(Received for publication, December 11, 1995; and in revised form, February 15, 1996)
From the
Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains: 1) an ErbB-2-specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enables sequence-specific high affinity binding to DNA. Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro. Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner. Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex. Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer.
Somatic gene therapy is based upon the introduction of therapeutically active genes into individual cells. A great variety of effector genes have been shown to hold promise for the treatment of inherited or acquired diseases in humans, but the lack of optimal gene delivery methods still presents a major limitation of such approaches. Viral vectors are widely used in gene therapy applications due to their high level of DNA uptake and expression. However, major disadvantages of viral vectors for gene transfer include a lack of cell type specificity with regard to their infectivity, a restriction in the size of incorporated DNA, and safety considerations. Alternative strategies for the delivery of DNA into target cells are being developed that are based upon the construction of artificial viral-like particles incorporating only activities required for efficient DNA transport and expression, but avoiding viral genomic information(1, 2) .
Complexes of DNA with ligand-poly-L-lysine conjugates for cellular uptake via receptor-mediated endocytosis fulfill some of the requirements of such reconstructed pseudo-viral vectors and have been developed extensively during the last years(3, 4) . By covering plasmid DNA with poly-L-lysine molecules covalently attached to different ligand moieties, multimolecular toroid structures can be obtained which are small enough (80-100 nm) to be engulfed by endosomes(5) . However, these particles lack the ability to actively escape from the endosome before reaching the lysosomal compartment, which strongly decreases transfection efficiency(6, 7) . Therefore adenovirus particles have been incorporated to supply an endosome escape activity(8, 9, 10) . Also the addition of fusogenic peptides of viral origin to the DNA complex provides endosome escape activity in the absence of a viral genome, but is not able to fully reproduce the efficiency of the whole viral particle(11, 12) . Both approaches rely on natural mechanisms to escape from the endosome, which are triggered by a drop in the endosomal pH during the intracellular routing to the lysosome.
Several naturally occurring proteins of non-viral origin also utilize this acidification as a signal to activate an endosome escape function. A variety of bacterial toxins are able to bind specifically to receptors on the target cell surface and, after internalization via receptor-mediated endocytosis, facilitate the translocation of biologically active protein domains from the endosome to the cytosol. In addition to cell binding and enzymatic domains these proteins carry an internal domain with translocation function(13) . By replacing the cell recognition domain with antibodies or natural ligands, such bacterial toxins have been engineered to redirect toxicity to a restricted type of target cells (14) or, by modifying the enzymatic domain, have been used to transport heterologous protein domains into cells(15, 16) . We have described previously a recombinant single chain toxin consisting of an antibody domain specific for the ErbB-2 receptor protein overexpressed in a high percentage of human tumors and the translocation and enzymatic domains of Pseudomonas exotoxin A(17, 18) . This molecule is selectively cytotoxic in vitro and in vivo for human tumor cells overexpressing the ErbB-2 receptor protein.
Here we propose a novel approach for the target cell-specific transfer of DNA. Based on the multidomain structure of the bacterial Pseudomonas exotoxin A we have developed a fusion protein as a specific DNA carrier. It consists of three functional domains: 1) a scFv(FRP5) single chain antibody domain binding to the ErbB-2 receptor confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a sequence-specific DNA binding domain derived from the yeast transcriptional activator GAL4 enables high affinity binding to DNA molecules. Complexes of the multidomain fusion protein with plasmid DNA carrying a reporter gene and a GAL4-specific recognition sequence, after condensation and charge neutralization with poly-L-lysine, are able to transfect ErbB-2-expressing tumor cells in vitro in a cell-specific manner. Our data suggest that this system might be useful for the target cell-specific delivery of genes with therapeutical potential.
