Tightly regulated ‘all-in-one’ lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy

Abstract

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.

Introduction

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.

Results

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).

Figure 1
figure1

Dox-inducible ‘all-in-one’ lentiviral constructs for improved CDD expression in K562 cells. Self-inactivating lentiviral (SIN-LV) vectors for Dox-inducible transgene expression. (a) Third-generation SIN-LV vectors, expressing either the hCDD (Lv.TII.CDD) or eGFP (Lv.TII.GFP) in the presence of Dox (Dox-ON) from the pTet-11 promoter sequence. The pTet promoter consists of a TetO hexamer fused to the minimal promoter of cytomegalovirus (mCMV). Constitutive expression of the reverse Tet-responsive transactivator protein (rtTA2S-M2) is regulated by the human phosphoglycerate kinase (hPGK) promoter. SIN-LV vectors contain 5′ and 3′ long-terminal repeats with SIN deletion in the U3 region (LTRs, ΔU3, R, U5), splice-donor (SD) and splice-acceptor (SA) sites, the posttranscriptional regulatory element of woodchuck hepatitis virus (wPRE), a central polypurine tract (cPPT), the Rev-responsive element (RRE) and an extended encapsidation signal (Ψ) including the 5′ region of gag (ΔGA). (b) K562 cells transduced with lentiviral Lv.TII.CDD or Lv.TII.GFP constructs, regulating eGFP expression either in the absence or presence of Dox administration.

Figure 2
figure2

Tightly regulated transgene induction kinetics of transduced K562 by administration of Dox. Dox-ON kinetic of Lv.TII.CDD-transduced human K562 cells investigating 2.0 μg ml−1 of Dox at different time intervals by either (a) flow cytometry analysis by MFI of eGFP expression (mean±s.d., n=3, significance is relative to 0 h by one-way ANOVA with Dunnet's post hoc), (b) qRT-PCR for hCDD mRNA (normalized to human GAPDH expression and relative to time point 0) (mean±s.e.m., n=2, significance is relative to 0 h by one-way ANOVA with Dunnet's post hoc) or (c) western blot analysis for hCDD protein expression (vinculin used as a loading control). Kinetics of Dox withdrawal in Lv.TII.CDD-transduced human K562 cells at different time intervals by either (d) flow cytometry analysis by MFI of eGFP expression (mean±s.d., n=3, significance is relative to 0 h by one-way ANOVA with Dunnet's post hoc), (e) qRT-PCR for hCDD (normalized to human GAPDH expression and relative to time point 0) (mean±s.e.m., n=3, significance is relative to 0 h by one-way ANOVA with Dunnet's post hoc) or (f) western blot analysis for hCDD protein expression (vinculin used as a loading control). Background expression was measured by (g) flow cytometry (MFI) of eGFP (mean ±s.e.m., n=3, two-way ANOVA with Bonferroni post hoc) or (h) qRT-PCR of hCDD in untransduced (untrans.), Lv.TII.GFP- or Lv.TII.CDD-transduced K562 cells either in the presence or absence of Dox (normalized to human GAPDH expression and relative to untransduced cells; mean±s.e.m., n=6, two-way ANOVA with Bonferroni post hoc). (*P0.05; **P0.01; ***P0.001; ****P0.0001).

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).

Figure 3
figure3

Transgene expression can be regulated by different concentrations of Dox. Dox-dependent transgene expression was evaluated in Lv.TII.CDD-transduced human K562 cells either by (a) flow cytometry analysis by MFI of eGFP expression or (b) time-lapse studies indicated for different time points and different concentrations of Dox (one representative experiment is shown (scale bar: 100 μm)).

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).

