Design and in vivo characterization of self-inactivating human and non
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《核酸研究医学期刊》
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland and 1 Institute of Biomedical Engineering, ETH Zurich and University of Zurich, CH-8044 Zurich, Switzerland
* To whom correspondence should be addressed. Tel: +41 1 633 34 48; Fax: +41 1 633 12 34; Email: fussenegger@biotech.biol.ethz.ch
ABSTRACT
Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmySS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines.
INTRODUCTION
Based on their ability to transduce a wide variety of difficult-to-transfect quiescent as well as mitotically active cell types and target tissues without eliciting significant humoral immune responses, transgenic lentiviral particles are currently at the forefront of clinical engineering initiatives (1–7). Although stripped of some 60% of their genomes including all accessory genes and produced by reassembly using multiply attenuated split-genome packaging systems, safety concerns about the clinical use of human immunodeficiency virus (HIV)-derived transduction systems are ongoing. In their latest generation, HIV-1-based lentiviral transduction systems consist of (i) helper plasmid(s) encoding gag (encoding major structural proteins), pol (coding for lentivirus-specific enzymes) and rev (a regulator of gag/pol expression and nuclear export of virus RNA); (ii) a vsv-g expression vector promoting pantropic transduction of pseudotyped lentiviral particles; and (iii) the actual transgene(s)-encoding lentivector which remains the only genetic material transferred to desired target cells (8).
The lentivector typically contains the transgene expression cassette flanked by cis-acting elements required for encapsidation, reverse transcription and integration: the extended packaging signal (+), the polypurine tracts (PPT, cPPT), 5'- and 3'-long terminal repeats (5'- and 3'-LTRs) as well as env-derived sequences encompassing the Rev response element (RRE) (8–12). To prevent transcriptional interference with transgene expression and provirus-flanking chromosomal cistrons, most lentivectors are self-inactivating (SIN), characterized by a 3'-LTR-located enhancer deletion (3'LTRU3) resulting in the elimination of 5'-LTR-based transcriptional activities following reverse transcription and integration (13–16). Following the generic design principle of pioneering HIV-based lentiviral transduction systems, a variety of different non-human/non-primate lentivirus derivatives have recently been reported to transduce mammalian cells at similar efficiencies and tropism while successfully addressing HIV-associated safety concerns (17–22). Whatever lentiviral transduction system will eventually prevail in the clinics it will have to be compatible with transcription control modalities to titrate protein pharmaceuticals into their therapeutic window and/or adjust their levels to daily changing dosing regimes (23–25).
We have recently developed a human-compatible transcription control system (PIP) responsive to clinically licensed antibiotics of the streptogramin class (e.g. pristinamycin) (26). A streptogramin-dependent transactivator (PIT), designed by fusing the Streptomyces coelicolor pristinamycin-induced protein to the Herpes simplex-derived VP16 transactivation domain (PIP-VP16), binds and activates chimeric promoters (PPIR8), assembled by cloning tandem PIT-specific operator modules 5' of a minimal eukaryotic promoter, in a pristinamycin-adjustable manner (26–28).
Capitalizing on recent advances in lentivector design (29) we have engineered a variety of isogenic lentiviral particles for streptogramin-responsive transgene expression and validated their performance in different human primary cells, cell lines as well as in chicken embryos.
MATERIALS AND METHODS
Vector design
All plasmids and oligonucleotides used in this study are listed in Tables 1 and 2, respectively. Detailed information on plasmid construction is also provided in Table 1.
Table 1. Plasmids used and designed in this study
Table 2. Oligonucleotides designed for this study
Cell culture
Human embryonic kidney cells transgenic for simian virus 40 (SV40) large T antigen , human fibrosarcoma cells (HT-1080; ATCC CCL-121), human peripheral blood mononuclear cells (PBMC; kindly provided by Claudia Ferrara), mouse CD4+ cells (kindly provided by Ivo Sonderegger) and C2C12PIT/tTA, a mouse myoblast cell line (C2C12; CRL-1772) transgenic for constitutive expression of streptogramin (PIT)- and tetracycline (tTA)-dependent transactivators (30), were cultivated in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany; Cat. No. 3302-P231902), 100 U penicillin and 100 μg/ml streptomycin (Sigma Chemicals, St. Louis, MO), 800 μg/ml G418 (Invitrogen) (C2C12PIT/tTA only) and kept at 37°C in a 5% CO2-containing humidified atmosphere. Chinese hamster ovary cells (CHO-K1; ATCC CCL-61) and their derivative CHO-TWIN1108 (31) were cultivated in 10% FCS-containing FMX-8 medium (Cell Culture Technologies GmbH, Zurich, Switzerland) supplemented with 400 μg/ml G418. Normal human dermal fibroblasts (NHDFs; PromoCell GmbH, Heidelberg, Germany; Cat. No. C-12300) were cultivated in fibroblast basal medium (PromoCell GmbH; Cat. No. C-23210). Human chronic myelogenous leukemia cell line K-562 (ATCC-243) was cultivated in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% FCS.
Lentivirus production and infection
For production of replication-incompetent, SIN HIV-based lentiviral particles, a mixture containing 94 μl DMEM, 6 μl FuGENE6 (Roche Diagnostics AG, Rotkreuz, Switzerland), 25 μM chloroquine, 1 μg pLTR-G , 1 μg pCD/NL-BH* (helper construct) (32) and 1 μg of the desired transgene-encoding lentiviral expression vector was co-transfected into HEK293-T cells. Likewise, the human HIV-1-derived vectors were replaced by non-primate equine infectious anaemia virus (EIAV) counterparts: 1 μg of envelope-encoding pRV67 (19), 2 μg pONY3.1 (helper construct) (19) and 2 μg of the engineered EIAV-derived lentivector. The medium of HEK293-T cultures was replaced 6 h post-transfection and lentiviral particles were produced for another 48 h prior to collection from the supernatant by filtration through a 0.45 μm filter (Schleicher & Schuell GmbH, Dassel, Germany; FP 030/2), which typically yielded titers of 2 x 107 cfu/ml following titration on CHO-K1 cells. To prevent cross-contamination of secreted proteins from production supernatants and increase overall transduction/production efficiency, lentiviral particles were concentrated by ultracentrifugation at 25 000 r.p.m. for 2 h at 4°C (Beckman Quick-Seal centrifuge tubes; Beckman Instruments Inc., CA; Cat. No. 342413). The pellets were resuspended in FCS-containing DMEM (see above) to adjust viral concentrations to desired levels. Lentiviral particle titers were quantified by titration on CHO-K1 cells and/or using standard p24-specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (NEK-050; PerkinElmer, Boston, MA). Unless stated otherwise, standard transduction experiments included infection of 24 000 target cells seeded per well of a 12-well plate with 2 x 106 cfu lentiviral particles (10 ng of p24).
Quantification of reporter protein production
Product proteins were quantified in cell culture supernatants/lysates three days post-transduction. AmySS, SAMY and secreted alkaline phosphatase (SEAP) titers were assessed following protocols by Schlatter et al. (33) using the blue starch Phadebas? (AmySS, SAMY; Pharmacia Upjohn, Peapack, NJ) or a chemiluminescence-based assay (SEAP; Roche Diagnostics AG). Vascular endothelial growth factor (VEGF) production was quantified using the human VEGF-specific DuoSet ELISA System (R&D Systems, Minneapolis, MO) according to the manufacturer's protocol.
Characterization of adipogenic and osteogenic cell phenotypes
To visualize adipogenic cell phenotypes following transduction of C2C12PIT/tTA cells with pBM103-derived lentiviral particles, transduced cells were cultured to 80% confluence in FCS-supplemented DMEM (see above) and then transferred to the same medium also containing 1 μM dexamethasone, 50 μM ETYA and 5 μg/ml insulin . After DEI induction for 14 days, cells were fixed for 1 h in 37% aqueous formaldehyde solution. Excess formaldehyde was removed by rinsing three times with ddH2O for 30 s prior to immersion of cells in a Oil Red O working solution for 30 min . Subsequently, cells were washed three times with ddH2O for 30 s and the nuclei stained for 60 s with Mayer's hematoxylin (Sigma Chemicals). Finally, cells were rinsed in ddH2O for 10 min and covered with cover slips using 10% glycerol in PBS (35). Alkaline phosphatase-specific staining of osteogenic cell phenotypes was visualized following a five-day cultivation using the Sigma Kit 85 according to the manufacturer's instructions (Sigma Chemicals).
