A Modular DNA Carrier Protein Based on the Structure of Diphtheria Toxin Mediates Target Cell-specific Gene Delivery*

Christoph UherekDagger , Jesús Fominaya§, and Winfried WelsDagger

From the Dagger  Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Federal Republic of Germany and the § Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Campus Cantoblanco, Madrid E-28049, Spain

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vectors for targeted gene delivery. Here, we describe a novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis. GD5 carries at the N terminus the DNA-binding domain of the yeast transcription factor Gal4, which is connected to a C-terminal antibody fragment specific for the tumor-associated ErbB2 antigen via an internal DT translocation domain as an endosome escape activity. Bacterially expressed GD5 protein specifically bound to ErbB2-expressing cells and formed protein-DNA complexes with a luciferase reporter gene construct. These complexes, after compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells. Inhibitors of endosomal acidification drastically reduced GD5-mediated transfection, indicating that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment. Our results suggest that fusion proteins that employ the natural endosome escape mechanism of bacterial toxins might aid in the development of efficient nonviral vectors for applications in gene therapy.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The specific delivery of therapeutic genes to defined target cell populations is a major goal of gene therapeutic strategies. Viral vectors generally facilitate highly efficient transfer and expression of foreign genes, but attempts to modify their target cell specificity have proven difficult and can result in a significant reduction of infectivity (1). Therefore, nonviral vectors, while still less efficient in gene delivery than viral particles, are gaining increasing attention (2). Self-assembling systems such as complexes of DNA with polycationic polymers can be easily manipulated with respect to their target cell specificity, and they are safe due to the absence of viral genomic sequences and highly flexible in the size of the DNA incorporated.

Molecular conjugates of poly-L-lysine with natural or artificial ligands utilize the DNA-binding and -condensing properties of poly-L-lysine for the interaction with DNA (3-5). Upon formation of a DNA-poly-L-lysine-ligand complex (polyplex (6)), gene transfer is facilitated via receptor-mediated endocytosis. While these conjugates deliver the DNA to defined target cell populations, for efficient gene transfer, they require an additional mechanism that, upon internalization of the polyplex, facilitates the release of DNA from endosomal vesicles and prevents lysosomal degradation. To achieve this, adenoviral particles (7) or fusogenic peptides of viral origin (8) have been included in the polyplex, thereby exploiting the natural endosome escape mechanism of viruses, which is dependent on a drop in the endosomal pH during intracellular trafficking.

Similar to these viral activities, a number of bacterial toxins, upon cell binding and internalization, depend on endosomal acidification as a trigger to induce a conformational change within a specialized translocation domain. This promotes insertion into vesicular membranes and allows the delivery of an enzymatically active toxin fragment to the cytosol of target cells (9). By replacing the enzymatic domain with sequences of heterologous origin, fusion proteins have been derived that employ the natural internalization mechanism of such toxins for the intracellular delivery of nontoxic peptides or protein domains (10, 11). We recently described a modular fusion protein that is based on the molecular organization of Pseudomonas exotoxin A (ETA)1 and that facilitates target cell-specific gene delivery via receptor-mediated endocytosis (12). In this protein, the C-terminal enzymatic domain of the toxin was exchanged with a DNA-binding function, and specific binding to target cells was accomplished by replacing the toxin's natural cell recognition domain with a recombinant antibody fragment.

Here, we have extended this approach and describe the construction, bacterial expression, and functional characterization of a novel carrier protein for target cell-specific gene transfer. This chimeric protein, termed GD5, is based on the molecular organization of diphtheria toxin (DT), another bacterial toxin with functional similarities to Pseudomonas ETA. DT is a single chain polypeptide of 535 residues (13) that, in contrast to ETA, carries the catalytic domain at the N terminus followed by an internal translocation domain and the C-terminal cell-binding domain (14). The chimeric GD5 fusion protein mimics the structure of wild-type DT and connects, via the DT translocation domain, an N-terminal Gal4 DNA-binding region and a C-terminal ErbB2-specific single chain antibody fragment as heterologous effector and cell recognition functions, respectively. Thereby, the DNA-binding domain derived from the yeast Gal4 transcription factor allows complexation of the chimeric molecule with plasmid DNA containing the Gal4 recognition motif (15). The chosen scFv(FRP5) antibody domain facilitates binding to the tumor-associated antigen ErbB2, which is overexpressed in many human tumors of epithelial origin and has been proposed as a suitable target for directed cancer therapy (16, 17).

Upon bacterial expression and purification, the GD5 fusion protein spontaneously formed a protein-DNA complex with a luciferase reporter gene construct, which, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells. Thereby, the transfection efficiency of GD5-DNA complexes was strictly dependent on the transit through an acidic environment, suggesting that the bacterial translocation domain in the chimeric DNA carrier protein functions in a fashion very similar to the wild-type toxin.

    EXPERIMENTAL PROCEDURES
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Cells and Culture Conditions-- COS-1 SV40-transformed African green monkey kidney cells and SKBR3 and MDA-MB468 human breast carcinoma cells were maintained in Dulbecco's modified Eagle's medium (BioWhittaker, Inc.) containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. HC11 murine mammary epithelial cells and HC11-ErbB2 cells (HC11 R1#11) stably transfected with a human erbB2 cDNA were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 µg/ml bovine insulin, 10 ng/ml EGF, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 mg/ml G418 (HC11-ErbB2) as described (18).

