From the Department of Pediatrics, Ludwig-Maximilians
University, 80337 Munich, Germany, § Institute of
Experimental Oncology, Technische Universität München,
81675 Munich, Germany, and ¶ Institute of Pharmacy, Free
University of Berlin, 12169 Berlin, Germany
Received for publication, April 4, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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We constructed multimers of the
TAT-(47-57) peptide. This polycationic peptide is known to be a
protein and particle transduction domain and at the same time to
comprise a nuclear localization function. Here we show that oligomers
of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles
and stabilize DNA toward nuclease degradation. At optimized vector
compositions, these peptides mediated gene delivery to cells in culture
6-8-fold more efficiently than poly-L-arginine or
the mutant TAT2-M1. When DNA was precompacted with TAT
peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was
added, transfection efficiency was enhanced up to 390-fold compared
with the standard vectors. As early as after 4 h of transfection,
reporter gene expression mediated by TAT-containing complexes was
higher than the 24-h transfection level achieved with a standard PEI
transfection. When cells were cell cycle-arrested by serum starvation
or aphidicolin, TAT-mediated transfection was 3-fold more efficient
than a standard PEI transfection in proliferating cells. In primary
nasal epithelial cells and upon intratracheal instillation in
vivo, TAT-containing complexes were superior to standard PEI
vectors. These data together with confocal imaging of TAT-DNA complexes
in cells support the hypothesis that the TAT nuclear localization
sequence function is involved in enhancing gene transfer.
Poor escape of nonviral gene vectors from the endosomal
compartment after cellular uptake and inefficient translocation into the nucleus substantially limit their efficiency (1, 2). In this study,
the arginine-rich motif of the
HIV-11 TAT protein should be
used to overcome these obstacles. The 101-amino acid HIV-1 TAT protein
regulates transcriptional activation of the human immunodeficiency
virus type 1 long terminal repeat promoter element by binding to a
short nascent stem-bulge-loop leader RNA, trans-activation response
(TAR), recruiting a positive transcription elongation complex (P-TEFb)
(3). Binding of HIV-1 TAT protein to the TAR RNA is substantially
mediated through the arginine-rich motif (amino acids 47-57), which
represents a basic stretch of amino acids located to domain 4 of the
HIV-1 TAT protein (3). Besides its importance for binding to the TAR
RNA, the arginine-rich motif of the HIV-1 TAT protein (TAT peptide) has
been shown to function as a protein transduction domain, penetrating
cell membranes in a manner different from classic endocytosis (4-6).
This uptake mechanism was also observed for heterologous proteins when
they were chemically coupled (7) or genetically fused (8) to the TAT
peptide. In addition, the conjugation of the TAT peptide to various
structures of nanometric size, such as superparamagnetic nanoparticles
(9), liposomes (10), and The objective of the present study was to examine whether the unique
features of the TAT peptide (protein transduction domain and nuclear
localization sequence) and its polycationic nature are suitable to
enhance nonviral gene delivery. Chemical properties of cationic
polymers, such as the degree of polymerization, the type of cationic
groups present in the polymer, or the amino acid composition of
cationic oligopeptides influence the biophysical properties of their
polyelectrolyte complexes with plasmid DNA. DNA binding affinity,
surface charge, and particle size have pronounced effects on their
efficacy in gene delivery and on biodistribution in vivo
(13-15). Since compaction of DNA into stable microparticulate structures by oligopeptides suitable for gene transfer requires a
minimum chain length of 6-10 cationic amino acids (13), we synthesized
the TAT peptide as oligomers. The dimer, trimer, and tetramer of
identical repeats with intervening glycine residues comprise 16, 24, and 32 positive charges, respectively.
We attempted to find a correlation between the degree of
oligomerization of the TAT peptide, the resulting biophysical
characteristics of peptide-DNA complexes, and their gene delivery
efficiency in context with the unique functions of the TAT peptide.
Peptide Synthesis
The following peptides were synthesized. Peptide
C(YGRKKRRQRRRG)2-4 (TAT2-4) contained
the arginine-rich motif of the human immunodeficiency virus type 1 TAT protein. Peptides were synthesized on an Applied Biosystems 431 A
automatic synthesizer according to a standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) protocol. Peptide
C(YGRKERRQERRG)2 (TAT2-M1) was synthesized by the Department of Medicine (Charité) (Institute of Biochemistry, Humboldt-University, Berlin). The free sulfhydryl groups were modified
by dithiodipyridine reaction (13).