Transduction of chicken embryos
Experiments involving chicken embryos were conducted following the shell-free cultivation protocols by Djonov et al. (36). After three days of incubation at 37°C, Brown Leghorn eggs were opened and their contents were carefully poured into 80 mm plastic Petri dishes. The chicken embryos were incubated at 37°C in a humidified atmosphere. Recombinant lentiviral particles (2 μl of pBM104 (8.7 x 106 cfu)- and 1 μl of pMF392 (1.5 x 105 cfu)-derived) were co-applied locally on top of the growing chorioallantoic membrane (CAM) at embryonic day 9, together with 0.5 μl CellTracker Orange CMTMR (Molecular Probes Inc., Eugene, OR; Cat.No. C-2927) in 100 μl DMEM (final concentration 0.5 nM). To modulate heterologous VEGF121 expression, different amounts (0, 6, 30, 60 μg) of pristinamycin were administered for 1 h post-transduction. On embryonic day 12, the CAMs were examined by in vivo fluorescence microscopy following intravenous injection of 100 μl of 2.5% fluorescein isothiocyanate dextran (FITC, 2 000 000; Sigma Chemicals) (37).
Fluorescence microscopy
EYFP expression in cell culture as well as FITC-stained CAM blood vessels were visualized at 50x and 100x magnifications using a Leica DM-RB fluorescence microscope equipped with a Leica digital fluorescence camera DC300 FX (Leica Microsystems AG, Heerbrugg, Switzerland) and a XF114 filter (Omega Optical Inc., Brattleboro, VT). EYFP-mediated fluorescence was quantified using a GeniusPro (Tecan Group Ltd, Maennedorf, Switzerland) and/or the ImageJ software (Wayne Rasband, National Institutes of Health, http://rsb.info.nih.gov/ij/).
Regulating antibiotics
The pristinamycin antibiotic Pyostacin? (PI, Aventis Inc., Paris, France: lot no. 27404) was prepared as stock solution of 500 μg/ml in dimethyl sulfoxide and used at a final concentration of 2 μg/ml unless indicated otherwise.
RESULTS
HIV- and EIAV-derived lentivector configurations for streptogramin-responsive EYFP expression
Based on the recently developed HIV-1-derived (modular) SIN lentivector design (29), we have constructed pBM57 (5'LTR-+-oriSV40-PPT-RRE-MCSI-EYFP-MCSII-3'LTRU3I-MCSIII-3'LTRU3II), which provides convenient multiple cloning sites (MCSI and MCSIII) for integration of promoter elements driving an EYFP transgene (Figure 1). The optimized streptogramin-responsive promoter was either placed within the U3 region to support directional copy into the 5'-LTR following lentivector reverse transcription and integration (pBM60; 5'LTR-+-oriSV40-PPT-RRE-MCSI-EYFP-MCSII-3'LTRU3I-PPIR8-3'LTRU3II) or cloned immediately 5' of EYFP (pBM94; 5'LTR-+-oriSV40-PPT-RRE-PPIR8-EYFP-MCSII-3'LTRU3I-MCSIII-3'LTRU3II) (Figure 2A). pBM60- and pBM94-derived lentiviral particles were transduced into CHO-TWIN1108 constitutively expressing the streptogramin-dependent transactivator , and cultivated in the presence or absence of the regulating streptogramin antibiotic pristinamycin I (PI). Both lentivectors provided high-level EYFP expression in the absence of PI whereas transgene production was completely repressed following addition of the regulating antibiotic (Figure 2B). Although both promoter-transgene configurations enabled tight regulation, qualitative assessment of EYFP expression profiles revealed higher maximum expression levels from PPIR8 placed 5' of the desired transgene rather than in the U3 region (Figure 2B). Following the direct PPIR8-EYFP design concept, a non-primate EIAV-based pONY8.0G-derived lentivector (19) (pBM80; ECMV-R-U5-MCS-Env-3'LTRU3;Figure 1) was engineered for streptogramin-responsive EYFP expression (pBM99; ECMV-R-U5-PPIR8-EYFP-Env-3'LTRU3) and transduced into CHO-TWIN1108. Fluorescence micrograph-based analysis suggested EIAV-mediated EYFP expression to be significantly lower compared to levels resulting from HIV-based transduction (Figure 2B), which likely confirms reports on decreased transduction efficiency of this non-human lentivector type in CHO cells (18). Nevertheless, this EIAV-based lentivector design pioneers transcription modulation in a non-human lentivirus configuration and combines tight regulation with maximum expression levels acceptable for most applications.
Figure 1. Plasmid maps of basic SIN lentiviral expression vectors derived from HIV (pBM57) and EIAV (pBM80). pBM57 contains a 5'-LTR, an extended packaging signal (+), SV40 origins of replication (oriSV40), a central polypurine tract (cPPT), the RRE, anEYFP and an enhancer-free 3'LTRU3). pBM80 harbors a modified 5'-LTR consisting of a CMV-derived enhancer element (ECMV) cloned adjacent to the R-U5 module, an extensive polylinker, a truncated envelope-encoding gene (Env) and an enhancer-free 3'-LTR (3'LTRU3). pBM57 is fully compatible with previously reported lentiviral expression vectors and enable straightforward module swapping (29).
Figure 2. Lentivector-based regulated expression of fluorescent proteins. (A) Detail representation of streptogramin-responsive transgene modules of HIV- (pBM60; pBM94) and EIAV- (pBM99) derived lentivectors. 3'LTRU3; enhancer-less 3'-LTR; 3'LTRU3I/3'LTRU3II, modified 3'LTRU3; PPIR8, streptogramin-responsive promoter; Env, truncated EIAV envelope-encoding gene. Abbreviation of restriction endonucleases: A, AscI; B, BamHI; Bs, BssHII; C, ClaI; E, EcoRI; F, FseI; H, HpaI; K, KpnI; M, MluI; N, NheI; No, NotI; Nr, NruI; P, PacI; Pm, PmeI; S, SacII; Sb, SbfI; Sl, SalI; Sg, SgfI; Sh, SphI; Sp, SpeI; Sr, SrfI; Sw, SwaI; X, XbaI; and Xh, XhoI. (B) EYFP-specific fluorescence micrographs of PIT (pristinamycin-dependent transactivator)-expressing CHO-TWIN1108 transduced with pBM60, pBM94 and pBM99 cultivated for 48 h in the presence (+PI) and absence (–PI) of the streptogramin antibiotic PI.
Comparative transgene expression profiling of different promoter positions
To quantify PPIR8-driven transgene regulation profiles associated with promoter positions immediately 5' of the transgene or within the 3'LTRU3, we replaced EYFP of pBM60 and pBM94 by the intracellular Bacillus stearothermophilus-derived heat-stabile -amylase . pBM84 (5'LTR-+-oriSV40-PPT-RRE-MCSI-amySS-MCSII-3'LTRU3I-PPIR8-3'LTRU3II)- and pBM97 (5'LTR-+-oriSV40-PPT-RRE-PPIR8-amySS-MCSII-3'LTRU3I-MCSIII-3' LTRU3II)-derived lentiviral particles were transduced into CHO-TWIN1108 and intracellular -amylase levels quantified 72 h post-transduction (Figure 3A). Both lentivector configurations enabled tight -amylase control and showed similar induction factors . Yet, the PPIR8-amySS configuration outperformed maximum expression levels of 3'LTRU3-located PPIR8 80-fold (Figure 3B). Therefore, we chose to use direct promoter-transgene configurations for further analysis.
Figure 3. Comparative streptogramin-responsive expression profiling of lentivectors with different promoter-transgene configurations. (A) Detail projection of isogenic lentivectors pBM84 and pBM97. Although pBM84 contains the streptogramin-responsive promoter (PPIR8) within a modified 3'-LTR (3'LTRU3; 3'LTRU3I; 3'LTRU3II) and requires reverse transcription and chromosomal integration for PPIR-driven expression of the intracellular reporter gene amySS (signal peptide-free B.stearothermophilus-derived -amylase), pBM97 contains a central PPIR8- amySS expression configuration. (see caption of Figure 2 for abbreviation of restriction endonucleases). (B) Quantitative -amylase profiling of PIT-producing CHO-TWIN1108 transduced with pMF84-/pMF97-derived lentiviral particles and cultivated for 48 h in the presence (+) and absence (–) of PI. pBM84-derived lentiviral particles reached 32-fold induction whereas cells transduced with pBM97 lentivectors showed an induction factor of 35.
Lentivectors engineered for streptogramin-responsive control of secreted protein production
Since tight control of secreted protein therapeutics is a standard challenge in current gene therapy scenarios, we have evaluated conditional expression performance of lentivectors engineered for streptogramin-responsive expression of the B. stearothermophilus-derived secreted -amylase , human VEGF121 (29) and the human model glycoprotein SEAP (38). pBM96 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SAMY-3'LTRU3)-, pBM104 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-VEGF121-3'LTRU3)- and pBM105 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SEAP-3'LTRU3)-derived lentiviral particles were transduced into CHO-TWIN1108 and respective reporter protein secretion quantified 72 h post-transduction cultivation in the presence and absence of regulating streptogramin antibiotics. All reporter proteins showed equivalent tight pristinamycin-responsive secretion substantiating the fact that this lentivector design enables robust expression control . Furthermore, pBM392/pBM104-derived lentiviral particles enabled transduction and VEGF121 regulation in human PBMC (induction factor 11, –PI: 1.2 ± 0.1 ng/ml, +PI: 0.1 ± 0.0 ng/ml) and mouse CD4+ cells (induction factor 13, –PI 0.8 ± 0.1 ng/ml, +PI: 0.1 ± 0.0 ng/ml).