Figure 1:
Construction and bacterial expression
of DNA-binding carrier proteins. A, schematic representation
of chimeric multidomain carrier proteins. The bacterially expressed
fusion protein 5EG consists of an N-terminal E. coli ompA
signal peptide (SP), the synthetic FLAG epitope (F),
a polyhistidine tag (H), the single chain antibody (scFv) domain derived from the ErbB-2-specific monoclonal
antibody FRP5, amino acids 252-366 of Pseudomonas exotoxin A (translocation domain), amino acids 2-147 of the
yeast GAL4 protein (DNA binding domain), and a C-terminal KDEL
endoplasmic reticulum retention signal. 5G is a similar chimeric
protein lacking the translocation domain and the KDEL signal, while EG
is the corresponding molecule without the cell recognition domain. B, SDS-polyacrylamide gel electrophoresis analysis of the 5EG
protein purified from bacterial lysates. Lane 1, bacterial
lysate cleared by ultracentrifugation; lane 2, eluate from a
Ni-saturated chelating Sepharose column with 250
mM imidazole; lane 3, sample 2 after concentration by
ultrafiltration; lane 4, immunoblot analysis of sample 2 with
a monoclonal antibody specific for the GAL4 DNA binding domain; M, molecular weight standards.
The multiple cloning site of plasmid pSW50 (18) was modified by inserting a double-stranded oligonucleotide containing HindIII, XbaI, KpnI, SacI, EcoRI, BglII, and XhoI restriction sites between the original HindIII and XhoI sites of pSW50. The resulting plasmid pWF50 was used for the construction of 5EG-derived fusion genes lacking either the scFv(FRP5) or the ETA domain. The HindIII restriction site between the ETA and the GAL4 domain in plasmid pWW35 was destroyed and replaced by a KpnI site via HindIII digestion of pWW35 and insertion of a double-stranded sequence derived by self-annealing the oligonucleotide 5`-AGCTGGGTACCC-3`. A KpnI/SacI fragment containing the GAL4 domain without the C-terminal KDEL sequence and a XbaI/EcoRI fragment containing the ETA-GAL4 sequences were derived from the resulting plasmid. The XbaI/EcoRI fragment was inserted into XbaI/EcoRI-digested pWF50 giving rise to plasmid pWF50-EG. pWF50-EG encodes a fusion protein consisting of the ETA and GAL4 domains but lacking the ErbB-2-specific scFv(FRP5) antibody domain. The plasmid pWF50-5G encoding a fusion of the scFv(FRP5) and the GAL4 domains was constructed by subsequently inserting the scFv(FRP5) domain as a HindIII/XbaI fragment from pSW50-5 into pWF50 (pWF50-5) followed by inserting into pWF50-5 the GAL4 domain as a KpnI/SacI fragment.
A
similar experiment was performed using ErbB-2-overexpressing SKBR3
human breast carcinoma cells. The cells were seeded on 96-well tissue
culture plates at a density of 1 10
cells/well and
grown for 16 h at 37 °C. The cells were fixed with 3.7%
formaldehyde, and nonspecific binding sites were blocked with 3% bovine
serum albumin. 5EG fusion protein was added to the cells, and specific
binding was analyzed as described above.
The protein-DNA complex
was added to the cells. The final concentration of the components in
the complex during cell incubation was 1 nM DNA, 4 nM fusion protein, and 50 nM pL. The cells
were incubated at 37 °C for 16 h, then the medium was exchanged,
and the cells were incubated for another 36 h before they were
harvested for analysis.
The fusion proteins were
expressed in E. coli strain CC118. Total bacterial lysates
were prepared in 8 M urea, cleared by centrifugation, and
recombinant proteins were purified via binding to
Ni-saturated chelating Sepharose and elution with 250
mM imidazole. Fractions containing the recombinant fusion
proteins were pooled, imidazole and denaturant were removed by
dialysis, and the proteins were concentrated by ultrafiltration. Fig. 1B shows a SDS-polyacrylamide gel electrophoresis
analysis of the 5EG fusion protein (>90% pure) after a single round
of purification. The protein was detected as a single band after
immunoblotting with a monoclonal antibody specific for the GAL4 DNA
binding domain (Fig. 1B, lane 4). The same band
appeared when a polyclonal exotoxin A antiserum (17) or a
polyclonal anti-scFv antibody (25) were used for detection
(data not shown).