Figure 4
figure4

Dox-mediated hCDD expression confers protection against Ara-C and prevents transduced cells from Ara-C-mediated cell cycle arrest. Functional hCDD expression is assessed by (a) analysis of Ara-C-induced cell death in Lv.TII.GFP- or Lv.TII.CDD-transduced cells in the absence (−Dox) or presence (+Dox) of 2.0 μg ml−1 Dox (mean ±s.d., n=3, significance is relative to Lv.TII.CDD-Dox by two-way ANOVA with Bonferroni post hoc n=3) (normalized to 0 nm Ara-C as 100% living cells) and (b) by LD50 values of Ara-C in Lv.TII.CDD-transduced cells exposed for 72 h to different concentrations of Dox (mean ±s.d., n=3, significance is relative to 0 μg ml−1 Dox by one-way ANOVA with Dunnet's post hoc). (c) Cell proliferation capacity in the absence of chemotherapeutic agent Ara-C was evaluated by flow cytometric analysis using e670 dye (FL4) of untransduced (black curve), Lv.TII.GFP- (blue curve) or Lv.TII.CDD- (red curve) transduced K562 cells. (d) Protection from cell cycle arrest by inducible hCDD overexpression. The assay is performed using e670 dye followed by an administration of 200 nm Ara-C in untransduced, Lv.TII.GFP- or Lv.TII.CDD-transduced K562 cells (blue curve=day 0 of e670 treatment; red curve=day 2 postadministration of e670 and 0 nm Ara-C; black curve=day 2 postadministration of e670 with 200 nm Ara-C (***P0.001).

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).

Figure 5
figure5

Tightly regulated transgene induction kinetics in umbilical human cord blood-derived transduced CD34+ cells and differentiated myeloid cells. (a) Dox-mediated eGFP (MFI) induction kinetic was evaluated in umbilical cord blood-derived CD34+ cells transduced with Lv.TII.GFP in the presence 2.0 μg ml−1 of Dox (mean±s.d., n=3) or (b) with different concentrations of Dox and different time intervals (MFI of eGFP (FL1), n=1). (c) In addition, GFP (33 kDa) expression was analysed for different Dox concentrations at 96 h by western blot analysis of Lv.TII.GFP-transduced CD34+ cells. (d) Similarly, CDD expression was analysed in Lv.TII.CDD- or Lv.TII.GFP-transduced CD34+ cells 144 h after administration of 2 μg ml−1 of Dox (c and d: vinculin (116 kDa) was used as a loading control). (e) GFP transgene expression was evaluated by fluorescence microscopy of Lv.TII.GFP-transduced CD34+-derived myeloid cells differentiated with GM-CSF for 10 days (two different magnifications shown; scale bars: 50 μm (left column) and 20 μm (right column)). (f) Similarly, CDD expression was evaluated by western blot analysis of Lv.TII.GFP- or Lv.TII.CDD-transduced CD34+-derived myeloid cells in the presence of GM-CSF/G-CSF/IL-3 and 2.0 μg ml−1 Dox for 10 days. (g) Drug resistance was analysed using Lv.TII.CDD- and Lv.TII.GFP-transduced CD34+-derived myeloid cells in the presence of 2 μg ml−1 Dox and Ara-C for 96 h. Cell survival for the different Ara-C concentrations was evaluated by flow cytometry using life cell exclusion by SSC/FSC (side scatter/forward scatter) gating (normalized to 0 nm Ara-C as 100% living cells; mean±s.d., n=2). Cell morphology and surface marker analysis of Lv.TII.CDD- and Lv.TII.GFP-transduced CD34+-derived myeloid cells was evaluated by (h) cytospins (sorted for GFP expression prior cytospin) or (i) flow cytometric analysis 144 h after administration of Dox in the presence of GM-CSF/G-CSF and IL-3 (pregated on GFP-positive population; Lv.TII.CDD (red), Lv.TII.GFP (blue)).

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).

Table 1 Differential count of CD34+-derived myeloid cells transduced with Lv.TII.GFP or Lv.TII.CDD

Discussion

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

Ethical statement

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.

Vector design

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.

Flow cytometry

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).

Western blot

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

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.

References

  1. 1

    Dropulic B . Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther 2011; 22: 649–657.

    CAS  Article  Google Scholar 

  2. 2

    Sakuma T, Barry MA, Ikeda Y . Lentiviral vectors: basic to translational. Biochem J 2012; 443: 603–618.