Transgene expression switching and adjustability of streptogramin-responsive SAMY-encoding lentivectors
Switch kinetics, reversibility of expression status and adjustability of transgene expression are key characteristics of any heterologous transcription control configuration. We have therefore subjected CHO-TWIN1108 cultures transduced with pBM96 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SAMY-3'LTRU3)-derived lentiviral particles to changing streptogramin dosing regimes (+PI/+PI/+PI; +PI/+PI/–PI; –PI/–PI/–PI; –PI/–PI/+PI; +PI/–PI/–PI; +PI/–PI/+PI; –PI/+PI/+PI; –PI/+PI/–PI) and SAMY expression readouts were taken at corresponding days 4/7/10. pMF364-derived lentiviral particles (5'LTR-+-oriSV40-cPPT-RRE-PhEF1-SAMY-3'LTRU3) were used as constitutive control. SAMY expression was characterized by tight expression switches as well as complete reversibility of transgene control (Figure 4A). Also, streptogramin-responsive SEAP expression encoded on pBM105-derived lentiviral particles can be precisely titrated to desired levels by defined PI dosing (Figure 4B).
Figure 4. Lentivector-based streptogramin-responsive transgene expression switching kinetics and adjustability in CHO-K1 cell derivatives. (A) SAMY expression of different cultures of CHO-TWIN1108 transduced with pBM96-derived lentiviral particles (1–8) was switched at days 4, 7 and 10 post-transduction (maximum induction factor 209). CHO-TWIN1108 transduced with pMF364-derived lentiviral particles was used as constitutive control. (B) Adjustable SEAP expression of CHO-TWIN1108 transduced with pBM105-derived lentiviral particles and cultivated for 48 h in the medium supplemented with indicated PI concentrations (maximum induction factor 20).
Streptogramin-controlled adipocyte versus osteoblast differentiation following lentivector-based transduction of muscle precursor cells
Current gene therapy and tissue engineering initiatives evaluate the potential of heterologous gene control systems for rational reprogramming of desired cell phenotypes following conditional expression of differentiation-modulating transgenes. Transdifferentiation studies require particularly tight control and set a high standard for artificial transcription control modalities. Following a multi-step design strategy, we have configured pBM57 for streptogramin-responsive expression of the human CCAAT/enhancer-binding protein alpha (C/EBP-) (pBM103; 5'LTR-+-oriSV40-PPT-RRE-PPIR8-c/ebp--MCSII-3'LTRU3I-MCSIII-3'LTRU3II) (Figure 5A). To induce differential differentiation pBM103-transduced C2C12PIT/tTA, myoblasts were cultivated in the presence (repression of c/ebp-) and absence (induction of c/ebp-) of PI. Indeed, following repression of c/ebp-, C2C12PIT/tTA differentiated into adipocytes following 14 days cultivation period in DEI-containing medium . In contrast, maintenance in streptogramin-free medium resulted in ongoing C/EBP- expression, which differentiated engineered C2C12PIT/tTA cell populations into osteoblasts (Figure 5C).
Figure 5. Streptogramin-responsive differential adipocyte versus myoblast versus osteoblast differentiation of C2C12-derived cell lines transduced with pBM103-derived lentiviral particles engineered for streptogramin-responsive expression of the CCAAT/enhancer-binding protein alpha (C/EBP-). (A) Schematic detail representation of pBM103 harboring a c/ebp- cassette driven by the streptogramin-responsive promoter (PPIR8) and terminated by an enhancer-free 3'-LTR (3'LTRU3) (see caption of Figure 2 for abbreviation of restriction endonucleases). (B) Light micrographs of C2C12PIT/tTA transduced with pBM103-derived lentiviral particles and cultivated for 14 days in DEI-supplemented PI-containing (+PI) or PI-free medium. Cultures were stained with Oil Red O to visualize triglyceride droplets exclusively produced by adipocytes (–PI, induction of c/ebp-). (C) Microscopic analysis of C2C12PIT/tTA transduced with pBM103-derived lentiviral particles and cultivated for 5 days in the presence (+) and absence (–) of PI prior to specific staining of osteoblasts (blue; –PI, induction of c/ebp-). C/EBP- has recently been shown to induce osteogenesis in C2C12-derived myoblast cultures (30).
Transduction of chicken embryos using lentiviral particles transgenic for streptogramin-adjustable expression of human VEGF
To validate lentivector-based streptrogramin-responsive transgene expression in vivo, we co-transduced chicken CAM by distinct lentiviral particles encoding (i) constitutive PIT (pMF392) and (ii) PIT-responsive PPIR8-driven VEGF121 (pBM104) expression, and scored neovascularization as well as vessel morphology after administration of different amounts of pristinamycin. CAMs cultivated under PI-free conditions exhibited VEGF-mediated induction of new blood vessels exemplified by increased formation of numerous arterioles and venules (Figure 6A and B). Also, the hierarchical, tree-like structure of the supplying vessels was disturbed and the multitude of arterioles and venules adopted an irregular tortuous shape associated with atypical delta- or brush-like vessel end points. The VEGF-induced effect was confined to a 4 mm radius around the lentiviral particle application sites and could not be observed on the same CAM beyond this perimeter (Figure 6C). Following administration of increasing amounts of PI (6, 30, 60 μg), VEGF121 expression was gradually decreased to full repression that resulted in native vascularization profiles and vessel morphologies at the application site (Figure 6D–F).
Figure 6. In vivo examination of microvascular growth in the CAM of 12-day-old chicken embryos 72 h post co-transduction with pBM104- and pMF392- (1 μl; 1.5 x 105 cfu) derived lentiviral particles encoding the human VEGF121. Lentivirus application induced an angiogenic response with atypical (brush- and delta-like) end point patterns (arrows) and irregular tortuous vessel shape (arrowhead) within a perimeter of 4 mm when no antibiotic was present . Beyond this boundary, the VEGF121-based effects fade in a distance-dependent manner and are undetectable at 15 mm (C). Following administration of increasing amounts of VEGF121-repressing PI concentrations post-transduction VEGF121-induced angiogenesis gradually decreased to full repression which resulted in native vascularization profiles and vessel morphologies at the application site .
Autoregulated streptogramin-responsive lentivectors
Owing to the binary nature of the streptogramin-responsive transcription control modality lentivector-based engineering of desired target cells requires co-transduction of lentiviral particles transgenic for constitutive PIT (pMF392) and PPIR8-driven EYFP expression (pBM60, pBM94). However, as exemplified by infection of primary NHDF and CHO-K1 cells co-transduction provided excellent regulation profiles but at lower efficiencies (Figure 7) compared to mono-transductions (Figure 2B) (24). In addition to compromising transduction efficiencies, co-integration of transactivator- and transgene-encoding proviruses may result in deregulated transgene expression owing to interference between the operator contained in the transactivator-driving promoter and the antibiotic-responsive promoter. To provide interference-free streptogramin-responsive transgene expression following transduction of a single lentiviral particle type, we designed the following classes of bidirectional autoregulated lentiviral expression vectors: (i) pBM93 (5'LTR-+-oriSV40-cPPT-RRE-pAI-EYFPPhCMVmin-PTR-Phsp70minPIT-pAII-3'LTRU3) and pBM126 (5'LTR-+-oriSV40-cPPT-RRE-pAI-SEAPPhCMVmin-PTR-Phsp70minPIT-pAII-3'LTRU3), (ii) pBM98 (5'LTR-+-oriSV40-cPPT-RRE-pAI-EYFPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and pBM127 (5'LTR-+-oriSV40-cPPT-RRE-pAI-SEAPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and (iii) pBM106 (5'LTR-+-oriSV40-cPPT-RRE-EYFPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and pBM128 (5'LTR-+-oriSV40-cPPT-RRE-SEAPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) (Figure 8A). All autoregulated lentiviral expression vectors contain a central asymmetric bidirectional streptogramin-responsive promoter consisting of a central PIT-specific PTR operator site flanked by minimal versions of the human cytomegalovirus immediate early (PhCMVmin) and the Drosophila heat-shock protein 70 (Phsp70min) promoters. The stronger PhCMVmin was arranged to drive EYFP or SEAP expression whereas Phsp70min was set to maintain the autoregulatory circuit via PIT expression. pBM93/pBM126, pBM98/pBM127 and pMF106/pBM128 differ in their configuration of synthetic minimal polyadenylation sites. Although the divergent expression units of pBM93 and pBM126 are terminated by synthetic size-optimized pA sites (pAI, pAII), only the EYFP/SEAP expression unit of pBM98 and pBM127 harbor a pA (pAI) site whereas pBM106 and pBM128 are devoid of any pA. Autoregulated expression is kicked off by leaky expression of the bidirectional promoter which produces PIT at regulation-effective concentrations which is either inactivated in the presence of PI or may bind and activate the bidirectional promoter in the absence of regulating streptogramins thereby initiating a positive feed-forward circuit expressing PIT as well as divergently encoded EYFP/SEAP. Following transduction of human HT-1080 and CHO-K1 with pBM93-, pBM98- and pBM106-derived lentiviral particles, streptogramin-responsive EYFP expression could be observed for all lentivectors (Figure 8B and C; Table IV). Furthermore, streptogramin-regulatable SEAP expression was quantified following transduction of pBM126-, pBM127- and pBM128- derived lentiviral particles into K-562 .