Plasmid
pCMV-Luc containing firefly luciferase cDNA driven by the
cytomegalovirus promoter was generously provided by Dr. E. Wagner (Department of Pharmacy, Ludwig-Maximilians-University Munich) and
amplified as described in Ref. 16.
Size and The particle sizes were determined by dynamic light scattering,
and Fluorescence Quenching Assay
Plasmid DNA (pCMVLuc) was labeled with TOTO-1 (dye/base pair
ratio of 1:20). The TAT oligomers were serially diluted in HBS in a
96-well plate corresponding to the indicated charge ratios. 100 µl of
a solution of TOTO-1-labeled DNA (0.25 µg of DNA in HBS) was pipetted
to either 100 µl of TAT oligomer solution or 100 µl of HBS (100%)
and thoroughly mixed. The excitation filter was set at 485 nm, and the
emission filter was set at 535 nm (SPECTRAFluor Plus, Tecan, Germany).
Measurements were performed in quadruples.
Cell Culture
Human bronchoepithelial cells (16HBE14o Preparation of Gene Vector Complexes
Gene vector complexes for one well were formulated as follows. 1 µg of DNA and the corresponding amount of vector were diluted in HBS
(150 mM NaCl, 10 mM HEPES, pH 7.4) to 75 µl,
respectively. The DNA solution was pipetted to the vector solution and
mixed vigorously by pipetting up and down. The complexes were incubated for 20 min at ambient temperature before use. Ternary gene vector complexes for one well were generated in the same manner, but DNA, TAT
oligomer, and standard cationic transfection agent (PEI, average
molecular mass of 25 kDa; Aldrich, Deisenhofen, Germany; dialyzed against water, 12-14-kDa molecular mass cut-off and adjusted to pH 7; fractured dendrimers; SuperFect (Qiagen, Hilden, Germany) or
LipofectAMINE (Invitrogen, GmbH) were diluted in HBS to 50 µl,
respectively. The DNA solution was pipetted to the TAT oligomer or
poly-L-arginine (pLa; Sigma catalog no. P 4663;
Mr 5000-15,000) solution, mixed vigorously, and
incubated at ambient temperature for 10 min, and then the standard
cationic vector solution was added, again incubated at ambient
temperature for 10 min. Alternatively, DNA was first pipetted to the
standard vector solution, and the TAT oligomer solution was then added.
Transfection Procedure and Luciferase Activity Measurement
150 µl of gene vector solution, corresponding to 1 µg of
DNA, were pipetted onto cells (COS-7, 30,000 cells/well; 16HBE, 100,000 cells/well), which had been seeded into 24-well plates 1 day before, and then covered with 850 µl of medium in the absence of FCS. After
4 h of incubation at 5% CO2 and 37 °C, the medium
was replaced with 10% FCS-containing medium supplemented with 0.1%
(v/v) penicillin/streptomycin and 0.5% (v/v) gentamycin (Invitrogen).
When transfections were performed at 4 °C, cells were incubated for
1 h at 4 °C before transfection and incubation with the gene
vectors was performed at 4 °C. When transfections were performed in
the presence of a mixture of endocytosis inhibitors, the transfection
medium was supplemented with antimycin A (1 µg/ml; Sigma), sodium
fluoride (10 mM; Sigma), and sodium azide (0.1% (m/v);
Sigma), respectively. After 4 h, the medium was replaced with 10%
FCS-containing medium supplemented with antibiotics (see above).
Transfections of growth-arrested cells were performed as follows. Cells
were incubated for 24 h in the absence of FCS (serum starvation)
or subjected to incubation followed by a 12-h incubation period
with aphidicolin (25 µM, with FCS) before transfection.
Transfections were then performed either in the presence of aphidicolin
(25 µM) followed by further incubation of the cells for
24 h in aphidicolin-supplemented medium or in the absence of FCS.
24 h later, cells were lysed by the addition of 200 µl of lysis
buffer (250 mM Tris, 0.1% Triton X-100, pH 7.8) per well,
and luciferase activity was measured (Promega kit, Lumat LB 9507;
Berthold, Bad Wildbach, Germany). Protein content was determined by a
standard Bio-Rad protein assay (Bradford method).
Fluorescence in Situ Hybridization and Confocal Laser-scanning
Microscopy
A three-dimensional fluorescence in situ
hybridization was done following the protocol of Solovei and Cremer
(18) with several modifications for our specific needs.