    CAS  Article  Google Scholar 

  3. 3

    Schambach A, Baum C . Clinical application of lentiviral vectors—concepts and practice. Curr Gene Ther 2008; 8: 474–482.

    CAS  Article  Google Scholar 

  4. 4

    Schambach A, Zychlinski D, Ehrnstroem B, Baum C . Biosafety features of lentiviral vectors. Hum Gene Ther 2013; 24: 132–142.

    CAS  Article  Google Scholar 

  5. 5

    Milsom MD, Jerabek-Willemsen M, Harris CE, Schambach A, Broun E, Bailey J et al. Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells. Cancer Res 2008; 68: 6171–6180.

    CAS  Article  Google Scholar 

  6. 6

    Gentner B, Visigalli I, Hiramatsu H, Lechman E, Ungari S, Giustacchini A et al. Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy. Sci Transl Med 2010; 2: 58ra84.

    CAS  Article  Google Scholar 

  7. 7

    Gossen M, Bujard H . Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 5547–5551.

    CAS  Article  Google Scholar 

  8. 8

    Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H . Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 1766–1769.

    CAS  Article  Google Scholar 

  9. 9

    Koponen JK, Kankkonen H, Kannasto J, Wirth T, Hillen W, Bujard H et al. Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. Gene Therapy 2003; 10: 459–466.

    CAS  Article  Google Scholar 

  10. 10

    Pluta K, Luce MJ, Bao L, Agha-Mohammadi S, Reiser J . Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J Gene Med 2005; 7: 803–817.

    CAS  Article  Google Scholar 

  11. 11

    Vieyra DS, Goodell MA . Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator. Stem Cells 2007; 25: 2559–2566.

    CAS  Article  Google Scholar 

  12. 12

    Yang WH, Yang C, Xue YQ, Lu T, Reiser J, Zhao LR et al. Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro. PLoS One 2013; 8: e64389.

    Article  Google Scholar 

  13. 13

    Barde I, Zanta-Boussif MA, Paisant S, Leboeuf M, Rameau P, Delenda C et al. Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector. Mol Ther 2006; 13: 382–390.

    CAS  Article  Google Scholar 

  14. 14

    Centlivre M, Zhou X, Pouw SM, Weijer K, Kleibeuker W, Das AT et al. Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo. Gene Ther 2010; 17: 14–25.

    CAS  Article  Google Scholar 

  15. 15

    Giry-Laterriere M, Cherpin O, Kim YS, Jensen J, Salmon P . Polyswitch lentivectors: 'all-in-one' lentiviral vectors for drug-inducible gene expression, live selection, and recombination cloning. Hum Gene Ther 2011; 22: 1255–1267.

    CAS  Article  Google Scholar 

  16. 16

    Huang Y, Zhen R, Jiang M, Yang J, Yang Y, Huang Z et al. Development of all-in-one multicistronic Tet-On lentiviral vectors for inducible co-expression of two transgenes. Biotechnol Appl Biochem 2014; 62: 48–54.

    Article  Google Scholar 

  17. 17

    Markusic D, Oude-Elferink R, Das AT, Berkhout B, Seppen J . Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter. Nucleic Acids Res 2005; 33: e63.

    Article  Google Scholar 

  18. 18

    Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ et al. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol 2011; 29: 79–83.

    CAS  Article  Google Scholar 

  19. 19

    Yamaguchi T, Hamanaka S, Kamiya A, Okabe M, Kawarai M, Wakiyama Y et al. Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming. PLoS One 2012; 7: e41007.

    CAS  Article  Google Scholar 

  20. 20

    Tian X, Wang G, Xu Y, Wang P, Chen S, Yang H et al. An improved tet-on system for gene expression in neurons delivered by a single lentiviral vector. Hum Gene Ther 2009; 20: 113–123.

    CAS  Article  Google Scholar 

  21. 21

    Paulus W, Baur I, Boyce FM, Breakefield XO, Reeves SA . Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J Virol 1996; 70: 62–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Vigna E, Amendola M, Benedicenti F, Simmons AD, Follenzi A, Naldini L . Efficient Tet-dependent expression of human factor IX in vivo by a new self-regulating lentiviral vector. Mol Ther 2005; 11: 763–775.