Figure 2:
Functional characterization of the 5EG
multidomain carrier protein. A, the ErbB-2-specific binding of
5EG was analyzed in an ELISA experiment. Immobilized recombinant
protein comprising the extracellular domain of ErbB-2 was incubated
with increasing concentrations of the 5EG fusion protein ranging from
240 pM to 1 µM. Specifically bound protein was
detected with a polyclonal rabbit antiserum raised against purified Pseudomonas exotoxin A followed by alkaline
phosphatase-coupled goat anti-rabbit antibody and photometrical
detection of the conversion of the phosphatase substrate p-nitrophenyl phosphate at 405 nm. B, the binding of
the 5EG protein to DNA was analyzed in a band shift assay. 1 pmol of
5EG protein was incubated with 50 fmol of P-labeled GAL4
2 double-stranded oligonucleotide containing a tandem repeat of
the GAL4 recognition motif 5`-CGGAGGACAGTCCTCCG-3` and separated on a
nondenaturing polyacrylamide gel (lane 1). The positions of
two bands with decreased electrophoretic mobility and the free probe
are indicated by arrows. The more intense higher molecular
weight complex represents two 5EG dimers bound to the GAL4-specific
probe (complex 4:1), the lower molecular weight complex represents only
one bound dimer (complex 2:1). To show the specificity of the binding,
increasing amounts of the nonradioactive probe ranging from 50 fmol to
12.8 pmol were added to the binding mixture as a competitor (lanes
2-6). C, correlation between the amount of
nonradioactive competitor added and the relative amount of the
radioactive probe in the higher molecular weight complexes in B. The relative radioactivity was quantified using a FUJIX
BAS1000 phosphorimager.
Binding of a 5EG protein-DNA complex containing digoxigenin-labeled GAL4-specific oligonucleotide to ErbB-2 was also analyzed. In an ELISA experiment with immobilized ErbB-2 overexpressing SKBR-3 cells specifically bound complex could be detected with an anti-digoxigenin AP-coupled antibody, indicating that both binding domains of 5EG are active simultaneously (data not shown).
The final complex was added to COS-1 SV40-transformed
monkey kidney cells in standard growth medium. Control cells were
treated with a complex containing plasmid DNA and
poly-L-lysine, but lacking the 5EG fusion protein. COS-1 cells
express approximately 2 10
ErbB-2 molecules/cell.
Monoclonal antibody FRP5 which is specific for human ErbB-2,
cross-reacts with the ErbB-2 homolog of COS-1 cells (data not shown).
Cells were harvested after 48 h, and the expression of the luciferase
reporter gene was analyzed in cell lysates (Fig. 3). A clear
linear correlation (r = 0.991) between the amount of
protein and the level of reporter gene expression was observed, when a
constant amount of plasmid DNA was used in the complex. Control cells
treated with a complex lacking the 5EG carrier protein displayed only
background luciferase activity.
Figure 3:
5EG fusion protein mediated gene transfer.
COS-1 cells were seeded in 12-well tissue culture plates at a density
of 4 10
cells/well and grown overnight. Samples
containing 4 µg of pSV2G4LUC luciferase reporter plasmid DNA,
increasing amounts of the multidomain fusion protein 5EG ranging from
19 to 75 ng, and 2.5 µg of poly-L-lysine pL
(average degree of polymerization, 236 residues) were added to
the cells. As a negative control, a sample without 5EG was used. Cells
were harvested and reporter gene expression was analyzed 48 h later.
Luciferase activity is expressed in relative light units/mg of total
protein. The inset shows the linear correlation between the
amount of fusion protein added and the luciferase activity as a measure
for the efficiency of DNA transfer.
Due to its large size and high
negative charge, spontaneous uptake of DNA into cells is prevented.
Both, neutralization and condensation of the DNA can be achieved by the
interaction with polycationic reagents (28, 29) . A
strong correlation between DNA condensation and cellular uptake of
ligand-DNA complexes has been shown previously(5) . In the 5EG
protein-DNA complex, the presence of poly-L-lysine was
essential, since neither plasmid DNA alone nor 5EG protein-DNA complex
in the absence of the polycation were able to successfully transfect
COS-1 cells and result in significant luciferase activity (data not
shown). Also protection of the DNA from nuclease activity is important.