Construction of the DNA Carrier Protein GD5-- Plasmid pSW50-G encoding the DNA-binding domain of the yeast Gal4 transcription factor (Gal4 amino acids 2-147) was derived by HindIII cleavage of plasmid pSW50-5EG (12) and subsequent religation of the vector fragment. A double-stranded oligonucleotide adapter with SacI and SalI compatible ends and containing an internal NheI restriction site was constructed by annealing the oligonucleotides 5'-CGCTAGCTGGTGGTG-3' and 5'-TCGACACCACCAGCTAGCGAGCT-3' and inserted into SacI/SalI-digested pSW50-G. A gene fragment encoding the DT translocation domain (Val195-Gly383) was derived by polymerase chain reaction using, as a template, plasmid pJV127, which contains a DT-interleukin-2 fusion gene (19), and the primers 5'-CGTGTCAGGCTAGCAGTAGGTAGC-3' and 5'-CATGCGTGTCGACACCCGGAGAGTAAGC-3', which introduce NheI and SalI restriction sites at the 5'- and 3'-ends of the polymerase chain reaction fragment, respectively. Plasmid pSW50-GD was obtained by cleavage of the resulting DT polymerase chain reaction product with NheI and SalI and insertion into NheI/SalI-digested pSW50-G containing the adapter sequence. A cDNA fragment encoding the ErbB2-specific single chain antibody scFv(FRP5) was obtained by SalI/BglII digestion of plasmid pWW152-5 (20) and inserted in-frame 3' of Gal4 and DT sequences in SalI/BglII-digested plasmid pSW50-GD, resulting in construct pSW50-GD5. Subsequently, HindIII/KpnI and KpnI/XhoI fragments of the fusion gene were isolated and reassembled in the HindIII/XhoI-digested bacterial expression vector pSW55, which is similar to pSW50, but lacks the bacterial ompA signal sequence. The resulting pSW55-GD5 plasmid encodes, under the control of the inducible tac promoter, the GD5 fusion protein consisting of a synthetic FLAG epitope and six histidine residues at the N terminus followed by the Gal4 DNA-binding domain, the DT translocation domain, and the ErbB2-specific antibody fragment at the C terminus.

Bacterial Expression and Purification of GD5-- Escherichia coli BL21(DE3) trxB- cells (kindly provided by A. Plückthun) (21) carrying plasmid pSW55-GD5 were grown at 37 °C to A550 = 0.7 in LB medium containing 100 µg/ml ampicillin and 0.6% glucose. Protein expression was induced for 90 min at room temperature by the addition of 0.25 mM isopropyl-beta -D-thiogalactopyranoside. Cells were harvested by centrifugation; resuspended in 50 mM Tris-HCl, pH 8.0, containing 8 M urea, 150 mM NaCl, and 0.3 mM phenylmethylsulfonyl fluoride; and lysed by sonication. Cell lysates were cleared by centrifugation at 30,000 × g for 30 min at 4 °C and loaded onto a Ni2+-saturated chelating Sepharose Fast Flow column (Amersham Pharmacia Biotech) under denaturing conditions (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 8 M urea). Unbound proteins were removed by washing the column with equilibration buffer containing 50 mM imidazole. Specifically bound proteins were eluted by increasing the imidazole concentration of the buffer to 250 mM. For complete denaturation and reduction of disulfide bonds, fractions containing the GD5 fusion protein were pooled and treated with 0.3 M dithioerythritol for 2 h at room temperature. The samples were then diluted 50-fold in refolding buffer containing 100 mM Tris-HCl, pH 8.0, 8 mM oxidized glutathione, 0.5 M L-arginine, 2 mM EDTA, and 150 mM NaCl. Refolding of the protein was allowed to take place for 2 days at 10 °C. Subsequently, the refolded protein was concentrated and dialyzed against 50 mM Tris-HCl, pH 8.0, containing 50 mM KCl, 5 mM MgCl2, 20 µM ZnCl2, and 20% glycerol.

Binding of the GD5 Protein to ErbB2-- The binding of the GD5 fusion protein to ErbB2 was determined by fluorescence-activated cell sorter analysis using ErbB2-expressing HC11-ErbB2 cells and parental HC11 cells as a control. Trypsinized cells (5 × 105) in phosphate-buffered saline were incubated for 40 min at 4 °C with 3 µg of purified GD5 protein. Unbound proteins were removed, and cells were washed twice with phosphate-buffered saline and then treated with 1 µg of monoclonal antibody RK5C1 (Santa Cruz Biotechnology Inc.) specific for the Gal4 DNA-binding domain for another 40 min followed by phycoerythrin-labeled goat anti-mouse IgG (Rockland Inc.) as a secondary antibody. Fluorescence of cells was analyzed with a FACScan (Becton Dickinson).

