From the Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom
Received for publication, November 13, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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VP22, a structural protein of herpes simplex
virus, exhibits unusual trafficking properties which we proposed might
be exploited in gene and protein delivery applications. To pursue the
use of the protein itself for cargo delivery into cells, we developed an expression system for the C-terminal half of VP22, residues 159-301
(VP22.C1), and purified the protein in high yields. Addition of short
oligonucleotides (ODNs) induced the assembly of novel particles, which
were regular spheres with a size range of 0.3 to 1.0 µm in diameter,
incorporating both protein and ODN. Following the particles in living
cells using fluorescently tagged ODNs, we show that they enter
efficiently within 2-4 h, and reside stably in the cell cytoplasm for
up to several days. Remarkably, however, light activation induced
particle disruption and release of the protein and ODN to the nucleus
and cytoplasm within seconds, a process that we have captured by time
lapse microscopy. In addition to delivering antisense ODNs, ribozymes,
and RNA/DNA hybrids, the VP22.C1 protein could also be modified to
include peptides or proteins. These particles have the potential for
delivery of a wide range of therapeutic agents in gene therapy and
vaccine development.
The herpes simplex virus protein VP22, encoded by the
UL49 gene (1), is a major structural component of the
virus (2) recruited into the tegument compartment in ~2000 molecules
per virion (3). We previously reported the observations of unusual trafficking properties of VP22 whereby after being synthesized in a
subpopulation of cells, in which it appeared largely cytoplasmic, the
protein could also be observed in adjacent cells where it accumulated
mainly in the nucleus (4). Similar observations have been made for a
number of the homologues of VP22 in other We also observed that when extracts containing VP22 were applied to the
medium of tissue culture cells, the protein could subsequently be
detected within cells, accumulating in the nuclei (4). Therefore we
further proposed that VP22 protein itself could be additionally
exploited for the delivery of different types of cargoes. One
particular class of molecules upon which much research has focused is
that of the small oligonucleotides (ODNs),1 including antisense
ODNs, ribozymes, triple helix forming ODNs, RNA/DNA hybrids, and
recently small interfering RNAs. While significant successes have been
reported in the development and application of such compounds, delivery
both in vitro where they may be applicable for,
e.g. target validation, and in vivo in a
therapeutic setting, remains a major issue (for a review, see Ref. 9).
As a result considerable effort has been directed into developing
strategies to overcome the different factors, such as the activity of
serum nucleases, cellular uptake, endosomal release, intracellular
distribution, and stability, that limit the activity of such ODNs. A
nonexhaustive list of the many approaches being pursued includes the
use of cationic lipids or liposomes, polycations, conjugation with
sterols or the use of fusigenic peptides, and while each approach
offers certain advantages, each also has distinct limitations (9, 10).
Proteins with unusual trafficking properties such as VP22, TAT (11),
and the Antennapaedia homeodomain (12) offer the prospect of protein
and peptide delivery, but proteins generally have not been developed as
agents for ODN delivery. In this work we present results on additional
unusual properties of VP22 in recruiting ODNs into spherical particles
which efficiently enter cells. A novel feature of the VP22/ODN
particles is that after cell entry they remain stable in the cytoplasm,
but can be activated by light, whereupon the ODN and protein diffuse
throughout the cell, with the ODN accumulating in the nuclei. While we
do not yet understand the mechanism promoting this activity, we have utilized the VP22/ODN particles to deliver candidate ODNs, and demonstrated light dependent activity. The assembly of such particles, which we have termed Vectosomes, may offer dual delivery applications, with the protein recruiting and delivering candidate therapeutic ODNs,
or the ODN nucleating the assembly of a particle for the delivery of
variants of VP22 carrying additional functional proteins. We propose
that Vectosomes may represent a versatile new type of delivery agent
for protein and ODN delivery.
Oligonucleotides--
HPLC purified fluorescent phosphorothioate
or phosphodiester oligonucleotides (F-ODN) were purchased from Genosys,
Cambridge, United Kingdom. A biotinylated fluorescent oligonucleotide
was obtained from the same supplier. The sequences of the F-ODNs used in this study were as follow: 5'-CCCCCACCACTTCCCCTCTC-3', corresponding to the 3'-untranslated region of ICAM-1; 5'-TCCCGCCTGTGACATGCATT-3', corresponding to the 3'-untranslated region of human
c-raf kinase gene;
5'-TCCCGCGCACTTGATGCATT-3', a control ODN for the
anti-c-raf ODN containing 7 base mismatches (underlined).