    CAS  Article  Google Scholar 

  23. 23

    Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W . Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000; 97: 7963–7968.

    CAS  Article  Google Scholar 

  24. 24

    Agha-Mohammadi S, O'Malley M, Etemad A, Wang Z, Xiao X, Lotze MT . Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness. J Gene Med 2004; 6: 817–828.

    CAS  Article  Google Scholar 

  25. 25

    Heinz N, Schambach A, Galla M, Maetzig T, Baum C, Loew R et al. Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum Gene Ther 2011; 22: 166–176.

    CAS  Article  Google Scholar 

  26. 26

    Loew R, Heinz N, Hampf M, Bujard H, Gossen M . Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol 2010; 10: 81.

    Article  Google Scholar 

  27. 27

    Adair JE, Beard BC, Trobridge GD, Neff T, Rockhill JK, Silbergeld DL et al. Extended survival of glioblastoma patients after chemoprotective HSC gene therapy. Sci Transl Med 2012; 4: 133ra57.

    PubMed  PubMed Central  Google Scholar 

  28. 28

    Flasshove M, Moritz T, Bardenheuer W, Seeber S . Hematoprotection by transfer of drug-resistance genes. Acta Haematol 2003; 110: 93–106.

    CAS  Article  Google Scholar 

  29. 29

    Lachmann N, Brennig S, Phaltane R, Flasshove M, Dilloo D, Moritz T . Myeloprotection by cytidine deaminase gene transfer in antileukemic therapy. Neoplasia 2013; 15: 239–248.

    CAS  Article  Google Scholar 

  30. 30

    Moritz T, Williams DA . Marrow protection—transduction of hematopoietic cells with drug resistance genes. Cytotherapy 2001; 3: 67–84.

    CAS  Article  Google Scholar 

  31. 31

    Neff T, Beard BC, Peterson LJ, Anandakumar P, Thompson J, Kiem HP . Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood 2005; 105: 997–1002.

    CAS  Article  Google Scholar 

  32. 32

    Flasshove M, Frings W, Schroder JK, Moritz T, Schutte J, Seeber S . Transfer of the cytidine deaminase cDNA into hematopoietic cells. Leuk Res 1999; 23: 1047–1053.

    CAS  Article  Google Scholar 

  33. 33

    Neff T, Blau CA . Forced expression of cytidine deaminase confers resistance to cytosine arabinoside and gemcitabine. Exp Hematol 1996; 24: 1340–1346.

    CAS  Google Scholar 

  34. 34

    Momparler RL, Eliopoulos N, Bovenzi V, Letourneau S, Greenbaum M, Cournoyer D . Resistance to cytosine arabinoside by retrovirally mediated gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells. Cancer Gene Ther 1996; 3: 331–338.

    CAS  Google Scholar 

  35. 35

    Bardenheuer W, Lehmberg K, Rattmann I, Brueckner A, Schneider A, Sorg UR et al. Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells. Leukemia 2005; 19: 2281–2288.

    CAS  Article  Google Scholar 

  36. 36

    Brennig S, Rattmann I, Lachmann N, Schambach A, Williams DA, Moritz T . In vivo enrichment of cytidine deaminase gene-modified hematopoietic cells by prolonged cytosine-arabinoside application. Cytotherapy 2012; 14: 451–460.

    CAS  Article  Google Scholar 

  37. 37

    Lachmann N, Brennig S, Pfaff N, Schermeier H, Dahlmann J, Phaltane R et al. Efficient in vivo regulation of cytidine deaminaseexpression in the haematopoietic system using a doxycycline-inducible lentiviral vector system. Gene Therapy 2013; 20: 298–307.

    CAS  Article  Google Scholar 

  38. 38

    Rattmann I, Kleff V, Sorg UR, Bardenheuer W, Brueckner A, Hilger RA et al. Gene transfer of cytidine deaminase protects myelopoiesis from cytidine analogs in an in vivo murine transplant model. Blood 2006; 108: 2965–2971.