Figure 7. Fluorescence micrographs of streptogramin-responsive EYFP expression in CHO-K1 and primary NHDF following co-transduction of pMF392- and pBM60- or pBM94- (harboring a direct PPIR8-EYFP expression unit) derived lentiviral particles and cultivation in the presence (+) and absence (–) of PI.
Figure 8. Autoregulated streptogramin-responsive transgene expression following transduction of a single lentiviral particle type into CHO-K1 and human HT-1080 cells. (A) Schematic representation of the bidirectional streptogramin-responsive autoregulated EYFP expression units of lentivectors pBM93, pBM98 and pBM106. At the center of these lentivectors resides an asymmetric bidirectional promoter containing a PIT (pristinamycin-dependent transactivator)-specific operator module (PTR) flanked by minimal versions of the human CMV immediate early (PhCMVmin) and the Drosophila heat-shock protein 70 (Phsp70min) promoters. Although PTR-Phsp70min initiates and maintains the autoregulated feedback circuit by driving PIT expression, PTR-PhCMVmin modulates EYFP transcription. pBM93, pBM98 and pBM106 differ in the configuration of their synthetic size-optimized polyadenylation sites (pAI, pAII). Whereas pBM93 contains two pAs flanking the bidirectional expression unit, only pAI terminates EYFP transcripts in pBM98 and pBM106 remains pA-free (see caption of Figure 2 for abbreviation of restriction endonucleases). pBM93-, pBM98- and pBM106-derived lentiviral particles were transduced into CHO-K1 (B) and HT-1080 cells (C) and grown for 48 h in the absence (–) or presence (+) of the regulating streptogramin antibiotic PI prior to microscopical and quantitative analyses of EYFP expression profiles: CHO-K1 (pBM93, –PI: 12.3 ± 1.0, +PI: 0.9 ± 0.3 RLU; pBM98, –PI: 15.6 ± 1.7, +PI: 0.9 ± 0.2 RLU; pBM106, –PI: 9.2 ± 0.2, +PI: 1.0 ± 0.2 RLU), HT-1080 (pBM93, –PI: 11.8 ± 0.8, +PI: 1.3 ± 0.3 RLU; pBM98, –PI: 21.1 ± 1.8, +PI: 1.9 ± 0.1 RLU; pBM106, –PI: 17.4 ± 0.5, +PI: 2.1 ± 0.5 RLU).
DISCUSSION
In the past decade, viral transduction systems have witnessed dramatic advances including implementation of heterologous transgene modulation (25,39–48). For lentivirus-based transduction arrangements, systems responsive to tetracycline antibiotics (the TET systems) have been most popular and were used in the following basic/optimized configurations: (i) classic TETOFF system (24,49–54), (ii) reverse tetracycline-dependent TETON system (55–59) and (iii) the TetR-based TETON technology (14,60,61). Lentiviral transduction systems have been engineered for tetracycline-responsive gene expression to (i) enable construction of stable helper cell lines producing pseudotyping VSV-G in a timely manner and/or at subtoxic doses (15,52,62), (ii) design chimeric adeno-lentiviral vectors which produce lentiviral particles from transgenic lentiviruses in a tetracycline-responsive manner (57), (iii) generate conditionally replicating HIV-based vectors for vaccination (56,59), (iv) mediate tetracycline-inducible/repressible transgene expression (24,50,51,53,54,58,60,63) and (v) modulate siRNA-based knockdown of endogenous genes (14,63).
Pioneering tetracycline-responsive expression configurations were based on two separate vectors one of which included a constitutive tetracycline-dependent transactivator and the other contained a tetracycline-responsive promoter-driven transgene expression unit engineered into a non-SIN lentiviral backbone. Owing to their non-SIN structure first-generation transgene-controlled lentivectors exhibited increased leakiness resulting from LTR-associated enhancer interference with tetracycline-responsive promoters (50–52). SIN-based tetracycline-responsive lentivectors containing an enhancer deletion in the 3' LTRU3 provided improved induction factors based on interference-free repression.
Following the proven design concept of the tetracycline-responsive expression technology, we have recently developed a human-compatible mammalian transcription control system responsive to clinically licensed streptogramin antibiotics (26). This so-called PIP system included several inducible/repressible configurations all of which showed excellent regulation performance in a variety of different cell lines and human primary cells as well as in mice (26–28,31,42,64). Also, PIP systems were fully compatible with TET and other antibiotic-adjustable transcription control systems and could be used for differential fine-tuning of different transgene activities (28,31,42). Following configuration into a lentiviral expression context, the PIP system showed excellent streptogramin-responsive transgene regulation performance, including tight repression, high-level expression, reversibility and adjustability in a variety of mammalian/human cell lines and primary cells. Efficiency of the streptogramin-responsive lentivector design was exemplified by differential differentiation reprogramming of muscle cells into fat or bone phenotypes as well as conditional vascularization of chicken embryos. Comparative regulation profiling of isogenic lentivectors either encoding a direct PPIR8-transgene expression unit or harboring PPIR8 within a SIN-modified 3'LTRU3 revealed the former configuration to enable almost two orders of magnitude higher transgene expression levels at comparable induction factors. Direct comparison of isogenic HIV- and EIAV-derived lentiviral vectors engineered for direct PPIR8-EYFP expression suggested lower EIAV-mediated transduction rates although both systems provided excellent regulation performance in the CHO-K1-derived cell lines. Although outperformed by HIV-derived lentivectors at first sight, this pioneering example of regulated transgene expression in non-primate lentiviruses may hold great promise for clinical application because of minimal risk associated with EIAV-based therapeutic interventions.
Owing to the binary nature of currently available transcription control systems, transduction of regulated transgene expression is typically realized by simultaneous co-infection of two separate lentiviral particles, one encoding the antibiotic-responsive transactivator and the second one harboring the transactivator-responsive transgene expression unit. Since co-transduction rates are typically lower and require a higher multiplicity of infection compared to transduction using a single lentiviral particle type design of single lentivector-based strategies for adjustable therapeutic interventions is the current clinical priority. First-generation one-vector-based regulated lentiviral transgene configurations included placement of independent transactivator- and transgene-encoding expression units on the same lentivector (51). However, this configuration mediated significant leaky expression due to combinatorial interference of the enhancers contained in the transactivator-driving promoter and the 3'-LTR. Autoregulated expression configurations in which transactivator and transgene are co-expressed in a coordinated manner represent the only a priori interference-free regulated expression configuration (42,65). Recently, Ogueta et al. (60) have engineered an autoregulatory lentiviral one-vector design by cloning the tetracycline repressor (TetR) and the gene of interest into a dicistronic configuration driven by the tetR-repressible promoter (PhCMV-tetO2-EGFP-IRES-TetR; tetO2, dimeric TetR-specific operator; EGFP, enhanced green fluorescent protein; and IRES, internal ribosome entry site). Although this inducible autoregulated lentiviral expression system enabled excellent regulation performance, the restricted tissue tropism of IRES elements as well as the lag phase required to accumulate repression-effective TetR concentrations may limit some applications. The bidirectional autoregulated cassette presented here addresses aforementioned limitations by enabling streptogramin-responsive transgene expression in the most compact and efficient format.
All of the currently available HIV-1-based expression vectors tailored for adjustable transgene expression in mammalian cells exhibit residual transcription activity under repressed conditions (42,65). Although such basal expression remains an inherent characteristic of any heterologous transcription control system (42,65), leaky expression is accentuated by HIV-1's integration preference for transcriptionally active hot spots, which may interfere with desired transgene control to a significant extent (42,65).
Combination of robust transgene control modalities with latest generation lentiviral transduction systems will further advance molecular interventions for gene therapy and tissue engineering.
ACKNOWLEDGEMENTS
We thank Valeria Nicolini-Gonzalez for critical comments on the manuscript, Martin Ehrbar for key advice on CAM experiments as well as Claudia Ferrara and Ivo Sonderegger for isolation of primary cells. This work was supported by the Swiss National Science Foundation (grant no. 631-065946), the Swiss Federal Office for Education and Science (BBW) within EC Framework 6 and a special research grant by the ETH Vice President for Research.