Transfection of Cells with GD5-Polyplex-- Transfection complexes were prepared by incubation of 2 µg of pSV2G4LUC plasmid DNA (12) with variable amounts of the GD5 fusion protein in a buffer containing 50 mM Hepes, pH 7.5, 50 mM KCl, 5 mM MgCl2, and 100 µM ZnCl2 for 15 min at room temperature. To facilitate condensation of the protein-DNA complex, poly-L-lysine HBr (Sigma) with an average degree of polymerization of 236 residues (pL236) was added slowly to the complex to a final concentration of 250 nM, and the mixture (total volume of 100 µl) was incubated for an additional 15 min. For transfection, cells were seeded in 12-well tissue culture plates at a density of 5 × 104 cells/well and grown overnight at 37 °C. The growth medium was exchanged with 1 ml/well fresh medium 30 min before the addition of protein-DNA complexes (100 µl/well). The cells were incubated with protein-DNA complexes for 4 h at 37 °C; then the medium was exchanged, and the cells were grown for another 40 h before they were harvested for analysis.

Luciferase Assay-- Luciferase assays were performed as described (12). The growth medium was removed, and the cells were washed twice with phosphate-buffered saline and lysed for 15 min at room temperature in 100 µl of buffer containing 25 mM glycylglycine, pH 7.8, 1 mM dithiothreitol, 15% glycerol, 8 mM MgSO4, 1 mM EDTA, and 1% Triton X-100. The lysates were cleared by centrifugation, and protein content was determined by the Bradford method (22). Fifty µl of the lysate were mixed with an identical volume of dilution buffer containing 25 mM glycylglycine, pH 7.8, 10 mM MgSO4, and 5 mM ATP. Luciferase activity was monitored for 30 s in a AutoLumat LB 953 luminometer (Berthold) with automatic injection of 300 µl of luciferin solution containing 250 mM luciferin (Sigma), 25 mM glycylglycine, pH 7.8, and 0.5 mM coenzyme A (Boehringer Mannheim). Luciferase activity was determined as relative light units/mg of cellular protein.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Bacterial Expression of the Modular DNA Carrier Protein GD5-- DNA fragments encoding the sequence-specific DNA-binding domain of the Gal4 transcription factor from the yeast Saccharomyces cerevisiae, the translocation domain of the bacterial diphtheria toxin, and the ErbB2-specific single chain antibody scFv(FRP5) were assembled into a single open reading frame in the bacterial expression vector pSW55. The resulting plasmid (pSW55-GD5) encodes, under the control of the isopropyl-beta -D-thiogalactopyranoside-inducible tac promoter, the modular fusion protein GD5, which is shown schematically in Fig. 1A. It consists of an N-terminal synthetic FLAG epitope, a polyhistidine tag facilitating the purification of the chimeric protein via Ni2+ affinity chromatography, amino acids 2-147 of the yeast Gal4 protein (15), amino acids 195-383 of diphtheria toxin (13), and the C-terminal scFv(FRP5) antibody domain (23).


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Fig. 1.   Construction and bacterial expression of the GD5 DNA carrier protein. A, schematic representation of the GD5 fusion gene in the E. coli expression plasmid pSW55-GD5. The inducible tac promoter controls the expression of the chimeric GD5 protein, which consists of an N-terminal synthetic FLAG epitope (F), a polyhistidine tag (H), amino acids 2-147 of the yeast Gal4 protein (DNA-binding domain), amino acids 195-383 of the bacterial diphtheria toxin (translocation domain (DT-T)), and the C-terminal ErbB2-specific scFv(FRP5) antibody domain. B, SDS-polyacrylamide gel electrophoresis analysis of GD5 fusion protein purified from bacterial lysates. Lane 1, bacterial lysate cleared by ultracentrifugation; lanes 2 and 3, 50 mM imidazole wash and 250 mM imidazole eluate, respectively, from a Ni2+-saturated chelating Sepharose column; lanes 4 and 5, immunoblot analysis of GD5-containing fractions with a monoclonal antibody specific for the synthetic FLAG epitope. M, molecular mass standards (in kilodaltons).

The recombinant protein was expressed in E. coli strain BL21(DE3) trxB- and purified from cleared bacterial lysates under denaturing conditions via binding to Ni2+-saturated chelating Sepharose and elution with 250 mM imidazole. Fractions containing the recombinant fusion protein were pooled, and the protein was refolded and dialyzed as described under "Experimental Procedures." A typical result of the purification is shown in Fig. 1B. GD5 fusion protein with a purity of >80% was obtained after a single round of purification as determined by SDS-polyacrylamide gel electrophoresis and Coomassie staining (Fig. 1B, lane 3) and was detected as a single band with calculated molecular mass of 67.9 kDa after immunoblotting with the FLAG-specific antibody M2 (Fig. 1B, lanes 4 and 5).

ErbB2-specific Binding of GD5-- The C-terminal scFv(FRP5) antibody domain was included in the chimeric GD5 protein to direct the protein specifically to ErbB2-expressing target cells and, upon binding to the ErbB2 receptor, to facilitate uptake of protein-DNA complexes via receptor-mediated endocytosis. The functionality of the scFv(FRP5) domain in the GD5 protein was investigated by fluorescence-activated cell sorter analysis. HC11 murine mammary epithelial cells or HC11-ErbB2 cells stably transfected with a human erbB2 cDNA (18) were incubated with GD5, and specifically bound fusion protein was detected with a monoclonal antibody directed against the Gal4 DNA-binding domain and with phycoerythrin-labeled goat anti-mouse IgG. The results are shown in Fig. 2. Strong binding of GD5 to HC11-ErbB2 cells, but no significant binding to HC11 control cells, was observed, showing that the scFv(FRP5) domain in the GD5 protein is functionally active and directs the fusion protein to ErbB2-expressing cells. In an enzyme-linked immunosorbent assay experiment, also binding of GD5 to recombinant ErbB2 protein with half-maximal saturation at a concentration of 12 nM was observed (data not shown). The DNA-binding activity of GD5 was confirmed in a gel retardation assay. As described previously for a fusion protein containing an identical Gal4 fragment (12), the addition of purified GD5 to a 32P-labeled Gal4-specific oligonucleotide resulted in the formation of a protein-DNA complex with lower electrophoretic mobility on a nondenaturing polyacrylamide gel (data not shown).