At the poly-L-lysine pL:DNA molar ratio of 50:1
established experimentally to achieve an electroneutral complex, no DNA
degradation was observed after incubation of the complex with 10% FCS
at 37 °C for 1 h. In addition, no difference in transfection
efficiency was observed when COS-1 cells were incubated with
5EG-DNA-pL
complex in tissue culture medium in the
presence or absence of 10% FCS (data not shown).
Figure 4:
Gene transfer mediated by the carrier
protein 5EG is target cell-specific. A, ErbB-2 expressing
COS-1 and SKBR3 cells and MDA-MB468 cells basically lacking the
receptor protein were seeded in 12-well tissue culture plates at a
density of 4 10
cells/well and grown overnight.
Samples containing 4 µg of pSV2G4LUC luciferase reporter plasmid
DNA, 240 ng of 5EG fusion protein, and 2.5 µg of
poly-L-lysine pL
were added to the cells (5EG). A sample lacking the 5EG fusion protein was used in
each case as a negative control (pL). Cells were harvested and
luciferase activity was analyzed 48 h later and expressed in relative
light units per mg of total protein. B, the dependence of
5EG-mediated DNA transfer on the binding to ErbB-2 was also determined
using a control protein (EG) which lacks the ErbB-2-specific
scFv domain. COS-1 cells were treated with protein-DNA complexes and
the expression of the luciferase reporter gene was analyzed as
described above.
Highest luciferase activity was observed in COS-1 cells, although
SKBR-3 cells express approximately five times more ErbB-2 protein. This
might be explained by episomal replication and amplification of the
pSV2G4LUC reporter plasmid in the SV40 T-antigen expressing COS-1
cells, facilitated by the plasmid's SV40 origin of
replication(30) . The level of nonspecific transfection with
the pSV2G4LUC-pL complex lacking the 5EG protein varied
among the cell lines tested and seemed to be cell type-dependent. Also
in this case COS-1 cells showed luciferase activities around 1 order of
magnitude higher than the other cell lines tested.
The dependence of
successful gene transfer on the binding to ErbB-2 was further confirmed
using a chimeric DNA-binding fusion protein termed EG which lacks the
N-terminal scFv(FRP5) domain. In contrast to the 5EG containing
complex, a similar complex containing EG, pSV2G4LUC plasmid DNA, and
poly-L-lysine pL did not result in an increase
of luciferase activity in COS-1 cells over background levels achieved
with a pL
-DNA control (Fig. 4B).
Figure 5:
Efficient gene transfer depends upon the
presence of the translocation domain. A, an ErbB-2-specific
DNA-binding protein lacking the translocation domain (5G) was
constructed in order to analyze the importance of this domain in the
gene transfer process. ErbB-2-specific binding of 5G was analyzed in
ELISA experiments. Samples containing 25 nM 5EG and increasing
concentrations from 0 to 166 nM of the 5G protein as a
competitor were added to formaldehyde-fixed SKBR3 cells. Specific
binding of 5EG was detected with a polyclonal rabbit antiserum raised
against purified Pseudomonas exotoxin A. B, SKBR3
cells were treated with 5G or 5EG containing protein-DNA complexes and
the expression of the luciferase reporter gene was analyzed as
described in the legend of Fig. 4. Competition between both
proteins was determined by coincubation of 5EG with a 3-fold molar
excess of 5G. C, the effect of the specific inhibitor of
vacuolar H-ATPases bafilomycin A1 on 5EG fusion
protein mediated gene transfer was analyzed in COS-1 cells. Bafilomycin
A1 was added to the cells at a concentration of 200 nM 30 min
before the treatment with the protein-DNA complex and maintained during
the time of the experiment.
The
transfection efficiency of complexes containing the truncated 5G
carrier protein was analyzed in SKBR-3 cells (Fig. 5B).
The cells were incubated with complexes containing pSV2G4LUC plasmid
DNA, poly-L-lysine pL and either 5G or 5EG
carrier protein. In addition, a complex containing 5EG and a 3-fold
molar excess of 5G was used. Control cells received
pSV2G4LUC-pL
complex. Luciferase expression as an
indicator for gene transfer activity could be detected in cells treated
with 5G containing complex. However, in comparison with 5EG-mediated
gene transfer the activity of 5G was reduced 6-fold. When a 3-fold
molar excess of 5G protein was added to the 5EG-containing protein-DNA
complex, 5EG-mediated gene transfer activity was completely abolished.