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* To whom correspondence should be addressed. Tel: +41 1 633 34 48; Fax: +41 1 633 12 34; Email: fussenegger@biotech.biol.ethz.ch
ABSTRACT
Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmySS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines.
INTRODUCTION
Based on their ability to transduce a wide variety of difficult-to-transfect quiescent as well as mitotically active cell types and target tissues without eliciting significant humoral immune responses, transgenic lentiviral particles are currently at the forefront of clinical engineering initiatives (1–7). Although stripped of some 60% of their genomes including all accessory genes and produced by reassembly using multiply attenuated split-genome packaging systems, safety concerns about the clinical use of human immunodeficiency virus (HIV)-derived transduction systems are ongoing. In their latest generation, HIV-1-based lentiviral transduction systems consist of (i) helper plasmid(s) encoding gag (encoding major structural proteins), pol (coding for lentivirus-specific enzymes) and rev (a regulator of gag/pol expression and nuclear export of virus RNA); (ii) a vsv-g expression vector promoting pantropic transduction of pseudotyped lentiviral particles; and (iii) the actual transgene(s)-encoding lentivector which remains the only genetic material transferred to desired target cells (8).
The lentivector typically contains the transgene expression cassette flanked by cis-acting elements required for encapsidation, reverse transcription and integration: the extended packaging signal (+), the polypurine tracts (PPT, cPPT), 5'- and 3'-long terminal repeats (5'- and 3'-LTRs) as well as env-derived sequences encompassing the Rev response element (RRE) (8–12). To prevent transcriptional interference with transgene expression and provirus-flanking chromosomal cistrons, most lentivectors are self-inactivating (SIN), characterized by a 3'-LTR-located enhancer deletion (3'LTRU3) resulting in the elimination of 5'-LTR-based transcriptional activities following reverse transcription and integration (13–16). Following the generic design principle of pioneering HIV-based lentiviral transduction systems, a variety of different non-human/non-primate lentivirus derivatives have recently been reported to transduce mammalian cells at similar efficiencies and tropism while successfully addressing HIV-associated safety concerns (17–22). Whatever lentiviral transduction system will eventually prevail in the clinics it will have to be compatible with transcription control modalities to titrate protein pharmaceuticals into their therapeutic window and/or adjust their levels to daily changing dosing regimes (23–25).
We have recently developed a human-compatible transcription control system (PIP) responsive to clinically licensed antibiotics of the streptogramin class (e.g. pristinamycin) (26). A streptogramin-dependent transactivator (PIT), designed by fusing the Streptomyces coelicolor pristinamycin-induced protein to the Herpes simplex-derived VP16 transactivation domain (PIP-VP16), binds and activates chimeric promoters (PPIR8), assembled by cloning tandem PIT-specific operator modules 5' of a minimal eukaryotic promoter, in a pristinamycin-adjustable manner (26–28).
Capitalizing on recent advances in lentivector design (29) we have engineered a variety of isogenic lentiviral particles for streptogramin-responsive transgene expression and validated their performance in different human primary cells, cell lines as well as in chicken embryos.
MATERIALS AND METHODS
Vector design
All plasmids and oligonucleotides used in this study are listed in Tables 1 and 2, respectively. Detailed information on plasmid construction is also provided in Table 1.
Table 1. Plasmids used and designed in this study
Table 2. Oligonucleotides designed for this study
Cell culture
Human embryonic kidney cells transgenic for simian virus 40 (SV40) large T antigen , human fibrosarcoma cells (HT-1080; ATCC CCL-121), human peripheral blood mononuclear cells (PBMC; kindly provided by Claudia Ferrara), mouse CD4+ cells (kindly provided by Ivo Sonderegger) and C2C12PIT/tTA, a mouse myoblast cell line (C2C12; CRL-1772) transgenic for constitutive expression of streptogramin (PIT)- and tetracycline (tTA)-dependent transactivators (30), were cultivated in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany; Cat. No. 3302-P231902), 100 U penicillin and 100 μg/ml streptomycin (Sigma Chemicals, St. Louis, MO), 800 μg/ml G418 (Invitrogen) (C2C12PIT/tTA only) and kept at 37°C in a 5% CO2-containing humidified atmosphere. Chinese hamster ovary cells (CHO-K1; ATCC CCL-61) and their derivative CHO-TWIN1108 (31) were cultivated in 10% FCS-containing FMX-8 medium (Cell Culture Technologies GmbH, Zurich, Switzerland) supplemented with 400 μg/ml G418. Normal human dermal fibroblasts (NHDFs; PromoCell GmbH, Heidelberg, Germany; Cat. No. C-12300) were cultivated in fibroblast basal medium (PromoCell GmbH; Cat. No. C-23210). Human chronic myelogenous leukemia cell line K-562 (ATCC-243) was cultivated in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% FCS.
Lentivirus production and infection
For production of replication-incompetent, SIN HIV-based lentiviral particles, a mixture containing 94 μl DMEM, 6 μl FuGENE6 (Roche Diagnostics AG, Rotkreuz, Switzerland), 25 μM chloroquine, 1 μg pLTR-G , 1 μg pCD/NL-BH* (helper construct) (32) and 1 μg of the desired transgene-encoding lentiviral expression vector was co-transfected into HEK293-T cells. Likewise, the human HIV-1-derived vectors were replaced by non-primate equine infectious anaemia virus (EIAV) counterparts: 1 μg of envelope-encoding pRV67 (19), 2 μg pONY3.1 (helper construct) (19) and 2 μg of the engineered EIAV-derived lentivector. The medium of HEK293-T cultures was replaced 6 h post-transfection and lentiviral particles were produced for another 48 h prior to collection from the supernatant by filtration through a 0.45 μm filter (Schleicher & Schuell GmbH, Dassel, Germany; FP 030/2), which typically yielded titers of 2 x 107 cfu/ml following titration on CHO-K1 cells. To prevent cross-contamination of secreted proteins from production supernatants and increase overall transduction/production efficiency, lentiviral particles were concentrated by ultracentrifugation at 25 000 r.p.m. for 2 h at 4°C (Beckman Quick-Seal centrifuge tubes; Beckman Instruments Inc., CA; Cat. No. 342413). The pellets were resuspended in FCS-containing DMEM (see above) to adjust viral concentrations to desired levels. Lentiviral particle titers were quantified by titration on CHO-K1 cells and/or using standard p24-specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (NEK-050; PerkinElmer, Boston, MA). Unless stated otherwise, standard transduction experiments included infection of 24 000 target cells seeded per well of a 12-well plate with 2 x 106 cfu lentiviral particles (10 ng of p24).
Quantification of reporter protein production
Product proteins were quantified in cell culture supernatants/lysates three days post-transduction. AmySS, SAMY and secreted alkaline phosphatase (SEAP) titers were assessed following protocols by Schlatter et al. (33) using the blue starch Phadebas? (AmySS, SAMY; Pharmacia Upjohn, Peapack, NJ) or a chemiluminescence-based assay (SEAP; Roche Diagnostics AG). Vascular endothelial growth factor (VEGF) production was quantified using the human VEGF-specific DuoSet ELISA System (R&D Systems, Minneapolis, MO) according to the manufacturer's protocol.
Characterization of adipogenic and osteogenic cell phenotypes
To visualize adipogenic cell phenotypes following transduction of C2C12PIT/tTA cells with pBM103-derived lentiviral particles, transduced cells were cultured to 80% confluence in FCS-supplemented DMEM (see above) and then transferred to the same medium also containing 1 μM dexamethasone, 50 μM ETYA and 5 μg/ml insulin . After DEI induction for 14 days, cells were fixed for 1 h in 37% aqueous formaldehyde solution. Excess formaldehyde was removed by rinsing three times with ddH2O for 30 s prior to immersion of cells in a Oil Red O working solution for 30 min . Subsequently, cells were washed three times with ddH2O for 30 s and the nuclei stained for 60 s with Mayer's hematoxylin (Sigma Chemicals). Finally, cells were rinsed in ddH2O for 10 min and covered with cover slips using 10% glycerol in PBS (35). Alkaline phosphatase-specific staining of osteogenic cell phenotypes was visualized following a five-day cultivation using the Sigma Kit 85 according to the manufacturer's instructions (Sigma Chemicals).
Transduction of chicken embryos
Experiments involving chicken embryos were conducted following the shell-free cultivation protocols by Djonov et al. (36). After three days of incubation at 37°C, Brown Leghorn eggs were opened and their contents were carefully poured into 80 mm plastic Petri dishes. The chicken embryos were incubated at 37°C in a humidified atmosphere. Recombinant lentiviral particles (2 μl of pBM104 (8.7 x 106 cfu)- and 1 μl of pMF392 (1.5 x 105 cfu)-derived) were co-applied locally on top of the growing chorioallantoic membrane (CAM) at embryonic day 9, together with 0.5 μl CellTracker Orange CMTMR (Molecular Probes Inc., Eugene, OR; Cat.No. C-2927) in 100 μl DMEM (final concentration 0.5 nM). To modulate heterologous VEGF121 expression, different amounts (0, 6, 30, 60 μg) of pristinamycin were administered for 1 h post-transduction. On embryonic day 12, the CAMs were examined by in vivo fluorescence microscopy following intravenous injection of 100 μl of 2.5% fluorescein isothiocyanate dextran (FITC, 2 000 000; Sigma Chemicals) (37).