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Fig. 2.   Binding of the GD5 fusion protein to ErbB2. The binding of GD5 to the surface of HC11-ErbB2 cells stably transfected with a human erbB2 cDNA and ErbB2-negative parental HC11 cells was detected by fluorescence-activated cell sorter analysis. Trypsinized cells (5 × 105) in phosphate-buffered saline were incubated with 3 µg of purified GD5 protein. Unbound proteins were removed; cells were washed; and cell-bound GD5 was detected with monoclonal antibody RK5C1 specific for the Gal4 DNA-binding domain followed by phycoerythrin-labeled goat anti-mouse IgG (filled areas). Control cells were treated with the secondary antibodies in the absence of GD5 (open areas).

GD5 Facilitates Gene Transfer into COS-1 Cells-- For the analysis of GD5-mediated gene transfer, we used the reporter gene plasmid pSV2G4LUC, which encodes the firefly luciferase gene under the control of the SV40 early promoter and contains two consecutive Gal4 recognition motifs in the 3'-untranslated region of the expression cassette for interaction with Gal4 fusion proteins (12). Increasing concentrations of purified GD5 protein were mixed with circular pSV2G4LUC plasmid DNA to allow complex formation. Subsequently, poly-L-lysine with an average chain length of 236 residues (pL236) was added to the protein-DNA complex to neutralize excess negative charge and to achieve condensation of the DNA (4). We used a pL236/DNA molar ratio of 50:1, which results in an electroneutral complex as confirmed by gel retardation assays (data not shown).

The GD5-DNA-poly-L-lysine complex (GD5-polyplex) was added to COS-1 SV40-transformed monkey kidney cells at a final concentration of 2 µg/ml pSV2G4LUC plasmid DNA, 0.5-4.5 µg/ml GD5 fusion protein, and 25 nM pL236 in standard growth medium containing 10% fetal bovine serum. Cells incubated with a similar polyplex lacking the GD5 fusion protein served as a control. COS-1 cells express high levels of ErbB2 protein on their surface and have previously been used as a model system for the analysis of ErbB2-specific gene transfer (12). The cells were incubated with the transfection complexes for 4 h; then the medium was exchanged, and the cells were grown for an additional 40 h before they were harvested and analyzed for luciferase activity. The results are shown in Fig. 3A. Treatment of cells with GD5-containing polyplex resulted in efficient expression of the luciferase reporter gene, which was up to 25-fold higher than in cells treated with polyplex alone. Thereby, luciferase activity correlated with the amount of DNA carrier protein in the transfection complex.


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Fig. 3.   A, GD5-mediated gene transfer in COS-1 cells. The cells were seeded in 12-well tissue culture plates at a density of 5 × 104 cells/well and grown overnight. Transfection complexes containing the indicated amounts of the GD5 fusion protein, pSV2G4LUC luciferase reporter plasmid, and poly-L-lysine (GD5) were prepared as described under "Experimental Procedures" and added to the cells in normal growth medium. Control cells were treated with pSV2G4LUC polyplex lacking the fusion protein (pL). After 4 h, the medium was exchanged, and the cells were grown for another 40 h before they were harvested for analysis. Luciferase activity is expressed in relative light units (RLU)/mg of total protein. B, dependence of gene transfer on specific interaction of the Gal4 DNA-binding domain. COS-1 cells were treated with polyplex containing 4.5 µg of GD5 and either pSV2G4LUC or the similar pSV2LUC plasmid, which lacks a Gal4-specific DNA recognition sequence. Control cells were incubated with pSV2G4LUC or pSV2LUC polyplex lacking the fusion protein. Cells were harvested, and reporter gene expression was analyzed as described above.

To analyze the dependence of GD5-mediated gene transfer on the interaction of the Gal4 DNA-binding domain with its DNA recognition sequence, COS-1 cells were treated with GD5-polyplex containing either pSV2G4LUC or the similar pSV2LUC plasmid, which lacks Gal4-specific DNA recognition sequences (24). As shown in Fig. 3B, in the absence of a Gal4-specific recognition sequence in the plasmid DNA, the efficiency of GD5-mediated gene transfer was drastically reduced, demonstrating the requirement of specific protein-DNA interaction for carrier protein-mediated gene transfer.