A 36-mer fluorescent hammerhead ribozyme (HPLC purified) directed
against c-myb (13) was as follows:
5'-guuuucccUGAuGaggccgaaaggccGaaAuucucc-3' (normal lowercase indicates 2'-O-methyl nucleotides,
uppercase indicates 2'-hydroxyl (ribo) nucleotides, U indicates
2'-O-allyl uridine, backbone linkages were phosphodiester with five
phosphorothioate linkages at each end). A HPLC purified 68-mer
fluorescein-labeled RNA/DNA hybrid molecule (RDO) directed against the
tyrosinase gene (14) had the following sequence;
5'-ACTGCGGAAACTGTAAGTTTGGATTTTTTaauccaaacuTACAGuuuccgcaguGCGCGTTTTCGCGC-3', where DNA residues are capitalized and the 2'-O-methyl
RNA residues are in lowercase. The ribozyme and RDO were purchased from
Cruachem, Glasgow, UK.
Expression and Purification of VP22 Protein--
A fragment
corresponding to the C-terminal amino acids 159-301 of HSV VP22 was
amplified with 5' and 3' primers designed for insertion of the fragment
into the prokaryotic expression vector pET24b (Novagen) to yield the
expression vector pVP24. This strategy results in the addition of an
extra 14 residues from the vector to the N terminus of the VP22 coding
region and a 6xHis tag to the C terminus. The coding region of the VP22
open reading frame in pVP24 was confirmed by sequencing. Expression was
performed in the Escherichia coli strain BL21pLysS.
Overnight cultures grown in the presence of kanamycin (10 µg/ml) and
chloramphenicol (34 µg/ml) were diluted (1:100) and incubated until
the A600 reached ~0.4. Expression was induced
by the addition of
isopropyl-1-thio-
The supernatant was further clarified by incubation (1 h, 4 °C) with
DEAE-Sepharose (3.5 ml of a 50% slurry in lysis buffer), and the
unbound fraction then loaded onto a 5-ml Ni-NTA (Qiagen) column
(equilibrated in lysis buffer). The column was then washed in buffer
(lysis buffer omitting DNase and RNase and containing 40 mM
imidazole, pH 8.0, 10% glycerol, 0.1% Nonidet P-40), and bound
material eluted in either a step elution at 500 mM
imidazole or a gradient of imidazole from 0 to 500 mM.
VP22.C1 eluted at ~200 mM imidazole. Samples were stored
frozen directly or after dialysis in PBS. Protein concentrations were
measured by the Bradford assay using bovine serum albumin as standard
(Pierce Protein Plus Assay kit). Mass Spectrometry was performed by the
Protein and Nucleic Acid Facility, University of Cambridge. A fraction
of the dialyzed protein was labeled with fluorescein using a
succinimidyl ester of fluorescein supplied in the Fluorescein-EX
Protein Labeling kit (Molecular Probes). VP22.C1 was incubated with the
reactive dye for 3 h at room temperature and unconjugated
fluorescein was separated from labeled protein by gel filtration.
ODN Binding by Gel Retardation Analysis--
To examine VP22.C1
binding to ODN, a standard amount of a fluorescently labeled 20-mer
phosphorothioate (0.6 nmol) was incubated with increasing amounts of
purified VP22.C1 (0.06, 0.12, and 1.2 nmol) in PBS (20 µl) for 10 min
at room temperature. Complexes were then separated in nondenaturing
gels (6% NuSieve GTG-agarose) in 1 × TBE. Free ODN and VP22.C1
complexes were visualized by shortwave illumination of the gel.