Such a strong competition effect at a molar ratio of 3:1 was
unexpected. It might be explained by differences in 5G and 5EG DNA
binding activities and/or an inability of heterologous 5EG/5G-DNA
complexes to translocate efficiently through endosomal membranes.
Endosomal acidification results in a conformational change of
internalized Pseudomonas exotoxin A, a step required for the
functional activation of the translocation domain and, after cleavage
by a cellular protease, successful transfer of the enzymatically active
C-terminal fragment into the cytosol(31) . In order to
characterize the activity of the exotoxin A translocation domain in the
5EG fusion protein, transfection experiments were carried out in COS-1
cells in the presence of reagents which block endosomal acidification.
When COS-1 cells were treated with complexes containing pSV2G4LUC
plasmid DNA, poly-L-lysine pL and 5EG carrier
protein in the presence of the acidotropic reagent chloroquine (100
µM), instead of a reduction we observed a
1.5-10-fold increase in transfection efficiency (data not shown).
Similar results have been reported previously when the effect of
chloroquine on gene transfer mediated by
transferrin-poly-L-lysine conjugates in the presence of the
fusogenic peptide Influ 1 was investigated(11) . This could be
explained by the dual activity of chloroquine. Chloroquine is a
vacuologenic amine which is freely membrane permeable in its
unprotonated form, but becomes trapped inside the vesicle when it binds
a proton(32) . This leads to an inhibition of endosomal
acidification, but due to an increase of the intravesicular
concentration of the molecule, it also leads to osmotic swelling, which
could result in the disruption of the endocytic vesicles and the
release of internalized DNA.
Therefore in a second approach the
macrolide antibiotic bafilomycin A1 was used, a potent inhibitor of
vacuolar H-ATPases that selectively blocks endosomal
acidification(33) . COS-1 cells were incubated with complexes
containing pSV2G4LUC plasmid DNA, poly-L-lysine pL
and 5EG carrier protein with or without the addition of 200
nM of bafilomycin A1. Luciferase expression as an indicator
for gene transfer activity was measured. The results are shown in Fig. 5C. The presence of bafilomycin A1 resulted in a
4-fold reduction of luciferase activity showing that 5EG-mediated DNA
transfer is dependent on endosomal acidification. Similar results have
been reported previously when endosome-disruptive peptides were used to
enhance gene transfer(12) . COS-1 cells were also incubated
with the protein-DNA complex in the presence of carboxylic ionophores,
another group of reagents which elevate vacuolar pH. Either the
addition of monensin (10 µM) or nigericin (5
µM) to the cells decreased the transfection efficiency to
background levels (data not shown). These data suggest that the
activity of the translocation domain in the 5EG protein is very similar
to its activity in the parental exotoxin A molecule.
Endocytosis is a major cellular mechanism for the uptake of macromolecules from the environment. Therefore a rational way to develop a system for the introduction of genetic material into cells in a nonaggressive manner is to take advantage of this natural mechanism. Receptor-mediated gene transfer has been successfully used to deliver reporter genes to cultured cells (6, 34) and to target either marker genes (35, 36) or therapeutical genes (37, 38, 39) to specific organs in an adult animal. Although still not as efficient as viral vectors, it has several advantages, which include high target cell specificity, lack of infection risk, no restriction in DNA size, and possibly lower immunogenicity. Most current approaches for receptor-mediated gene transfer are based on a ligand moiety chemically conjugated to poly-L-lysine that confers DNA binding capacity and facilitates the formation of condensed DNA-conjugate complexes of toroid structure. In addition endosome disrupting activities of viral origin have been included either in trans or via a second poly-L-lysine conjugate in order to improve the transfection efficiency(3) .