GD5-mediated Transfection of Cells Is Dependent on the Presence of ErbB2-- The target cell specificity of GD5-mediated gene transfer was investigated using COS-1 and SKBR3 human breast carcinoma cells, which express ~2 × 105 and 1 × 106 ErbB2 molecules/cell, respectively (12, 20); MDA-MB468 human breast carcinoma cells, which do not express detectable ErbB2 levels (20); and HC11 cells, which do express the murine ErbB2 homologue, but are not recognized by the FRP5 antibody, which is specific for the human ErbB2 molecule (25). The cells were treated with polyplex containing pSV2G4LUC plasmid DNA and 4.5 µg/ml GD5 fusion protein for 4 h as described above, and luciferase activity was determined 40 h later. Control cells were treated with polyplex lacking the carrier protein. High levels of luciferase activity were detected in ErbB2-expressing COS-1 and SKBR3 cells after treatment with GD5-polyplex, whereas only low nonspecific DNA uptake and reporter gene expression were found in cells treated with polyplex alone (Fig. 4). In contrast, the ErbB2-negative cell lines MDA-MB468 and HC11 could not be transfected with GD5-polyplex. These results indicate that GD5-mediated gene transfer is cell type-specific and dependent on the accessibility of ErbB2 receptors on the surface of target cells.


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Fig. 4.   Specificity of GD5-mediated gene transfer. ErbB2-expressing COS-1 and SKBR3 cells and MDA-MB468 and HC11 cells lacking the expression of human ErbB2 were seeded in 12-well tissue culture plates at a density of 5 × 104 cells/well and grown overnight. The cells were then treated with GD5-pSV2G4LUC polyplex (GD5) or with pSV2G4LUC polyplex lacking the fusion protein (pL). After 4 h, the medium was exchanged, and the cells were grown for another 40 h before they were harvested for analysis. Luciferase activity is expressed in relative light units (RLU)/mg of total protein.

Effect of Chloroquine on GD5-mediated Gene Transfer-- The chimeric GD5 molecule contains the translocation domain of the bacterial diphtheria toxin as a means to facilitate endosome escape of protein-DNA complexes upon internalization into target cells. Previously, the acidotropic reagent chloroquine (26) has been shown to serve as an endosome escape activity and to dramatically enhance the efficiency of various nonviral gene transfer vectors that utilize receptor-mediated endocytosis for DNA delivery (27-29). Chloroquine blocks endosomal acidification, but also accumulates in intracellular vesicles and induces osmotic swelling of the endosomes, which could result in endosome destabilization and the release of internalized DNA (30). The effect of chloroquine on GD5-mediated gene delivery was investigated using COS-1 cells. The cells were treated with GD5-polyplex as described above in the presence or absence of 100 µM chloroquine. Control cells were treated with polyplex lacking the fusion protein. As shown in Fig. 5, chloroquine had a drastic effect on gene transfer mediated by polyplex alone, which resulted in an ~20-fold increase in reporter gene expression. In contrast, GD5-mediated gene transfer was enhanced only 2-fold in the presence of chloroquine, suggesting that GD5 already provides an efficient endosome escape mechanism.


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Fig. 5.   Effect of chloroquine on GD5-mediated gene transfer. COS-1 cells were seeded in 12-well tissue culture plates at a density of 5 × 104 cells/well and grown overnight. The cells were then treated with GD5-pSV2G4LUC polyplex (GD5) for 4 h as described in the legend of Fig. 4 either in the presence or absence of 100 µM chloroquine as indicated. Control cells were incubated with pSV2G4LUC polyplex lacking the fusion protein (pL). Cells were harvested, and reporter gene expression was analyzed 40 h later. Luciferase activity is expressed in relative light units (RLU)/mg of total protein.

GD5-mediated Gene Transfer Is Dependent on the Activity of the Diphtheria Toxin Translocation Domain-- Intact diphtheria toxin binds to the heparin-binding EGF-like growth factor precursor on the surface of target cells (31). Upon receptor-mediated internalization, the DT translocation domain integrates into the endosomal membrane and facilitates translocation of the N-terminal enzymatic domain of the toxin across the membrane to the cytosol. For this membrane insertion, a conformational change of the translocation domain is required, which is induced by a drop in the endosomal pH below 5.5, resulting in the exposure of membrane-interacting hydrophobic helices (14, 32, 33). Due to its dependence on a low pH environment, the cytotoxic activity of wild-type diphtheria toxin can be reduced significantly if endosomal acidification is prevented with specific inhibitors (34).

In the GD5 fusion protein, the N-terminal enzymatic domain of the toxin was replaced by the Gal4 DNA-binding domain, and the C-terminal cell recognition domain was exchanged with the ErbB2-specific scFv(FRP5) antibody fragment. To confirm that the DT translocation domain in the chimeric GD5 protein is functionally active and contributes to transfection efficiency, GD5-mediated gene transfer in the presence of various inhibitors of endosomal acidification was investigated. COS-1 cells were transfected with GD5-pSV2G4LUC polyplex in the presence of 50 mM ammonium chloride, 50 mM methylamine, 5 µM nigericin, or 200 nM bafilomycin A1. These drugs inhibit vacuolar acidification by different mechanisms: ammonium chloride and methylamine are weak acidotropic bases, nigericin is a carboxylic ionophore that mediates exchange of monovalent cations through the membrane, and bafilomycin A1 is a potent inhibitor of vacuolar H+-ATPases (26, 35). In control cells treated with polyplex in the absence of GD5, reporter gene expression remained unaffected by the inhibitors or was slightly enhanced (up to 2-fold) (Fig. 6A). In contrast, GD5-mediated gene transfer was strongly reduced when endosomal acidification was inhibited. Thereby, treatment with nigericin had the most significant effect and reduced luciferase expression to 11% of that in untreated control cells, followed by ammonium chloride (reduction to 23%), methylamine (reduction to 31%), and bafilomycin A1 (reduction to 33%).