Formation and Cellular Uptake of VP22.C1 Complexes--
After
preliminary evaluation of conditions, VP22.C1·F-ODN complexes
were routinely assembled and analyzed as follows. The oligonucleotide
(10 µM) in 25 µl of PBS was incubated for 10 min at
room temperature with VP22.C1 (20 µM) in 25 µl of PBS
(final volume 50 µl at a VP22/oligonucleotide molar ratio of 2)
theoretically resulting in overall neutral charge with respect to VP22
and the ODN. The sample was then diluted into 0.5 ml of Dulbecco's
modified minimal essential medium containing 10% calf serum and added
to monolayers of cells (usually seeded at 105 cells/ml) in
4-well coverglass incubation chambers (Nunc, Life Technologies). After
incubation at 37 °C live cells were either observed directly or
after washing with PBS. For any prolonged observation cultures were
maintained on a heated stage in the presence of 5% CO2 or
transferred to CO2 independent medium (Life Technologies,
Inc., Catalogue number 18045-054).
Cultures were usually examined by epifluorescence (HBO 50 mercury lamp)
and particles would redistribute within 10 s after initial
illumination. To capture initial particle localization without ODN
redistribution, cells were observed and focused by phase-contrast
microscopy and then localization captured immediately by confocal
microscopy using a Zeiss LSM 410 inverted confocal microscope set at
low laser power (1:100 or 1:300 attenuation, 15 mW 488 nm argon laser).
Broad range illumination was achieved using a fiber optic halogen lamp
(Schott KL2500LCD, Fiber Optics Ltd., UK) incorporating a 250-W halogen
reflector bulb. Light intensity reaching the cells was recorded using a
Spectra-Physics Lightmeter (Thermopile Detector, Model 407A).
Routinely, after particle uptake, cultures were removed from the
incubator, washed in PBS, and illuminated for ~3 min at a distance of
12 cm. Light intensity reaching the cells was ~170
mW/cm2. Alternatively cultures were detached from
monolayers with trypsin, washed in PBS, and then illuminated in
suspension prior to replating. Numerous control experiments
demonstrated that neither mode of illumination had any detectable
cytotoxic effect nor any effect on cell proliferation rates.
Complexes containing streptavidin-Alexa 594 (Molecular Probes), were
formed as follow. The F-ODN containing biotin at the 3' end was
suspended in 12.5 µl of PBS at a concentration of 20 µM, and incubated with streptavidin-Alexa 594 (400 nM) in 12.5 µl of PBS for 2 h at 25 °C with
occasional mixing. VP22.C1 (20 µM) in 25 µl of PBS was
added to the mixture to make a final volume of 50 µl and the mixture
incubated for 10 min. The complexes were then diluted in medium
containing 10% serum as above and added to the cells. Images were
annotated and presented using Adobe Photoshop software.
Biological Activity of an Antisense Oligonucleotide--
For
studies using antisense ODNs to inhibit cell proliferation we used a
20-mer ODN directed against the 3'-untranslated region of human
c-raf kinase previously reported by a number of workers to
inhibit proliferation in A549 cells (15, 16). An oligonucleotide with
mismatches at 7 bases served as a control. The A549 cells were plated
in 6-well plates at 4 × 105 cells/ml the day before
the experiment. The cells were then incubated overnight with
VP22.C1·F-ODN complexes. The following day the cells were
trypsinized, washed, and illuminated for 3 min with the fiber optic
cold light in suspension in cell culture medium containing 10% serum.
The cells (2 × 105) were then replated in 6-well
dishes. Two days later, the cells were trypsinized and counted.
Large Scale Production and Purification of VP22--
We previously
demonstrated that when crude soluble extracts containing VP22 were
applied to the medium of monolayer cells in tissue culture, the protein
could subsequently be detected in the cell nuclei (4). Based on these
observations we proposed that VP22 protein could be exploited for the
delivery of different types of cargoes into cells. Numerous attempts to
produce the intact protein in different bacterial systems were made but
resulted in low yields or cleaved products. However, we noted from
alignment analysis (ClustalX) of the VP22 homologues in other
Oligonucleotide Binding and Particle Formation by VP22.C1--
To
pursue the ability of VP22 to deliver heterologous cargoes into cells
we chose first to examine its potential with small oligonucleotides.
This class of molecules encompasses an increasingly wide range of
potentially useful agents including, e.g. antisense oligonucleotides, ribozymes, interfering RNAs, and chimeroplasts but
stability and delivery issues remain a major concern in their use. To
begin with, the interaction between VP22.C1 and a series of standard
20-mer oligonucleotides (ODNs) containing a phosphorothioate backbone
was assessed by gel retardation assays. The ODNs additionally contained
a fluorescein molecule at their 5' end to enable visualization in this
assay and also subsequently in cell import assays (see below). Similar
results were observed with a number of unrelated ODNs and typical
results are shown in Fig. 2a.