Fluorescence microscopy
EYFP expression in cell culture as well as FITC-stained CAM blood vessels were visualized at 50x and 100x magnifications using a Leica DM-RB fluorescence microscope equipped with a Leica digital fluorescence camera DC300 FX (Leica Microsystems AG, Heerbrugg, Switzerland) and a XF114 filter (Omega Optical Inc., Brattleboro, VT). EYFP-mediated fluorescence was quantified using a GeniusPro (Tecan Group Ltd, Maennedorf, Switzerland) and/or the ImageJ software (Wayne Rasband, National Institutes of Health, http://rsb.info.nih.gov/ij/).
Regulating antibiotics
The pristinamycin antibiotic Pyostacin? (PI, Aventis Inc., Paris, France: lot no. 27404) was prepared as stock solution of 500 μg/ml in dimethyl sulfoxide and used at a final concentration of 2 μg/ml unless indicated otherwise.
RESULTS
HIV- and EIAV-derived lentivector configurations for streptogramin-responsive EYFP expression
Based on the recently developed HIV-1-derived (modular) SIN lentivector design (29), we have constructed pBM57 (5'LTR-+-oriSV40-PPT-RRE-MCSI-EYFP-MCSII-3'LTRU3I-MCSIII-3'LTRU3II), which provides convenient multiple cloning sites (MCSI and MCSIII) for integration of promoter elements driving an EYFP transgene (Figure 1). The optimized streptogramin-responsive promoter was either placed within the U3 region to support directional copy into the 5'-LTR following lentivector reverse transcription and integration (pBM60; 5'LTR-+-oriSV40-PPT-RRE-MCSI-EYFP-MCSII-3'LTRU3I-PPIR8-3'LTRU3II) or cloned immediately 5' of EYFP (pBM94; 5'LTR-+-oriSV40-PPT-RRE-PPIR8-EYFP-MCSII-3'LTRU3I-MCSIII-3'LTRU3II) (Figure 2A). pBM60- and pBM94-derived lentiviral particles were transduced into CHO-TWIN1108 constitutively expressing the streptogramin-dependent transactivator , and cultivated in the presence or absence of the regulating streptogramin antibiotic pristinamycin I (PI). Both lentivectors provided high-level EYFP expression in the absence of PI whereas transgene production was completely repressed following addition of the regulating antibiotic (Figure 2B). Although both promoter-transgene configurations enabled tight regulation, qualitative assessment of EYFP expression profiles revealed higher maximum expression levels from PPIR8 placed 5' of the desired transgene rather than in the U3 region (Figure 2B). Following the direct PPIR8-EYFP design concept, a non-primate EIAV-based pONY8.0G-derived lentivector (19) (pBM80; ECMV-R-U5-MCS-Env-3'LTRU3;Figure 1) was engineered for streptogramin-responsive EYFP expression (pBM99; ECMV-R-U5-PPIR8-EYFP-Env-3'LTRU3) and transduced into CHO-TWIN1108. Fluorescence micrograph-based analysis suggested EIAV-mediated EYFP expression to be significantly lower compared to levels resulting from HIV-based transduction (Figure 2B), which likely confirms reports on decreased transduction efficiency of this non-human lentivector type in CHO cells (18). Nevertheless, this EIAV-based lentivector design pioneers transcription modulation in a non-human lentivirus configuration and combines tight regulation with maximum expression levels acceptable for most applications.
Figure 1. Plasmid maps of basic SIN lentiviral expression vectors derived from HIV (pBM57) and EIAV (pBM80). pBM57 contains a 5'-LTR, an extended packaging signal (+), SV40 origins of replication (oriSV40), a central polypurine tract (cPPT), the RRE, anEYFP and an enhancer-free 3'LTRU3). pBM80 harbors a modified 5'-LTR consisting of a CMV-derived enhancer element (ECMV) cloned adjacent to the R-U5 module, an extensive polylinker, a truncated envelope-encoding gene (Env) and an enhancer-free 3'-LTR (3'LTRU3). pBM57 is fully compatible with previously reported lentiviral expression vectors and enable straightforward module swapping (29).
Figure 2. Lentivector-based regulated expression of fluorescent proteins. (A) Detail representation of streptogramin-responsive transgene modules of HIV- (pBM60; pBM94) and EIAV- (pBM99) derived lentivectors. 3'LTRU3; enhancer-less 3'-LTR; 3'LTRU3I/3'LTRU3II, modified 3'LTRU3; PPIR8, streptogramin-responsive promoter; Env, truncated EIAV envelope-encoding gene. Abbreviation of restriction endonucleases: A, AscI; B, BamHI; Bs, BssHII; C, ClaI; E, EcoRI; F, FseI; H, HpaI; K, KpnI; M, MluI; N, NheI; No, NotI; Nr, NruI; P, PacI; Pm, PmeI; S, SacII; Sb, SbfI; Sl, SalI; Sg, SgfI; Sh, SphI; Sp, SpeI; Sr, SrfI; Sw, SwaI; X, XbaI; and Xh, XhoI. (B) EYFP-specific fluorescence micrographs of PIT (pristinamycin-dependent transactivator)-expressing CHO-TWIN1108 transduced with pBM60, pBM94 and pBM99 cultivated for 48 h in the presence (+PI) and absence (–PI) of the streptogramin antibiotic PI.
Comparative transgene expression profiling of different promoter positions
To quantify PPIR8-driven transgene regulation profiles associated with promoter positions immediately 5' of the transgene or within the 3'LTRU3, we replaced EYFP of pBM60 and pBM94 by the intracellular Bacillus stearothermophilus-derived heat-stabile -amylase . pBM84 (5'LTR-+-oriSV40-PPT-RRE-MCSI-amySS-MCSII-3'LTRU3I-PPIR8-3'LTRU3II)- and pBM97 (5'LTR-+-oriSV40-PPT-RRE-PPIR8-amySS-MCSII-3'LTRU3I-MCSIII-3' LTRU3II)-derived lentiviral particles were transduced into CHO-TWIN1108 and intracellular -amylase levels quantified 72 h post-transduction (Figure 3A). Both lentivector configurations enabled tight -amylase control and showed similar induction factors . Yet, the PPIR8-amySS configuration outperformed maximum expression levels of 3'LTRU3-located PPIR8 80-fold (Figure 3B). Therefore, we chose to use direct promoter-transgene configurations for further analysis.
Figure 3. Comparative streptogramin-responsive expression profiling of lentivectors with different promoter-transgene configurations. (A) Detail projection of isogenic lentivectors pBM84 and pBM97. Although pBM84 contains the streptogramin-responsive promoter (PPIR8) within a modified 3'-LTR (3'LTRU3; 3'LTRU3I; 3'LTRU3II) and requires reverse transcription and chromosomal integration for PPIR-driven expression of the intracellular reporter gene amySS (signal peptide-free B.stearothermophilus-derived -amylase), pBM97 contains a central PPIR8- amySS expression configuration. (see caption of Figure 2 for abbreviation of restriction endonucleases). (B) Quantitative -amylase profiling of PIT-producing CHO-TWIN1108 transduced with pMF84-/pMF97-derived lentiviral particles and cultivated for 48 h in the presence (+) and absence (–) of PI. pBM84-derived lentiviral particles reached 32-fold induction whereas cells transduced with pBM97 lentivectors showed an induction factor of 35.
Lentivectors engineered for streptogramin-responsive control of secreted protein production
Since tight control of secreted protein therapeutics is a standard challenge in current gene therapy scenarios, we have evaluated conditional expression performance of lentivectors engineered for streptogramin-responsive expression of the B. stearothermophilus-derived secreted -amylase , human VEGF121 (29) and the human model glycoprotein SEAP (38). pBM96 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SAMY-3'LTRU3)-, pBM104 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-VEGF121-3'LTRU3)- and pBM105 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SEAP-3'LTRU3)-derived lentiviral particles were transduced into CHO-TWIN1108 and respective reporter protein secretion quantified 72 h post-transduction cultivation in the presence and absence of regulating streptogramin antibiotics. All reporter proteins showed equivalent tight pristinamycin-responsive secretion substantiating the fact that this lentivector design enables robust expression control . Furthermore, pBM392/pBM104-derived lentiviral particles enabled transduction and VEGF121 regulation in human PBMC (induction factor 11, –PI: 1.2 ± 0.1 ng/ml, +PI: 0.1 ± 0.0 ng/ml) and mouse CD4+ cells (induction factor 13, –PI 0.8 ± 0.1 ng/ml, +PI: 0.1 ± 0.0 ng/ml).
