and incubated with the appropriate horseradish peroxidase-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Protein detection was performed using enhanced chemoluminescence reagents (SuperSignal West Femto/Pico; Thermo Scientific) and the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA).
In vitro proliferation assay
Cell proliferation analysis of (transduced) K652 cells was determined using cell proliferation dye eFluor 670 (eBioscience, San Diego, CA, USA) according to the manufacturer’s recommendation. In brief, 1 × 106 cells were washed with phosphate-buffered saline to remove any serum followed by staining with prewarmed 10 μm solution of cell proliferation dye eFluor 670 in phosphate-buffered saline for 10 min at 37 °C. Subsequently, stained cells were washed three times with K652 culture medium and analysed for the initial time point of dye uptake marking it as day 0 (d0). After cultivation for additional 48 h in 12-well suspension plates (Sarstedt, Nümbrecht, Germany), marked cells were analysed by flow cytometry in the FL4 channel (FACSCalibur; Becton Dickinson) for the dye dilution using the same instrument settings from day 0. In addition, proliferation experiments were performed including treatment with Ara-C.
Time-lapse video microscopy
Continuous live-cell imaging of Dox-dependent eGFP reporter expression was performed with Lv.TII.CDD-transduced K562 cells. For Dox-ON kinetic, transduced cells starved of Dox for 4 or more weeks were exposed to different concentrations of Dox (0, 0.002, 0.008, 0.04, 0.2, 1.0 and 2.0 μg ml−1) on day 0. To avoid floating of suspension cells during the image processing, 5 × 104 cells were immobilized in non-adhesive triangular microcavities. Brightfield and fluorescence images were captured in 20 min intervals for at least 65 h for Dox-ON using the AxioObserver Z1 fluorescence microscope (Zeiss, Jena, Germany) with a 37 °C humidity chamber and 5% CO2 levels. Video analysis was performed with Axiovision Software 4.70 (Zeiss).
Statistical analysis was performed using Prism 6 software (GraphPad, La Jolla, CA, USA). Unless otherwise noted, analysis of variance (ANOVA) with respective post hoc testing (see figure legend) was used.
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We thank Matthias Ballmaier (PhD) and his team from the Core Facility Cell Sorting of Hannover Medical School for cell sorting and Doreen Lüttge (Hannover Medical School) for excellent technical assistance. Furthermore, we thank Georg Kensah (Hannover Medical School, now Otto-von-Guericke University, Magdeburg, Germany) for help in performing the time-lapse video studies. This work was supported by grants from the Deutsche Forschungsgemeinschaft: Cluster of Excellence REBIRTH (Exc 62/1), SPP1230 Grant MO 886/3-1 (to TM), Grant MO 886/4-1 (TM), the EU framework program grant PERSIST (to CHB) and Hannover Biomedical Research School (HBRS; DFG, GSC 108).
The authors declare no conflict of interest.
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Lachmann, N., Brennig, S., Hillje, R. et al. Tightly regulated ‘all-in-one’ lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy. Gene Ther 22, 883–892 (2015). https://doi.org/10.1038/gt.2015.61
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