Here we present an alternative approach for a non-viral gene transfer vector that differs in several aspects from previously described systems. We have used molecular biology techniques to engineer a modular multidomain DNA carrier protein that combines in a single polypeptide chain all functions required for efficient target cell-specific gene transfer, i.e. target cell recognition, specific DNA binding, endosome escape, and a signal for nuclear transport. The fusion protein can be expressed as a recombinant molecule in bacteria and be purified to homogeneity. Chemical modification reactions which require relatively high amounts of purified material and sometimes result in products which are difficult to characterize can be avoided.
The 5EG carrier protein includes amino acids 2-147 of the yeast GAL4 transcriptional activator at the C terminus as a sequence-specific DNA binding domain. This type of domain was chosen because of the specificity and high affinity of its binding to DNA(20) . The presence of a natural nuclear localization signal provides an additional advantage. Other nuclear proteins conferring DNA binding activity and nuclear tropism have been used previously, but all of them were structural proteins (histones, protamines) with no sequence specificity (40, 41, 42, 43) . The introduction of specificity of DNA binding opens the possibility to insert the recognition motif at different nonvital positions in the DNA to be transferred and allows to predetermine the number of ligand moieties per DNA molecule and thereby to control the efficiency of its cellular uptake. As demonstrated in an electrophoretic mobility shift assay the 5EG fusion protein spontaneously forms complexes with double-stranded DNA carrying the GAL4 recognition motif. These complexes appeared to be of the expected stoichiometric composition, suggesting that well defined protein-DNA complexes could be generated simply by mixing appropriate amounts of DNA and carrier protein.
High affinity binding of the carrier protein to DNA appears desirable for two main reasons. First, the carrier protein might dissociate from the DNA more slowly which would extend the half-life of intact protein-DNA complexes. Second, after internalization by target cells it might allow to retain DNA binding in the endosomal environment. Endosome escape of the DNA still bound to the protein domain which carries a nuclear localization signal could support subsequent nuclear transport of the DNA molecule.
The 5EG fusion protein carries a single chain Fv domain derived from the ErbB-2-specific monoclonal antibody FRP5 as a target cell recognition domain. The ErbB-2 molecule is a member of the EGF receptor-related family of growth factor receptor tyrosine kinases that play an important role in the development of human malignancies. In particular overexpression of ErbB-2 has been observed in a high percentage of tumors of epithelial origin and could be correlated with an unfavorable patient prognosis(44) . Because of its enhanced expression on tumor cells, its extracellular accessibility and its ability to internalize after ligand binding, ErbB-2 presents a suitable target for directed tumor therapy. The scFv(FRP5) domain has been incorporated previously in fusion proteins to target enzymes or cytotoxic proteins to ErbB-2 expressing cells(17, 18, 25) .
Monoclonal antibodies chemically conjugated to poly-L-lysine have been used previously to target DNA to cells either alone (45) or with the help of cationic liposomes (46) or inactivated adenoviral particles(47) . The use of a single chain antibody domain for cell recognition allows the construction of smaller DNA carriers and could reduce the immunogenicity of the final protein-DNA complex in in vivo applications. The idea of reducing the size of the ligand (``minimal ligands'') has already been exploited for the development of gene transfer systems, especially in the case of asialoglycoprotein receptor targeted vectors(48, 49, 50, 51) . Recently a recombinant Fab molecule where the antibody heavy chain sequences were fused to protamine as a DNA binding domain has been used to target a suicide gene construct to cells expressing human immunodeficiency virus-1 glycoprotein 120(43) .
When ligand-poly-L-lysine conjugates were used for DNA transfer in the absence of an endosome escape function no or only low expression of transferred reporter genes was usually observed(49, 52) . The ligand-DNA complexes were able to bind to cells and could be internalized but most of them were transported to the lysosomal compartment where they were degraded by hydrolytic enzymes. The addition of acidotropic agents such as chloroquine or membrane destabilizing agents such as adenoviral particles or fusogenic peptides enhanced considerably the transfection efficiency(6, 8, 9, 10, 11, 12) . Intracellular transport of membrane-bound constituents during endocytosis is pH-dependent. Most natural mechanisms employed by viruses or bacterial toxins which avoid lysosomal degradation take advantage of endosomal acidification to trigger a reaction which facilitates the exit to the cytosol.