Probe Generation--
A DNA probe was generated from the pEGFP
plasmid (Clontech) by nick translation. A
digoxigenin hapten was inserted with Dig Nick Translation Mix (Roche
Diagnostics) generating a probe with a 300-bp median length. The
reaction mixtures were cleaned of small fragments and nucleotides with
the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany). 1 µg of
probe was precipitated with 50 µg of salmon testes DNA (Sigma). The
dried pellet was reconstituted in 20 µl of formamide at 37 °C for
2 h and stored until use at Hybridization--
The probe was brought to 37 °C and mixed
1:1 with "Fish Mix" (4× SSC, 40% dextran sulfate), resulting in a
hybridization solution of 25 ng/µl probe in 2× SSC, 20% dextran
sulfate, 50% formamide. The hybridization solution was denatured at
75 °C for 5 min and briefly kept at 37 °C prior to slide
application. The slides were removed one at a time from the storage
solution, 50 µl of hybridization solution was placed on each slide, a
coverslip was applied, and finally rubber cement was used as a sealant.
The sealed slides were heated to 75 °C for 3 min to denature the
cellular DNA, also further denaturing the probe DNA. The slides were
placed in a moist chamber within a 37 °C oven and were hybridized
for 2-3 days.
Image Acquisition--
Light optical sections were generated for
each nucleus with a three-channel confocal laser-scanning microscope
(TCS 4D; Leica Inc., Deerfield, IL) equipped with a Plan Apo 63×/1.32
oil immersion lens. Using the 488 and 567 lines of an argon/krypton
laser for visualization of the Sytox and rhodamine, signals
respectively, stacks of 256 × 256 equidistant 8-bit grayscale
images were generated at an axial distance of 250 nm, pixel size of 1 nm. Each series consisted of ~20-25 images. Lines were averaged
eight times. The different fluorochromes were imaged sequentially in
identical nuclear planes. Laser power and voltage for the rhodamine
channel were maintained at the same level for all conditions, whereas the voltage of the Sytox channel was adjusted for variations in nuclear
counterstain. Cells were randomly selected when a signal appeared to be
in the cell, and the midnuclear sections were used to detect the
distribution of plasmid DNA.
Animals and Delivery of Gene Vectors to the Lung
Ternary gene vector complexes used for in vivo
experiments were generated as described above, but the TAT oligomer and
DNA solution were diluted in double-distilled water (Fresenius AG, Bad
Homburg, Germany), respectively. 60 µl of gene vector solution containing 20 µg of DNA were applied per mouse. The gene vector application was performed as described in Ref. 16. In brief, mice were
anesthetized intraperitoneally with pentobarbital and directly
intubated with a single 60-µl bolus of the indicated gene vector
using a 22-gauge intravascular cannula sleeve, needle removed (25 mm,
0.9-mm outer diameter, 0.6-mm inner diameter; Baxter, Germany). At
24 h post-transfection, mice were anesthetized intraperitoneally
with pentobarbital, and mice peritoneum were opened by midline
incision. In order to wash blood from the lungs and to avoid
interference with the subsequent luciferase assay, a posterior vena
cava exit was cut, and 1 ml of an isotonic sodium chloride solution was
slowly perfused into the mice right cardiac ventricle. The lungs were
dissected from animals, frozen in liquid nitrogen, and stored at
Statistical Analysis
Results are reported as means ± S.D. The statistical
analysis between different groups has been determined with a nonpaired t test. p Biophysical Properties of the TAT Oligomer Gene Vectors--
The
condensation of DNA into particulate structures is a prerequisite for
gene delivery. For this reason, we first examined the capability of the
TAT oligomers to condense DNA and to form particulate complexes. Laser
light scattering showed that all of the TAT oligomer gene vectors had a
diameter of ~70 nm in water, which increased to 700 nm in HBS at
charge ratios of ±1 and 5, respectively (Table
I). pLa (control) gene vectors showed a
similar diameter. TAT oligomer and pLa gene vectors had a positive Transfection Efficiency of TAT Oligomer Gene Vectors--
Plasmid
DNA was complexed with each of the TAT oligomers at different charge
ratios and transfection efficiency was examined in vitro. In
addition, pLa gene vectors were prepared under the same conditions as
control to assess the possibility of a sequence-dependent process (Fig. 2A). For each
charge ratio tested, a significant improvement of transfection by
TAT2 and TAT3 gene vectors compared with pLa
gene vectors was observed. No statistical difference between
TAT4 and pLa gene vectors was observed. The transfection rates mediated by each of the oligomers did not correlate with the
degree of oligomerization of the TAT peptide. Intermediate length of
the TAT3-oligomer mediated the highest level of transgene expression. To further characterize sequence dependence, gene transfer
efficiency mediated by the nuclear transport-deficient TAT2-M1 peptide (12) was examined. Gene transfer efficiency mediated by the TAT2-M1 peptide (± charge = 10) was
6-fold lower as compared with the TAT2 peptide (± charge = 10, Fig. 2C, p < 0.01).