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Fig. 6.   GD5-mediated gene transfer depends on the activity of the translocation domain. A, COS-1 cells were treated with GD5-pSV2G4LUC polyplex (filled bars) or pSV2G4LUC polyplex lacking the fusion protein (open bars) for 4 h in the presence of 50 mM ammonium chloride, 50 mM methylamine, 5 µM nigericin, or 200 nM bafilomycin A1 as indicated. Cells were harvested, and reporter gene expression was analyzed 40 h later. Relative luciferase activities in comparison to cells treated with GD5-pSV2G4LUC polyplex in the absence of inhibitors are shown. B, to illustrate the contribution of the translocation domain to transfection efficiency, the ratios of relative luciferase activities upon treatment of cells with GD5-polyplex versus polyplex alone (pL) in the presence of the indicated inhibitors of endosomal acidification are shown. The data for chloroquine were taken from the experiment shown in Fig. 5.

To further illustrate the contribution of the translocation domain to transfection efficiency, the ratios of relative luciferase activities upon treatment of cells with GD5-polyplex versus polyplex alone in the presence of inhibitors of endosomal acidification were calculated (Fig. 6B). In the absence of drugs, the incorporation of GD5 in the polyplex resulted in a 25-fold increase in luciferase expression in COS-1 cells in comparison to treatment with polyplex alone, whereas transfection efficiency was decreased to less than five times over control values when inhibitors of endosomal acidification were present. This remaining enhancement of transfection is most likely due to the targeting effect mediated by the scFv domain of the GD5 fusion protein, which is not affected by the inhibitors. These results clearly show that endosomal acidification is required for efficient GD5-mediated gene transfer and suggest that the DT translocation domain in the fusion protein acts in a fashion similar to the wild-type toxin.

    DISCUSSION
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Abstract
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Procedures
Results
Discussion
References

Molecular conjugates that employ antibodies or other ligands chemically coupled to polycations to deliver DNA to cells constitute an important group of nonviral vectors that are investigated as tools for therapeutic gene delivery (3-5). The protein and DNA components of such artificial compounds assemble into stable complexes with defined target cell tropism. Thereby, interaction with DNA is mediated by the binding of a polycationic reagent such as poly-L-lysine to negatively charged plasmid DNA, whereas incorporation of a target cell-specific ligand facilitates cell recognition and uptake of the complex via receptor-mediated endocytosis. An important improvement of this concept was the incorporation of endosomolytic activities, which, upon internalization into cells, greatly enhances the release of DNA from endocytic vesicles and the expression of the transferred gene (7, 8).

An alternative approach for the design of modular self-assembling systems for gene delivery is based on fusion proteins engineered to incorporate different domains that account for the distinct activities required for DNA binding and the different steps of cell recognition, intracellular delivery, and nuclear transport (12, 24, 36-38). Here, we have chosen the bacterial diphtheria toxin as a molecular building plan for a fusion protein that combines target cell recognition, endosome escape, and DNA-binding activities in a single polypeptide chain. Analysis of the crystal structure of diphtheria toxin revealed that the different functions of the toxin that facilitate cell binding, endosome release, and inhibition of protein synthesis reside in distinct protein domains (14). The N-terminal domain represents the catalytic moiety that facilitates ADP-ribosylation of eukaryotic elongation factor 2; the C-terminal domain confers binding to the cellular receptor heparin-binding EGF-like growth factor precursor (31); and the internal domain mediates the release of the toxic domain from the endosome to the cytosol.

In the chimeric GD5 protein, the N-terminal effector domain was replaced by the sequence-specific DNA-binding domain of the yeast transcription factor Gal4, which also incorporates the natural nuclear localization signal of Gal4 located within the first 29 amino acid residues of the protein (39). The original cell recognition function of the toxin was exchanged with the ErbB2-specific scFv(FRP5) antibody domain. The internal DT translocation domain represented by DT amino acids 195-383 was retained as an endosome escape activity. Due to the different molecular organization of the respective parental toxin, in the GD5 fusion protein, DNA- and cell-binding functions are arranged in an orientation opposite to that of the previously described 5EG molecule, which is based on Pseudomonas ETA (12). Therefore, the scFv domain is located at the C terminus of the DT-derived fusion protein, but at the N terminus of the ETA-derived construct.

For GD5, the C-terminal location of the scFv domain did not affect cell-specific binding as demonstrated by flow cytometric analysis. Target cell specificity was also observed in transfection experiments with a luciferase reporter construct where only ErbB2-positive cells showed much higher luciferase expression when transfected with GD5-polyplex in comparison to cells transfected with polyplex alone. In ErbB2-negative cells, polyfection could not be enhanced by GD5. Unexpectedly, transfection of ErbB2-negative cell lines with GD5-polyplex resulted in even lower luciferase activities than transfection with polyplex alone (~10 times lower for HC11 cells and 20 times lower for MDA-MB468 cells), suggesting that nonspecific uptake of protein-DNA complexes is reduced when the GD5 protein is bound to the polyplex.