VP22.C1 was incubated in increasing amounts (0.06, 0.12, and 1.2 nmol)
with a fluorescein-conjugated 20-mer ODN (F-ODN, 0.6 nmol) in PBS and
the complexes resolved in nondenaturing agarose gels (Fig.
2a). With limiting amounts of protein, 2 complexes (marked
C1 and C2) with lower mobilities than the free F-ODN were observed
while at the highest dose tested all of the F-ODN was observed in a
complex which represented retention in the sample well (C3). We
observed no sequence specificity for this interaction and binding could
be competed by excess nonspecific DNA (data not shown), consistent with
formation of the VP22.C1·F-ODN complex being due to nonspecific
charge interactions.
While complexes were formed with limiting doses of VP22.C1, it was
possible that retention of the F-ODN in the well (C3) represented a
general aggregation at this ratio of protein to F-ODN (predicted to
result in overall neutrality of the two agents). Aliquots of the sample
were therefore analyzed by fluorescent microscopy. At this ratio of
VP22.C1 to F-ODN, we observed that the F-ODN was now present in
discrete spherical and intensely fluorescent particles (Fig.
2b), quite unlike the free F-ODN which was seen as a uniform
haze with no obvious particulate material (data not shown). The
particles were also examined by electron microscopy with representative
images of the EM grid and different magnifications shown in Fig.
2c. Consistent with the results from fluorescent microscopy,
the particles were observed to be uniform regular spheres, rather than
nonspecific aggregates. While there was some variation in size, the
majority of particles ( Cellular Uptake of VP22.C1·F-ODN Complexes and Intracellular
Redistribution--
The presence of the fluorescein molecule on the
ODN facilitated analysis by microscopy. We first determined that the
complexes were stable in serum and tissue culture medium (data not
shown). Thereafter all the analysis of cellular uptake and distribution were performed in living cells, and all images of the particles and
F-ODN reported throughout this work were from live cells.
Complexes were formed in PBS by mixing VP22.C1 and F-ODN at a 2:1 molar
ratio and the samples then added to tissue culture medium containing
10% calf serum. The medium containing the complexes was then added to
monolayers of different cell types (e.g. COS, Vero, or HeLa)
grown on chambered coverslips and the cells examined at various times
using a Zeiss Axiovert inverted confocal microscope. The final nominal
concentrations in the medium of the protein and ODN were 1 µM and 0.5 µM, respectively, and for the
sake of comparison, a concentration of 0.5 µM was used
for the free uncomplexed F-ODN.
The VP22.C1·F-ODN complexes could be readily observed in the medium
and after overnight incubation these complexes could also be observed
within cells. Typical representative images in COS and Hela cells are
shown in Fig. 3, b and
e, respectively. The intense fluorescent particles were
located exclusively within the cytoplasm, numbered ~2-10 per cell on
average and were of the same size distribution as the particles
observed before application to the cells. The particles were observed
in all cells in a population and confocal microscopy demonstrated that
they were inside cells, confirmed by results in the following sections
(see below). By comparison free F-ODN was observed in a faint speckled
cytoplasmic pattern (Fig. 3a), probably representing low
levels of endocytic uptake of free ODNs as described previously (17).
Thus the VP22.C1 particles appeared to be taken up by cells and located
within the cytoplasm, but the F-ODN was retained within the complexes with little being detected in a free diffuse pattern within the cytoplasm or nucleus.