The Influence of Endocytosis on Transfection Efficiency of TAT
Oligomer Gene Vectors and Comparison with Standard Cationic
Transfection Agents--
To examine endocytosis as an uptake
mechanism, transfections were performed in the presence of chloroquin
(19), at 4 °C (6, 10, 11) or in the presence of metabolic inhibitors (antimycin A, sodium azide, and sodium fluoride) (20-22). For each of
the TAT oligomer gene vectors, transfection efficiencies in the
presence of chloroquine strongly increased (Fig. 2B; compare with Fig. 2A). Gene transfer mediated by TAT2
and TAT3 gene vectors was significantly higher as compared
with pLa gene vectors. This was not the case for TAT4
vectors. Overall transfection efficiency increased in the following
order: TAT2 > TAT3 > TAT4. In the
presence of chloroquine, the transfection efficiency of
TAT2 gene vectors was enhanced 40-fold, whereas the
transfection efficiency of pLa gene vectors was only enhanced 3-fold.
Transfections performed at 4 °C led to a 20-90-fold decrease of
transgene expression (Fig. 2D). Transfection rates in the
presence of the endocytosis inhibitors decreased even more (Fig.
2E). Transgene expression mediated by TAT3-DNA
complexes in the presence of chloroquine was 3- and 4-fold higher as
compared with PEI and SuperFect polyplexes and at the same level as
LipofectAMINE lipoplexes (Fig. 2F). These data suggest that
the cellular uptake mechanism of the TAT oligomer gene vector complexes
was predominated by the endosomal pathway and could not be precisely
differentiated from endocytosis. However, due to the much stronger
increase of gene transfer of the TAT oligomer complexes in the presence
of chloroquin, as compared with pLa complexes, one could suggest that
gene transfer of the TAT oligomers, in particular TAT2, was
peptide sequence-dependent. This is supported by the higher
gene transfer efficiency of the TAT2 gene vectors as
compared with the TAT2-M1 gene vectors (Fig.
2C).
Intracellular Localization of Plasmid DNA after Transfection with
TAT Oligomer Gene Vectors--
To further investigate the localization
of plasmid DNA electrostatically bound to each of the TAT oligomers
within the cell, COS-7 cells were transfected with TAT oligomer
polyplexes or with pLa polyplexes, and plasmid DNA distribution was
visualized by fluorescence in situ hybridization.
Transfections were stopped after 4 h, and slides were analyzed by
confocal laser-scanning microscopy. Fig.
3 shows a single light optical section.
Numerous rounded red spots of DNA were seen in the cytoplasm, and a few were located in the cell nucleus and at the nuclear membrane for TAT2 gene vectors. The same distribution pattern could be
observed for TAT3 gene vectors, but the total number of
intracellular DNA spots seemed to be reduced. There were no DNA spots
found in the nucleus of cells transfected with TAT4 gene
vectors. In this case, DNA was rather found as rounded and irregular
structures restricted to only the outer side of the nuclear membrane. A
similar distribution pattern was observed for pLa gene vectors.
Ternary Gene Vector Complexes: Combination of TAT Oligomers with
Various Standard Cationic Transfection Agents--
The previous
experiments suggest that the TAT oligomers could facilitate the
transport of DNA into the nucleus. Next we examined whether the TAT
oligomers were able to enhance gene transfer efficiency of poly- or
lipoplexes that lack efficient nuclear plasmid transport (2, 23, 24).
Ternary gene vector complexes consisting of TAT oligomer, standard
cationic transfection agent, and DNA were formulated in two different
manners. Either TAT oligomer was added to preformed gene vectors
consisting of DNA and standard cationic transfection agent, or
complexes were generated vice versa through the addition of
standard cationic transfection agent to preformed TAT oligomer gene
vector complexes. When one charge equivalent of each of the TAT
oligomers or pLa was added to preformed standard cationic gene vector
complexes, the level of transgene expression remained approximately at
the same level as of the standard cationic gene vectors (Fig.