    CAS  Article  Google Scholar 

  39. 39

    Mizuguchi H, Xu Z, Ishii-Watabe A, Uchida E, Hayakawa T . IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 2000; 1: 376–382.

    CAS  Article  Google Scholar 

  40. 40

    Kustikova OS, Schwarzer A, Stahlhut M, Brugman MH, Neumann T, Yang M et al. Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells. Leukemia 2013; 27: 1127–1138.

    CAS  Article  Google Scholar 

  41. 41

    Reuss S, Sebestyen Z, Heinz N, Loew R, Baum C, Debets R et al. TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs. Eur J Immunol 2014; 44: 265–274.

    CAS  Article  Google Scholar 

  42. 42

    Zhou BY, Ye Z, Chen G, Gao ZP, Zhang YA, Cheng L . Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. Stem Cells 2007; 25: 779–789.

    CAS  Article  Google Scholar 

  43. 43

    Heinz N, Hennig K, Loew R . Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator. BMC Biotechnol 2013; 13: 5.

    CAS  Article  Google Scholar 

  44. 44

    Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 2010; 5: e10611.

    Article  Google Scholar 

  45. 45

    Benabdellah K, Cobo M, Munoz P, Toscano MG, Martin F . Development of an all-in-one lentiviral vector system based on the original TetR for the easy generation of Tet-ON cell lines. PLoS One 2011; 6: e23734.

    CAS  Article  Google Scholar 

  46. 46

    Kafri T, van Praag H, Gage FH, Verma IM . Lentiviral vectors: regulated gene expression. Mol Ther 2000; 1: 516–521.

    CAS  Article  Google Scholar 

  47. 47

    Modlich U, Navarro S, Zychlinski D, Maetzig T, Knoess S, Brugman MH et al. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther 2009; 17: 1919–1928.

    CAS  Article  Google Scholar 

  48. 48

    Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 2006; 24: 687–696.

    CAS  Article  Google Scholar 

  49. 49

    Cesana D, Ranzani M, Volpin M, Bartholomae C, Duros C, Artus A et al. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol Ther 2014; 22: 774–785.

    CAS  Article  Google Scholar 

  50. 50

    Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E et al. Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther 2008; 16: 718–725.

    CAS  Article  Google Scholar 

  51. 51

    Latta-Mahieu M, Rolland M, Caillet C, Wang M, Kennel P, Mahfouz I et al. Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther 2002; 13: 1611–1620.

    CAS  Article  Google Scholar 

  52. 52

    Markusic DM, de Waart DR, Seppen J . Separating lentiviral vector injection and induction of gene expression in time, does not prevent an immune response to rtTA in rats. PLoS One 2010; 5: e9974.

    Article  Google Scholar 

  53. 53

    Favre D, Blouin V, Provost N, Spisek R, Porrot F, Bohl D et al. Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol 2002; 76: 11605–11611.

    CAS  Article  Google Scholar 

  54. 54

    Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM et al. Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 1995; 332: 143–149.

    CAS  Article  Google Scholar 

  55. 55

    Hoyng SA, Gnavi S, de Winter F, Eggers R, Ozawa T, Zaldumbide A et al. Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve. Gene Therapy 21: 549–557.

  56. 56

    Seggewiss R, Einsele H . Immune reconstitution after allogeneic transplantation and expanding options for immunomodulation: an update. Blood 115: 3861–3868.

  57. 57

    Gregori S, Tomasoni D, Pacciani V, Scirpoli M, Battaglia M, Magnani CF et al. Differentiation of type 1T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway. Blood 116: 935–944.

  58. 58

    Gordon JR, Ma Y, Churchman L, Gordon SA, Dawicki W . Regulatory dendritic cells for immunotherapy in immunologic diseases. Front Immunol 5: 7.

  59. 59

    Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D et al. Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene Therapy 2006; 13: 641–645.

    CAS  Article  Google Scholar 

  60. 60

    Lachmann N, Jagielska J, Heckl D, Brennig S, Pfaff N, Maetzig T et al. MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy. Gene Therapy 2012; 19: 915–924.

    CAS  Article  Google Scholar 

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Acknowledgements

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).

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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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