The GD5 protein contains the DT translocation domain as an endosome escape activity. When wild-type DT becomes exposed to an acidic pH below 5.5, the translocation domain undergoes a conformational change that triggers its insertion into the endosomal membrane (9, 40). Thereby, ion-conductive channels are formed (41), and translocation of the catalytic domain is induced (42, 43). Despite the apparent correlation between channel formation and translocation, the relevance of these channels for the process of translocation remains uncertain. In one possible model, direct translocation of the extended catalytic domain through such a channel has been proposed (41, 44). Alternatively, pore formation could severely affect endosome integrity, which would result in bulk release of luminal contents (40, 45). The latter effect is more likely to be involved in GD5-mediated gene transfer, where a large DNA-poly-L-lysine complex must be released from the endosomes, and has also been suggested as a possible mechanism in a recent study where the DT translocation domain chemically coupled to poly-L-lysine was used to facilitate endosome escape of a polyplex (46).

Here, the DT translocation domain is provided as part of the DNA carrier protein. Its presence in the polyplex resulted in a 25-fold increase in transfection efficiency in comparison to the polyplex alone, which is similar to the 20-fold increase observed when cells were simultaneously treated with polyplex and chloroquine, a reagent with potent endosomolytic activity (27-29). When both GD5 and chloroquine were present in the transfection, only a further 2-fold enhancement of luciferase expression was observed, indicating that GD5 alone already mediates efficient endosomal release. However, whereas cytotoxic effects were observed upon prolonged exposure of cells to chloroquine, GD5 did not affect cell viability or inhibit proliferation at the concentrations used (data not shown).

The cytotoxic activity of wild-type DT is reduced significantly if endosomal acidification is prevented, which underscores the critical importance of an acidic environment for toxin translocation (34). Here, we have used different inhibitors of endosomal acidification to block the translocating properties of the DT translocation domain in the GD5 protein. Among the reagents used, chloroquine was the only one that enhanced GD5-mediated transfection. Ammonium chloride, methylamine, nigericin, and bafilomycin A1 drastically reduced luciferase expression, indicating that the DT translocation domain in the GD5 protein functions in a manner similar to wild-type DT. The enhancement of transfection by chloroquine is most probably not related to its pH-neutralizing activity, but due to secondary effects such as osmotic swelling and endosome disruption induced by the reagent (30). When the ratios of relative luciferase activities upon transfection of cells with GD5-polyplex versus polyplex alone were calculated, chloroquine did not differ from the other inhibitors and reduced GD5-mediated transfection efficiency to <5-fold over control values compared with a 25-fold increase in transfection by GD5 in the absence of these compounds. This indicates that like the other inhibitors, chloroquine, despite its enhancing effect on transfection, blocked the activation of the DT translocation domain of GD5.

We have shown that a chimeric molecule based on the structure of the bacterial diphtheria toxin facilitates efficient target cell-specific gene delivery. This concept of modular fusion proteins that combine a cell recognition function with DNA-binding and endosome escape activities in a single molecule allows them to easily adopt novel or improved functions. This might aid in the further development of artificial virus-like particles suitable for a broad range of applications in gene therapy.

    ACKNOWLEDGEMENTS

We thank Drs. J. R. Murphy and J. vanderSpek for providing plasmid pJV127 and Dr. R. Rüger (Boehringer Mannheim GmbH) for support of this work.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-761-2061630; Fax: 49-761-2061599; E-mail: wels{at}tumorbio.uni-freiburg.de.