The composition of the 5EG DNA carrier protein is based on the multidomain structure of the bacterial Pseudomonas exotoxin A. 5EG contains an internal translocation domain representing amino acids 252 to 366 of the toxin. Wild-type exotoxin A, after binding to target cells via its N-terminal cell binding domain, is internalized. In the acidic environment of the endosome a conformational change occurs, the toxin is proteolytically cleaved within the translocation domain between Arg-279 and Gly-280 followed by the reduction of a disulfide bond, and the C-terminal fragment carrying the enzymatically active effector domain translocates to the cytosol(31) . Exotoxin A has been used previously to facilitate the internalization and translocation to the cytosol of heterologous peptides or protein domains located C-terminal of the translocation domain(15, 16) . The C-terminal GAL4 domain of the 5EG protein could be released into the cytosol via a similar mechanism. Thereby the high affinity binding of the GAL4 domain to the DNA might allow a stable interaction. However, it was not clear whether a plasmid DNA as a second molecule noncovalently attached to the C-terminal domain could be released from the endosomal compartment simultaneously. Our data support the hypothesis that DNA transfer is mediated by the translocation domain, since both the use of a carrier protein lacking this domain and the blockage of endosomal acidification resulted in drastically reduced transfection efficiency.
The amino acid sequence REDLK, which is located at the very C terminus of exotoxin A and resembles the mammalian endoplasmic reticulum retention signal, has been shown to be important for toxin activity. Indeed, when the REDLK sequence was replaced by the mammalian KDEL signal, a toxin with enhanced activity was obtained, suggesting that this signal plays a role in intracellular routing(26) . Therefore the KDEL signal was also included at the C terminus of the 5EG fusion protein, although it is not clear at present whether this signal is absolutely required for efficient endosomal release of the GAL4-DNA complex.
Improvement
of transfection efficiency by endosomolytic agents used in trans
requires relative high concentrations: 50-100 µM chloroquine(11, 50, 52) ,
10-10
adenoviral particles
(approximately 10
viral
particles/cell)(47, 53, 54) , or 10-100
µM fusogenic
peptides(12, 48, 49) . This limits its
applicability in vivo and has therefore prompted researchers
to physically attach such agents directly to DNA transfer complexes (9, 10, 55) in order to increase the
effectivity of the system and reduce the amount of material required
and the risk of undesired secondary effects. In the case of the 5EG
multidomain fusion protein, all activities are contained within the
same molecule and are therefore used at identical molar concentrations
(nM order of magnitude). The translocation domain in the
natural exotoxin A molecule is highly effective considering its
purpose: translocation of only a few toxin molecules to the cytosol is
sufficient to kill a cell (31) . For DNA transfer the situation
is different. The translocation of a high number of DNA molecules is
desired in order to achieve maximal gene expression. Therefore a higher
number of carrier proteins per DNA molecule might further improve
transfection efficiency. For other receptor-mediated gene delivery
vectors the number of ligand moieties per DNA molecule varies from 12
to
hundreds(5, 34, 46, 54, 56) .
The minimal ligand/DNA molar ratio for efficient delivery has been
established in the case of transferrin-mediated DNA transfer to be
10-15(5) . In natural gene delivery systems such as
viruses the number of cell binding moieties (spikes) per particle is
several hundreds. Therefore the current protein/DNA molar ratio in our
system of 4:1 might still be suboptimal and the introduction of
additional protein binding sites in the DNA molecule could present one
way for further improvement.
Structural domains which are able to fold independently from other regions in the parental protein are likely to retain their functionality as isolated domains and are good candidates for the use in chimeric fusion proteins. We have shown that such heterologous protein domains of mammalian, bacterial, and yeast origin can be assembled into a functional chimeric protein that thereby acquires a novel biological activity. The 5EG DNA carrier protein contains in a single polypeptide chain all the activities required for the introduction of exogenous genetic material into mammalian cells and has been shown to facilitate efficient transfection of human tumor cells in a cell-specific manner. Due to the modular structure of the 5EG carrier protein also similar vectors with modified activities, e.g. different target cell specificity, could be obtained by replacing individual protein domains. Such carrier proteins might become useful in gene therapy protocols for the target cell-specific delivery of genes with therapeutical potential.