4). In contrast, when the ternary gene
vector complexes were generated vice versa (i.e.
the DNA was first complexed with one charge equivalent of each of the
TAT oligomers, and then the standard transfection agents were added), a
strong increase of transgene expression depending on the degree of
oligomerization and the type of standard transfection agent was
observed (Fig. 4). The presence of TAT2, TAT3,
and TAT4 enhanced transgene expression of PEI 130-, 80-, and 40-fold, respectively. pLa did not enhance PEI-mediated gene
transfer. An analog behavior could be observed when the TAT oligomers
were combined with either fractured dendrimers or LipofectAMINE.
Kinetics of Transgene Expression Mediated by Ternary
TATn-PEI-DNA Complexes--
To characterize in more
detail the transfection mechanism of the ternary TATn-PEI-DNA
complexes, the kinetics of transgene expression was examined on 16HBE
cells (Fig. 5). At the 4-h time point,
the transgene expression of transfected 16HBE cells was 220-, 170-, and
100-fold higher for the TAT2-, TAT3-, and
TAT4-derived ternary gene vectors as compared with PEI
polyplexes, respectively. At the 8-h time point, the enhancement in
transfection efficiency increased to 390-, 240-, and 140-fold,
respectively. After 24 h, the level of transfection efficiency
mediated by the ternary TAT2-4 gene vectors was 104-, 56-, and 45-fold higher as compared with PEI polyplexes, respectively. The
transfection efficiency of the TAT2-PEI-DNA complex at as
early as the 4-h time point was 4-fold higher as compared with the
transfection efficiency mediated by PEI polyplexes after 24 h
(p < 0.01). These data suggest rapid accumulation of
the DNA in the cell nucleus when TAT oligomers were incorporated into
the PEI polyplexes.
Transfection Efficiency of Ternary TATn-PEI-DNA on
Growth-arrested Cells--
To further assess whether the enhancement
of transfection efficiency mediated by the TAT oligomers could be due
to facilitated nuclear transport of the transgene, transfection
experiments were performed on growth-arrested cells. Transfection
experiments were performed on 16HBE cells either arrested by
aphidicolin incubation (25) or through serum starvation (26). Under
both conditions, the transfection efficiency of the ternary gene vector
complexes was significantly higher as compared with PEI polyplexes
(Fig. 6). The incorporation of
TAT2, TAT3, and TAT4 led to a 35-, 20-, and 12-fold higher transfection rate on aphidicolin-treated cells and a 12-, 10-, and 8-fold higher transfection rate on cells treated under conditions of serum starvation, respectively. In both cases, transgene expression mediated by the ternary complexes (e.g.
TAT2-PEI-DNA) was 2-3-fold higher as compared with
transgene expression mediated by PEI polyplexes under standard
conditions (i.e. no growth-arrested cells). pLa improved
PEI-mediated transfection 13- and 3.5-fold.
Transfection Efficiency of Ternary TATn-PEI-DNA on Primary
Cells and in Vivo--
To better mimic the in vivo
situation, transfection with the ternary TAT2 or pLa gene
vector complexes has been performed on primary nasal respiratory
epithelium (Fig. 7A).
Transgene expression mediated by the ternary TAT2-PEI-DNA
complexes was 10-fold higher as compared with transgene expression
mediated by PEI. The incorporation of pLa improved PEI-mediated
transfection as well, but only 1.6-fold. Gene transfer efficiency of
ternary TAT2 gene vectors has been examined in
vivo and compared with ternary pLa and standard PEI gene vectors.
Mice were intratracheally instilled with gene vector solutions, and
luciferase gene expression was measured after 24 h (Fig.
7B). Whereas luciferase expression mediated by ternary TAT2 gene vectors was 4-fold higher as compared with PEI
polyplexes, pLa induced only a 2-fold increase in luciferase gene
expression. These data indicate that TAT2 can improve gene
delivery efficiency of PEI polyplexes in vivo.
In this study, we examined whether the unique features of the
arginine-rich motif of the HIV-1 TAT protein (TAT peptide)
(i.e. protein transduction domain and nuclear localization
sequence) would enhance nonviral gene transfer. We examined these
unique features in the context of biophysical parameters of the gene vectors. The biophysical parameters of synthetic vectors have been
shown, on the one hand, to influence their gene transfer efficiency
(13, 27, 28); on the other hand, they are influenced by the molecular
weight of the cationic polymer (13, 14). Thus, to control the
biophysical parameters (i.e. also to control in parts the
gene transfer efficiency), the TAT peptide was synthesized as oligomers
of different molecular weights. Interestingly, the biophysical complex
parameters of the TAT2-4 gene vectors were similar. The
degree of oligomerization only correlated with the degree of DNA
condensation as examined by fluorescence quenching assay and increased
with the higher degree of oligomerization. Gene vectors formulated with
pLa exhibited similar biophysical parameters. These data show that the
size and surface characteristics of both the TAT oligomer and
pLa-derived gene vectors were similar, whereas DNA compaction of the
gene vectors slightly varied depending on the degree of
oligomerization. From this we conclude that differences in transfection
efficiency among the TAT oligomers and when compared with the pLa can
be primarily attributed to the TAT sequence itself rather than to
different biophysical gene vector parameters. The results obtained from
transfection experiments were further confirmed by confocal
laser-scanning microscopy, which showed higher accumulation of DNA in
the cell nucleus mediated by TAT2 and TAT3 than
by TAT4 and pLa. In addition, these data could suggest that
the affinity of the TAT oligomers to the nuclear import machinery
decreases with the higher degree of oligomerization and is minimal for
pLa.