Transgene expression switching and adjustability of streptogramin-responsive SAMY-encoding lentivectors
Switch kinetics, reversibility of expression status and adjustability of transgene expression are key characteristics of any heterologous transcription control configuration. We have therefore subjected CHO-TWIN1108 cultures transduced with pBM96 (5'LTR-+-oriSV40-cPPT-RRE-PPIR8-SAMY-3'LTRU3)-derived lentiviral particles to changing streptogramin dosing regimes (+PI/+PI/+PI; +PI/+PI/–PI; –PI/–PI/–PI; –PI/–PI/+PI; +PI/–PI/–PI; +PI/–PI/+PI; –PI/+PI/+PI; –PI/+PI/–PI) and SAMY expression readouts were taken at corresponding days 4/7/10. pMF364-derived lentiviral particles (5'LTR-+-oriSV40-cPPT-RRE-PhEF1-SAMY-3'LTRU3) were used as constitutive control. SAMY expression was characterized by tight expression switches as well as complete reversibility of transgene control (Figure 4A). Also, streptogramin-responsive SEAP expression encoded on pBM105-derived lentiviral particles can be precisely titrated to desired levels by defined PI dosing (Figure 4B).
Figure 4. Lentivector-based streptogramin-responsive transgene expression switching kinetics and adjustability in CHO-K1 cell derivatives. (A) SAMY expression of different cultures of CHO-TWIN1108 transduced with pBM96-derived lentiviral particles (1–8) was switched at days 4, 7 and 10 post-transduction (maximum induction factor 209). CHO-TWIN1108 transduced with pMF364-derived lentiviral particles was used as constitutive control. (B) Adjustable SEAP expression of CHO-TWIN1108 transduced with pBM105-derived lentiviral particles and cultivated for 48 h in the medium supplemented with indicated PI concentrations (maximum induction factor 20).
Streptogramin-controlled adipocyte versus osteoblast differentiation following lentivector-based transduction of muscle precursor cells
Current gene therapy and tissue engineering initiatives evaluate the potential of heterologous gene control systems for rational reprogramming of desired cell phenotypes following conditional expression of differentiation-modulating transgenes. Transdifferentiation studies require particularly tight control and set a high standard for artificial transcription control modalities. Following a multi-step design strategy, we have configured pBM57 for streptogramin-responsive expression of the human CCAAT/enhancer-binding protein alpha (C/EBP-) (pBM103; 5'LTR-+-oriSV40-PPT-RRE-PPIR8-c/ebp--MCSII-3'LTRU3I-MCSIII-3'LTRU3II) (Figure 5A). To induce differential differentiation pBM103-transduced C2C12PIT/tTA, myoblasts were cultivated in the presence (repression of c/ebp-) and absence (induction of c/ebp-) of PI. Indeed, following repression of c/ebp-, C2C12PIT/tTA differentiated into adipocytes following 14 days cultivation period in DEI-containing medium . In contrast, maintenance in streptogramin-free medium resulted in ongoing C/EBP- expression, which differentiated engineered C2C12PIT/tTA cell populations into osteoblasts (Figure 5C).
Figure 5. Streptogramin-responsive differential adipocyte versus myoblast versus osteoblast differentiation of C2C12-derived cell lines transduced with pBM103-derived lentiviral particles engineered for streptogramin-responsive expression of the CCAAT/enhancer-binding protein alpha (C/EBP-). (A) Schematic detail representation of pBM103 harboring a c/ebp- cassette driven by the streptogramin-responsive promoter (PPIR8) and terminated by an enhancer-free 3'-LTR (3'LTRU3) (see caption of Figure 2 for abbreviation of restriction endonucleases). (B) Light micrographs of C2C12PIT/tTA transduced with pBM103-derived lentiviral particles and cultivated for 14 days in DEI-supplemented PI-containing (+PI) or PI-free medium. Cultures were stained with Oil Red O to visualize triglyceride droplets exclusively produced by adipocytes (–PI, induction of c/ebp-). (C) Microscopic analysis of C2C12PIT/tTA transduced with pBM103-derived lentiviral particles and cultivated for 5 days in the presence (+) and absence (–) of PI prior to specific staining of osteoblasts (blue; –PI, induction of c/ebp-). C/EBP- has recently been shown to induce osteogenesis in C2C12-derived myoblast cultures (30).
Transduction of chicken embryos using lentiviral particles transgenic for streptogramin-adjustable expression of human VEGF
To validate lentivector-based streptrogramin-responsive transgene expression in vivo, we co-transduced chicken CAM by distinct lentiviral particles encoding (i) constitutive PIT (pMF392) and (ii) PIT-responsive PPIR8-driven VEGF121 (pBM104) expression, and scored neovascularization as well as vessel morphology after administration of different amounts of pristinamycin. CAMs cultivated under PI-free conditions exhibited VEGF-mediated induction of new blood vessels exemplified by increased formation of numerous arterioles and venules (Figure 6A and B). Also, the hierarchical, tree-like structure of the supplying vessels was disturbed and the multitude of arterioles and venules adopted an irregular tortuous shape associated with atypical delta- or brush-like vessel end points. The VEGF-induced effect was confined to a 4 mm radius around the lentiviral particle application sites and could not be observed on the same CAM beyond this perimeter (Figure 6C). Following administration of increasing amounts of PI (6, 30, 60 μg), VEGF121 expression was gradually decreased to full repression that resulted in native vascularization profiles and vessel morphologies at the application site (Figure 6D–F).
Figure 6. In vivo examination of microvascular growth in the CAM of 12-day-old chicken embryos 72 h post co-transduction with pBM104- and pMF392- (1 μl; 1.5 x 105 cfu) derived lentiviral particles encoding the human VEGF121. Lentivirus application induced an angiogenic response with atypical (brush- and delta-like) end point patterns (arrows) and irregular tortuous vessel shape (arrowhead) within a perimeter of 4 mm when no antibiotic was present . Beyond this boundary, the VEGF121-based effects fade in a distance-dependent manner and are undetectable at 15 mm (C). Following administration of increasing amounts of VEGF121-repressing PI concentrations post-transduction VEGF121-induced angiogenesis gradually decreased to full repression which resulted in native vascularization profiles and vessel morphologies at the application site .
Autoregulated streptogramin-responsive lentivectors
Owing to the binary nature of the streptogramin-responsive transcription control modality lentivector-based engineering of desired target cells requires co-transduction of lentiviral particles transgenic for constitutive PIT (pMF392) and PPIR8-driven EYFP expression (pBM60, pBM94). However, as exemplified by infection of primary NHDF and CHO-K1 cells co-transduction provided excellent regulation profiles but at lower efficiencies (Figure 7) compared to mono-transductions (Figure 2B) (24). In addition to compromising transduction efficiencies, co-integration of transactivator- and transgene-encoding proviruses may result in deregulated transgene expression owing to interference between the operator contained in the transactivator-driving promoter and the antibiotic-responsive promoter. To provide interference-free streptogramin-responsive transgene expression following transduction of a single lentiviral particle type, we designed the following classes of bidirectional autoregulated lentiviral expression vectors: (i) pBM93 (5'LTR-+-oriSV40-cPPT-RRE-pAI-EYFPPhCMVmin-PTR-Phsp70minPIT-pAII-3'LTRU3) and pBM126 (5'LTR-+-oriSV40-cPPT-RRE-pAI-SEAPPhCMVmin-PTR-Phsp70minPIT-pAII-3'LTRU3), (ii) pBM98 (5'LTR-+-oriSV40-cPPT-RRE-pAI-EYFPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and pBM127 (5'LTR-+-oriSV40-cPPT-RRE-pAI-SEAPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and (iii) pBM106 (5'LTR-+-oriSV40-cPPT-RRE-EYFPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) and pBM128 (5'LTR-+-oriSV40-cPPT-RRE-SEAPPhCMVmin-PTR-Phsp70minPIT-3'LTRU3) (Figure 8A). All autoregulated lentiviral expression vectors contain a central asymmetric bidirectional streptogramin-responsive promoter consisting of a central PIT-specific PTR operator site flanked by minimal versions of the human cytomegalovirus immediate early (PhCMVmin) and the Drosophila heat-shock protein 70 (Phsp70min) promoters. The stronger PhCMVmin was arranged to drive EYFP or SEAP expression whereas Phsp70min was set to maintain the autoregulatory circuit via PIT expression. pBM93/pBM126, pBM98/pBM127 and pMF106/pBM128 differ in their configuration of synthetic minimal polyadenylation sites. Although the divergent expression units of pBM93 and pBM126 are terminated by synthetic size-optimized pA sites (pAI, pAII), only the EYFP/SEAP expression unit of pBM98 and pBM127 harbor a pA (pAI) site whereas pBM106 and pBM128 are devoid of any pA. Autoregulated expression is kicked off by leaky expression of the bidirectional promoter which produces PIT at regulation-effective concentrations which is either inactivated in the presence of PI or may bind and activate the bidirectional promoter in the absence of regulating streptogramins thereby initiating a positive feed-forward circuit expressing PIT as well as divergently encoded EYFP/SEAP. Following transduction of human HT-1080 and CHO-K1 with pBM93-, pBM98- and pBM106-derived lentiviral particles, streptogramin-responsive EYFP expression could be observed for all lentivectors (Figure 8B and C; Table IV). Furthermore, streptogramin-regulatable SEAP expression was quantified following transduction of pBM126-, pBM127- and pBM128- derived lentiviral particles into K-562 .