1 The abbreviations used are: ETA, exotoxin A; DT, diphtheria toxin; EGF, epidermal growth factor; scFv, single chain antibody.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Schnierle, B. S., and Groner, B. (1996) Gene Ther. 3, 1069-1073[Medline] [Order article via Infotrieve]
  2. Cooper, M. J. (1996) Semin. Oncol. 23, 172-187[Medline] [Order article via Infotrieve]
  3. Wu, G. Y., and Wu, C. H. (1987) J. Biol. Chem. 262, 4429-4432[Abstract/Free Full Text]
  4. Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4255-4259[Abstract]
  5. Perales, J. C., Ferkol, T., Molas, M., and Hanson, R. W. (1994) Eur. J. Biochem. 226, 255-266[Abstract]
  6. Felgner, P. L., Barenholz, Y., Behr, J. P., Cheng, S. H., Cullis, P., Huang, L., Jessee, J. A., Seymour, L., Szoka, F., Thierry, A. R., Wagner, E., and Wu, G. (1997) Hum. Gene Ther. 8, 511-512[Medline] [Order article via Infotrieve]
  7. Curiel, D. T., Agarwal, S., Wagner, E., and Cotten, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8850-8854[Abstract]
  8. Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7934-7938[Abstract]
  9. Olsnes, S., van Deurs, B., and Sandvig, K. (1993) Med. Microbiol. Immunol. 182, 51-61[Medline] [Order article via Infotrieve]
  10. Donnelly, J. J., Ulmer, J. B., Hawe, L. A., Friedman, A., Shi, X. P., Leander, K. R., Shiver, J. W., Oliff, A. I., Martinez, D., Montgomery, D., and Liu, M. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3530-3534[Abstract]
  11. Novoa, I., Cotten, M., and Carrasco, L. (1996) J. Virol. 70, 3319-3324[Abstract]
  12. Fominaya, J., and Wels, W. (1996) J. Biol. Chem. 271, 10560-10568[Abstract/Free Full Text]
  13. Greenfield, L., Bjorn, M. J., Horn, G., Fong, D., Buck, G. A., Collier, R. J., and Kaplan, D. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6853-6857[Abstract]
  14. Choe, S., Bennett, M. J., Fujii, G., Curmi, P. M., Kantardjieff, K. A., Collier, R. J., and Eisenberg, D. (1992) Nature 357, 216-222[CrossRef][Medline] [Order article via Infotrieve]
  15. Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F., and Ptashne, M. (1989) J. Mol. Biol. 209, 423-432[Medline] [Order article via Infotrieve]
  16. Hynes, N. E., and Stern, D. F. (1994) Biochim. Biophys. Acta 1198, 165-184[CrossRef][Medline] [Order article via Infotrieve]
  17. Wels, W., Groner, B., and Hynes, N. E. (1996) Curr. Top. Microbiol. Immunol. 213, 113-128[Medline] [Order article via Infotrieve]
  18. Hynes, N. E., Taverna, D., Harwerth, I. M., Ciardiello, F., Salomon, D. S., Yamamoto, T., and Groner, B. (1990) Mol. Cell. Biol. 10, 4027-4034[Medline] [Order article via Infotrieve]
  19. vanderSpek, J. C., Mindell, J. A., Finkelstein, A., and Murphy, J. R. (1993) J. Biol. Chem. 268, 12077-12082[Abstract/Free Full Text]
  20. Wels, W., Beerli, R., Hellmann, P., Schmidt, M., Marte, B. M., Kornilova, E. S., Hekele, A., Mendelsohn, J., Groner, B., and Hynes, N. E. (1995) Int. J. Cancer 60, 137-144[Medline] [Order article via Infotrieve]
  21. Proba, K., Ge, L., and Plückthun, A. (1995) Gene (Amst.) 159, 203-207[CrossRef][Medline] [Order article via Infotrieve]
  22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  23. Wels, W., Harwerth, I. M., Zwickl, M., Hardman, N., Groner, B., and Hynes, N. E. (1992) Bio/Technology 10, 1128-1132[Medline] [Order article via Infotrieve]
  24. Fominaya, J., Uherek, C., and Wels, W. (1998) Gene Ther. 5, 521-530[CrossRef][Medline] [Order article via Infotrieve]
  25. Harwerth, I. M., Wels, W., Marte, B. M., and Hynes, N. E. (1992) J. Biol. Chem. 267, 15160-15167[Abstract/Free Full Text]
  26. Mellman, I., Fuchs, R., and Helenius, A. (1986) Annu. Rev. Biochem. 55, 663-700[CrossRef][Medline] [Order article via Infotrieve]
  27. Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3655-3659[Abstract]
  28. Cotten, M., Langle-Rouault, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4033-4037[Abstract]
  29. Fritz, J. D., Herweijer, H., Zhang, G., and Wolff, J. A. (1996) Hum. Gene Ther. 7, 1395-1404[Medline] [Order article via Infotrieve]
  30. Wagner, E., Curiel, D., and Cotten, M. (1994) Adv. Drug Delivery Rev. 14, 113-135
  31. Naglich, J. G., Metherall, J. E., Russell, D. W., and Eidels, L. (1992) Cell 69, 1051-1061[Medline] [Order article via Infotrieve]
  32. Sandvig, K., and Olsnes, S. (1981) J. Biol. Chem. 256, 9068-9076[Free Full Text]
  33. vanderSpek, J., Cassidy, D., Genbauffe, F., Huynh, P. D., and Murphy, J. R. (1994) J. Biol. Chem. 269, 21455-21459[Abstract/Free Full Text]
  34. Umata, T., Moriyama, Y., Futai, M., and Mekada, E. (1990) J. Biol. Chem. 265, 21940-21945[Abstract/Free Full Text]
  35. Bowman, E. J., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7972-7976[Abstract]
  36. Chen, S. Y., Zani, C., Khouri, Y., and Marasco, W. A. (1995) Gene Ther. 2, 116-123[Medline] [Order article via Infotrieve]
  37. Paul, R. W., Weisser, K. E., Loomis, A., Sloane, D. L., LaFoe, D., Atkinson, E. M., and Overell, R. W. (1997) Hum. Gene Ther. 8, 1253-1262[Medline] [Order article via Infotrieve]
  38. Wels, W., and Fominaya, J. (1998) in Self-assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial (Kabanov, A. V., Felgner, P., and Seymour, L. W., eds), pp. 351-369, John Wiley & Sons Ltd., Chichester, England
  39. Nelson, M., and Silver, P. (1989) Mol. Cell. Biol. 9, 384-389[Medline] [Order article via Infotrieve]
  40. London, E. (1992) Biochim. Biophys. Acta 1113, 25-51[Medline] [Order article via Infotrieve]
  41. Kagan, B. L., Finkelstein, A., and Colombini, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4950-4954[Abstract]
  42. O'Keefe, D. O., Cabiaux, V., Choe, S., Eisenberg, D., and Collier, R. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6202-6206[Abstract]
  43. Madshus, I. H., Wiedlocha, A., and Sandvig, K. (1994) J. Biol. Chem. 269, 4648-4652[Abstract/Free Full Text]
  44. Hoch, D. H., Romero-Mira, M., Ehrlich, B. E., Finkelstein, A., DasGupta, B. R., and Simpson, L. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1692-1696[Abstract]
  45. Jiang, G. S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429[Abstract/Free Full Text]
  46. Fisher, K. J., and Wilson, J. M. (1997) Biochem. J. 321, 49-58[Medline] [Order article via Infotrieve]


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