The presence of chloroquine during transfection has been shown to
increase the level of transgene expression of several nonviral gene
vectors due to reduced endolysosomal entrapment and degradation of the gene vector complexes (19). Since transgene expression strongly
increased in the presence of chloroquine, this indicated that endosomes
were involved in cellular uptake of the gene vectors. However, it is
conceivable that two separate uptake mechanisms (i.e.
endosomal uptake and direct membrane penetration) take place independently in parallel at the same time. To examine this
possibility, the transfection process has been analyzed when
endocytosis was blocked (i.e. at 4 °C (6, 10, 11)) or in
the presence of metabolic inhibitors (20-22). Both transfections
performed at 4 °C and in the presence of endocytosis inhibitors led
to a strong reduction of transgene expression. Therefore, the main
uptake mechanism of TAT oligomer gene vectors was apparently through endocytosis and not mediated by a proposed protein transduction domain.
A possible explanation could be due to the size of the gene vectors
(~700 nm). Constructs that have been delivered into cells via the
conjugation of the TAT peptide so far have been much smaller in size
(45-200 nm) (9, 10). Thus, simple electrostatic binding of DNA by the
TAT peptide might lead to particles inappropriate for membrane penetration.
The higher gene transfer efficiency mediated by the TAT2
peptide as compared with the nuclear transport-deficient
TAT2-M1 peptide (12) and the strong increase of gene
transfer of the TAT oligomer complexes in the presence of chloroquine
as compared with pLa complexes suggests that gene transfer of the TAT
oligomers was peptide sequence-dependent, which could be
due to facilitated nuclear translocation mediated by the nuclear
localization sequence function. This concept is supported by the
behavior of ternary gene vector constructs. Transfection efficiency of
PEI in the presence of the TAT oligomers was strongly enhanced on
growth-arrested and primary cells as well as in vivo and at
early time points after transfection. In all of these cases, only a
minor fraction of cells underwent mitosis (i.e. breakdown of
the nuclear membrane could be excluded as the major mechanism for
nuclear DNA localization). Consequently, these experiments suggest that
the TAT oligomers could promote nuclear translocation of the DNA. An
increase was observed when pLa was combined with LipofectAMINE and when
experiments were performed on growth-arrested cells and in
vivo. These observations could be due to partial activity of pLa
as a nuclear localization sequence, which has been reported recently
(29).
The formulation order of vector complexes was found to have an enormous
effect on the gene delivery efficiency. A 390-fold increase of
transgene expression was only observed when the DNA was first complexed
with the TAT oligomer and PEI was added afterward. When the complexes
were generated vice versa, transgene expression remained on
the same level as of PEI polyplexes. These differences could be
explained by analysis of the structure of the resulting gene vectors.
When DNA was first complexed with the TAT oligomers at a charge ratio
of ±1, the resulting intermediate complexes had a In conclusion, we showed that oligomers of the TAT peptide mediate
efficient gene delivery. In particular, their combination with powerful
standard cationic transfection reagents (e.g. PEI) resulted
in very efficient gene transfer. This effect correlated inversely with
the degree of oligomerization, and we suggest that facilitated nuclear
localization could be involved. Further studies will focus on improved
formulation methods of TAT-DNA complexes to overcome size restrictions
that could limit the functionality of the protein transduction domain
and nuclear localization sequence so far.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage (11), led to their uptake into cells
in a manner apparently different from endocytosis. Besides this unique
feature of the TAT-peptide, the TAT peptide represents a nuclear
localization sequence (12). Upon binding of the TAT peptide itself or
its conjugates to the nucleocytoplasmic shuttle protein importin
,
nuclear import through the nuclear pore has been observed (4,
6-10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Potential Measurement
potentials were measured electrophoretically (Zetasizer 3000HS; Malvern Instruments, Herrenberg, Germany). Gene vector solutions in distilled water or HBS were generated at a DNA
concentration of 10 µg/ml (17).