Figure 7. Fluorescence micrographs of streptogramin-responsive EYFP expression in CHO-K1 and primary NHDF following co-transduction of pMF392- and pBM60- or pBM94- (harboring a direct PPIR8-EYFP expression unit) derived lentiviral particles and cultivation in the presence (+) and absence (–) of PI.
Figure 8. Autoregulated streptogramin-responsive transgene expression following transduction of a single lentiviral particle type into CHO-K1 and human HT-1080 cells. (A) Schematic representation of the bidirectional streptogramin-responsive autoregulated EYFP expression units of lentivectors pBM93, pBM98 and pBM106. At the center of these lentivectors resides an asymmetric bidirectional promoter containing a PIT (pristinamycin-dependent transactivator)-specific operator module (PTR) flanked by minimal versions of the human CMV immediate early (PhCMVmin) and the Drosophila heat-shock protein 70 (Phsp70min) promoters. Although PTR-Phsp70min initiates and maintains the autoregulated feedback circuit by driving PIT expression, PTR-PhCMVmin modulates EYFP transcription. pBM93, pBM98 and pBM106 differ in the configuration of their synthetic size-optimized polyadenylation sites (pAI, pAII). Whereas pBM93 contains two pAs flanking the bidirectional expression unit, only pAI terminates EYFP transcripts in pBM98 and pBM106 remains pA-free (see caption of Figure 2 for abbreviation of restriction endonucleases). pBM93-, pBM98- and pBM106-derived lentiviral particles were transduced into CHO-K1 (B) and HT-1080 cells (C) and grown for 48 h in the absence (–) or presence (+) of the regulating streptogramin antibiotic PI prior to microscopical and quantitative analyses of EYFP expression profiles: CHO-K1 (pBM93, –PI: 12.3 ± 1.0, +PI: 0.9 ± 0.3 RLU; pBM98, –PI: 15.6 ± 1.7, +PI: 0.9 ± 0.2 RLU; pBM106, –PI: 9.2 ± 0.2, +PI: 1.0 ± 0.2 RLU), HT-1080 (pBM93, –PI: 11.8 ± 0.8, +PI: 1.3 ± 0.3 RLU; pBM98, –PI: 21.1 ± 1.8, +PI: 1.9 ± 0.1 RLU; pBM106, –PI: 17.4 ± 0.5, +PI: 2.1 ± 0.5 RLU).
DISCUSSION
In the past decade, viral transduction systems have witnessed dramatic advances including implementation of heterologous transgene modulation (25,39–48). For lentivirus-based transduction arrangements, systems responsive to tetracycline antibiotics (the TET systems) have been most popular and were used in the following basic/optimized configurations: (i) classic TETOFF system (24,49–54), (ii) reverse tetracycline-dependent TETON system (55–59) and (iii) the TetR-based TETON technology (14,60,61). Lentiviral transduction systems have been engineered for tetracycline-responsive gene expression to (i) enable construction of stable helper cell lines producing pseudotyping VSV-G in a timely manner and/or at subtoxic doses (15,52,62), (ii) design chimeric adeno-lentiviral vectors which produce lentiviral particles from transgenic lentiviruses in a tetracycline-responsive manner (57), (iii) generate conditionally replicating HIV-based vectors for vaccination (56,59), (iv) mediate tetracycline-inducible/repressible transgene expression (24,50,51,53,54,58,60,63) and (v) modulate siRNA-based knockdown of endogenous genes (14,63).
Pioneering tetracycline-responsive expression configurations were based on two separate vectors one of which included a constitutive tetracycline-dependent transactivator and the other contained a tetracycline-responsive promoter-driven transgene expression unit engineered into a non-SIN lentiviral backbone. Owing to their non-SIN structure first-generation transgene-controlled lentivectors exhibited increased leakiness resulting from LTR-associated enhancer interference with tetracycline-responsive promoters (50–52). SIN-based tetracycline-responsive lentivectors containing an enhancer deletion in the 3' LTRU3 provided improved induction factors based on interference-free repression.
Following the proven design concept of the tetracycline-responsive expression technology, we have recently developed a human-compatible mammalian transcription control system responsive to clinically licensed streptogramin antibiotics (26). This so-called PIP system included several inducible/repressible configurations all of which showed excellent regulation performance in a variety of different cell lines and human primary cells as well as in mice (26–28,31,42,64). Also, PIP systems were fully compatible with TET and other antibiotic-adjustable transcription control systems and could be used for differential fine-tuning of different transgene activities (28,31,42). Following configuration into a lentiviral expression context, the PIP system showed excellent streptogramin-responsive transgene regulation performance, including tight repression, high-level expression, reversibility and adjustability in a variety of mammalian/human cell lines and primary cells. Efficiency of the streptogramin-responsive lentivector design was exemplified by differential differentiation reprogramming of muscle cells into fat or bone phenotypes as well as conditional vascularization of chicken embryos. Comparative regulation profiling of isogenic lentivectors either encoding a direct PPIR8-transgene expression unit or harboring PPIR8 within a SIN-modified 3'LTRU3 revealed the former configuration to enable almost two orders of magnitude higher transgene expression levels at comparable induction factors. Direct comparison of isogenic HIV- and EIAV-derived lentiviral vectors engineered for direct PPIR8-EYFP expression suggested lower EIAV-mediated transduction rates although both systems provided excellent regulation performance in the CHO-K1-derived cell lines. Although outperformed by HIV-derived lentivectors at first sight, this pioneering example of regulated transgene expression in non-primate lentiviruses may hold great promise for clinical application because of minimal risk associated with EIAV-based therapeutic interventions.
Owing to the binary nature of currently available transcription control systems, transduction of regulated transgene expression is typically realized by simultaneous co-infection of two separate lentiviral particles, one encoding the antibiotic-responsive transactivator and the second one harboring the transactivator-responsive transgene expression unit. Since co-transduction rates are typically lower and require a higher multiplicity of infection compared to transduction using a single lentiviral particle type design of single lentivector-based strategies for adjustable therapeutic interventions is the current clinical priority. First-generation one-vector-based regulated lentiviral transgene configurations included placement of independent transactivator- and transgene-encoding expression units on the same lentivector (51). However, this configuration mediated significant leaky expression due to combinatorial interference of the enhancers contained in the transactivator-driving promoter and the 3'-LTR. Autoregulated expression configurations in which transactivator and transgene are co-expressed in a coordinated manner represent the only a priori interference-free regulated expression configuration (42,65). Recently, Ogueta et al. (60) have engineered an autoregulatory lentiviral one-vector design by cloning the tetracycline repressor (TetR) and the gene of interest into a dicistronic configuration driven by the tetR-repressible promoter (PhCMV-tetO2-EGFP-IRES-TetR; tetO2, dimeric TetR-specific operator; EGFP, enhanced green fluorescent protein; and IRES, internal ribosome entry site). Although this inducible autoregulated lentiviral expression system enabled excellent regulation performance, the restricted tissue tropism of IRES elements as well as the lag phase required to accumulate repression-effective TetR concentrations may limit some applications. The bidirectional autoregulated cassette presented here addresses aforementioned limitations by enabling streptogramin-responsive transgene expression in the most compact and efficient format.
All of the currently available HIV-1-based expression vectors tailored for adjustable transgene expression in mammalian cells exhibit residual transcription activity under repressed conditions (42,65). Although such basal expression remains an inherent characteristic of any heterologous transcription control system (42,65), leaky expression is accentuated by HIV-1's integration preference for transcriptionally active hot spots, which may interfere with desired transgene control to a significant extent (42,65).
Combination of robust transgene control modalities with latest generation lentiviral transduction systems will further advance molecular interventions for gene therapy and tissue engineering.
ACKNOWLEDGEMENTS
We thank Valeria Nicolini-Gonzalez for critical comments on the manuscript, Martin Ehrbar for key advice on CAM experiments as well as Claudia Ferrara and Ivo Sonderegger for isolation of primary cells. This work was supported by the Swiss National Science Foundation (grant no. 631-065946), the Swiss Federal Office for Education and Science (BBW) within EC Framework 6 and a special research grant by the ETH Vice President for Research.
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