) were
provided by Dieter C. Gruenert (University of Vermont, Burlington,
VT). Cells were grown in FCS (10%)-supplemented minimum essential
medium (Invitrogen GmbH, Karlsruhe, Germany) at 37 °C in a 5%
CO2 humidified air atmosphere. COS-7 cells were cultivated
in FCS (10%)-supplemented Dulbecco's modified Eagle's medium
(Invitrogen). Fresh nasal respiratory epithelium was obtained from
patients through surgery and immediately processed as follows. Briefly,
epithelial cells were detached from connective tissue after 1-h
incubation at 37 °C in the presence of dispase II (1 unit/ml
phosphate-buffered saline; Roche Diagnostics) and then filtered and
centrifuged (150 × g, 5 min, ambient temperature). The pellet was
resuspended in 8 ml of RPMI plus 10% FCS containing antibiotics, and
cells were seeded into 35-mm dishes. The next day, the medium was
replaced by bronchial epithelial cell growth medium (catalog no.
C21160; Promocell).
20 °C.
70 °C. At assay time, the tissue was thawed on ice, and 500 µl
of ice-cold lysis buffer (25 mM glycylglycine, 15 mM magnesium sulfate, 4 mM EDTA, 0.1% Triton
X-100 (m/V), 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml buffer aprotinin) was added to each sample and homogenized for
20 s using a Polytron Pt 2100 homogenizer (level 5 corresponding
to 26,000 rpm; Kinematica, Litau/Luzern, Switzerland). Samples were
centrifuged at 10,000 × g at 4 °C for 10 min, and
40 µl of the supernatant were measured for luciferase activity in a
Lumat LB 9507 instrument (Berthold) injecting 100 µl of luciferase
reagent (Promega) to each sample, and the light emitted over 10 s
was measured. The background was subtracted from the reported values.
1 × 106 relative luciferase units/10 s correspond to
1.25 ng of luciferase. All animal procedures were approved and
controlled by the local ethics committee and carried out according to
the guidelines of the German law of protection of animal life.
0.05 was considered
significant. All statistical analyses were performed using the program
StatView 5.0. (SAS Institute Inc., Cary, NC).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
potential of approximately +40 mV in water and negative
potential
of approximately
20 mV at ±1 in HBS, which changed into a
positive
potential of 20 mV at ± 5 and slightly
increased with the degree of oligomerization (Table I). The extent of
complexation was independent of the degree of polymerization as
examined by electrophoretic mobility shift assay and DNase I protection
assay (see Supplementary Material). The only difference among TAT
oligomer gene vectors depending on the oligomer size could be observed
when complexes were analyzed by a fluorescence quenching assay (Fig.
1). Fluorescence quenching strongly
increased at low charge ratios, reaching a plateau at approximately
±2. Higher charge ratios only slightly increased further DNA
condensation. Fluorescence quenching induced by pLa was shifted to a
higher charge ratio compared with the TAT peptides. Fluorescence
quenching at a given charge ratio increased with the degree of
oligomerization, indicating that the DNA was more tightly packed in the
complex when the size of the TAT oligomer increased.
Particle size (nm) and potential (mV) in water and HBS
potential (mV) was measured.
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Fig. 1.
TOTO-I-labeled DNA was complexed with each of
the TAT oligomers or pLa with increasing charge ratios. The
relative fluorescence intensity ratio depending on the charge ratio of
each of the TAT oligomer gene vector complexes is plotted with respect
to the fluorescence of DNA-TOTO-I before condensation.
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Fig. 2.
Transfection efficiency of TAT oligomers or
pLa gene vectors at different charge ratios under various conditions
(COS-7 cells). A, in the absence of chloroquin;
B, in the presence of chloroquine (300 µM);
D, at different temperatures; E, in the absence
or presence of endocytosis inhibitors; C, in comparison with
the mutant TAT2-M1 (16HBE14o cells), F, in
comparison with various nonviral gene vectors (data show a
representative experiment, n = 4, ± S.D.; transfection
rates of TAT oligomers significantly different from the pLa or
TAT2-M1 complexes are indicated by an asterisk
(p < 0.05), TAT2-M1 (p < 0.01)).
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Fig. 3.
Intracellular localization of plasmid DNA
(pEGFP). COS-7 cells were transfected with TAT oligomer or pLa
gene vectors, and transfections were stopped after 4 h. Images
were generated with a 63× objective by confocal laser-scanning
microscopy. Green emission signal represents the cell nuclei
stained with Sytox 16; red emission signal shows the
distribution of pEGFP in the same microscope field using a
digoxigenin-labeled DNA probe. The probe was detected with
anti-digoxigenin rhodamine antibody.
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Fig. 4.
Transfection of COS-7 cells with various
ternary TAT oligomer gene vector complexes. Either DNA was first
complexed with the TAT oligomer peptides at a charge ratio of 1 and PEI
(N/P = 10), fractured dendrimers
(N/P = 4.5), or LipofectAMINE (w/w = 10:1) were added afterward (A), or DNA was first complexed
with PEI (N/P = 10), fractured dendrimers
(N/P = 4.5), or LipofectAMINE (w/w = 10:1) and the TAT oligomer peptides (±1) were added (B)
(data show a representative experiment, n = 4, ± S.D.;
transfection rates significantly different from the standard vector are
indicated by an asterisk (p < 0.05)).
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Fig. 5.
Kinetics of transgene expression. 16HBE
cells were transfected with the ternary gene vector complexes or PEI
polyplexes, and luciferase activity was measured at the indicated time
points (n = 3, ± S.D.; transfection rates mediated by
the ternary complexes were significantly different from PEI at each
time point (p < 0.01)).
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Fig. 6.
Transfection efficiency of ternary gene
vector complexes on growth-arrested cells. 16HBE cells were either
blocked by aphidicolin incubation or by serum starvation and
transfected with ternary gene vector complexes or PEI polyplexes
(n = 3, ± S.D.; transfection rates significantly
different from PEI are indicated by an asterisk
(p < 0.05)).
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Fig. 7.
Transfection efficiency of ternary gene
vector complexes on primary nasal respiratory epithelium
(A) or in vivo
(B). A, data show a
representative experiment, n = 4, ± S.D.; transfection
rates significantly different from the standard vector are indicated by
an asterisk (p < 0.05). B,
n = 5; for PEI, n = 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
potential of
20 mV (Table I), whereas DNA complexed with PEI at a nitrogen of PEI
per phosphate of the DNA ratio of 10 resulted in a
potential of +32
mV. Thus, preformed negatively charged TAT gene vectors allow the
binding of positively charged PEI to their surface through
electrostatic interaction, which was indicated by a change of the
potential from negative to positive (+40 mV) and by size reduction to
600 nm. In contrast, such changes were not observed when gene vectors
were formulated vice versa. Thus, the formulation method
could result in a shell-like ternary complex with the TAT oligomers
bound to the DNA in the core of the complex and a layer of PEI at the
periphery of the complex (see Supplementary Material Fig.
3A). In contrast, binding of the TAT oligomers on the
surface of positively charged PEI polyplexes should not be possible due
to electrostatic repulsion. In this case, TAT oligomers could rather be
homogeneously distributed in the solution (Supplementary Material Fig.
3B). From these data, we infer the following model, which
could explain the exceptional efficiency of the ternary gene vector
complexes. The ternary complexes, which apparently look like a
plain PEI complex, could behave like a plain PEI polyplex concerning
the first steps that are involved in gene transfer (i.e. the
complexes are apparently taken up into the cell via receptor-mediated
endocytosis (heperan sulfate proteoglycan receptor) (30) and are
located to the endosomal compartment. In the endolysosomes, the
"proton-sponge" effect of PEI (31) could induce their disruption
and release the complexes into the cytosol. Within the next steps, the
PEI shell could be released, probably due to nonspecific interaction
with cytosolic components, and the core complex could slowly diffuse
toward the cell nucleus. Binding of the TAT oligomer to the
nucleocytoplasmic shuttle protein importin
could then facilitate
DNA translocation into the nucleus, resulting in high transgene
expression. In this model, PEI would function as an endosomal
disrupting agent such as chloroquin but incorporated into the complex itself.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants Ro994/2-1.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.
The on-line version of this article (available at
http://www.jbc.org) contains three additional figures.
To whom correspondence should be addressed: Division of
Molecular Pulmonology, Dept. of Pediatrics, University of Munich, Lindwurm 2a, Kubus, D-80337 Munich, Germany. Tel.: 49-89-5160-7524; Fax: 49-89-5160-4421.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211891200
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; TAR, trans-activation response; HBS, Hepes-buffered saline; FCS, fetal calf serum; pLa, poly-L-arginine; PEI, polyethyleneimine; RLU, relative light units.
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REFERENCES |
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