However, upon closer inspection we made some unusual observations on
the F-ODN distribution from the complexes. In certain fields a small
number of cells exhibited a diffuse distribution of the F-ODN
(e.g. Fig. 3b, arrowheads). Moreover during
systematic analysis scanning through numerous fields in the cultures,
we frequently encountered complete fields in which the fluorescence pattern was distributed throughout every cell, with pronounced accumulation in the nuclei. It became clear that such fields were ones
which had been previously visualized and in which, on initial examination, discrete cytoplasmic particles were observed. Upon re-examination, diffuse cytoplasmic and nuclear fluorescence was observed. A typical example of this is shown in Fig. 3, b
and d, which show the same field, initially upon examination
(panel b) and 2 min later (panel d). For
reference, intact arrows indicate the same cells in the two
panels. Thus initially the F-ODN was present in cytoplasmic particles
as described above, while in the same cells 2 min later pronounced
diffuse distribution together with nuclear accumulation could now be
observed. This activity of uptake, cytoplasmic stability, and release
was also observed in HeLa cells (Fig. 3, e and f)
and in a number of other cell types (data not shown). Parallel
examination of fields containing the control F-ODN alone, showed no
such alteration or diffuse distribution and this was never observed at
any time (Fig. 3, a and c, and data not shown).
To account for these results we proposed that the process of
epifluorescence illumination during visualization was itself promoting
release of the F-ODN, from the discrete localization in VP22.C1·F-ODN
particles into the cytoplasm and nucleus. To follow the progress of
this process, particles were applied as before. To capture initial
particle localization without ODN redistribution (Fig. 3g,
0 s), cells were observed and focused by phase-contrast microscopy
and then distribution recorded immediately by confocal microscopy using
a very low laser power (1:100 or 1:300 attenuation). The field was then
subjected to epifluorescence illumination once for ~10 s, and the
image of the same field recorded again by confocal microscopy (Fig.
3g, 10 s) and thereafter at various intervals up to
130 s (Fig. 3g). The results illustrate the dynamic
nature of fluorescence distribution, and redistribution in living
cells. Thus as indicated above, immediately upon examination (Fig.
3g, 0 s), discrete cytoplasmic particles were observed,
with little or no diffuse fluorescence within the cells. Within seconds
after the first observation the fluorescence could now be observed in a
broad diffuse cytoplasmic location, frequently showing a gradient of
intensity from the particles (Fig. 3g, 10 s).
Thereafter the pattern became more evenly distributed throughout the
cell until ~2 min after initial observation, when the F-ODN could be
observed accumulating in the nucleus, although with nucleolar sparing
(Fig. 3g, 130 s). We were also able to record the
process of release dynamically by time lapse microscopy and a movie
(Fig. 4) recording release can be located
for viewing at http://www.jbc.org. We conclude that a short
burst of light (in this case supplied through the microscope objective,
see also below) was sufficient to somehow induce release of the F-ODN
from the otherwise stable particles into the body of the cell,
whereupon it accumulated in the nucleus. We confirmed that neither the
cellular uptake of the VP22.C1·F-ODN particles, nor the release of
the F-ODN upon illumination was toxic to the cells (Fig.
5 and data not shown). In fact, after uptake and light induced redistribution of F-ODN, nuclear accumulation of the F-ODN could be observed for several days thereafter. An example
of nuclear accumulation of the F-ODN 3 days after application and
~2 days after redistribution can be seen in Fig.
6c.
Comparative Aspects of VP221.C1·F-ODN Complex Uptake and
Redistribution--
In this section we report results of a series of
experiments examining different aspects of the uptake of the
VP22.C1·F-ODN complexes and of release of the F-ODN (Figs. 5-7 and
data not shown). As indicated above, particle uptake and F-ODN release
was observed in a variety of cell types, including monkey kidney cells
(COS and Vero) and human epithelial cells (HeLa, Fig. 3, e
and f). Additional cell types, supporting uptake and release
include primary human cells (MRC-5), a macrophage cell line (RAW), and
primary dendritic cells (data not shown).
In terms of the requirements for light activation of F-ODN release,
cells in chambered coverslips were examined by standard epifluorescence
microscopy. Illumination was routinely performed using a HBO 50W lamp
and a fluorescein filter set, and an exposure of 5-10 s was sufficient
to induce F-ODN release from complexes within seconds thereafter. The
conditions for release had no detrimental effect on cell growth and are
standard conditions we frequently employ, e.g. for time
lapse microscopy of living cells containing green fluorescent protein
(18). Furthermore, broad range illumination on the bench was achieved
using a conventional fiber optic halogen lamp (Schott KL2500 LCD). In
this case, cells were grown in 96-, 24-, or 6-well tissue culture
trays, particles applied in medium and the cultures then removed from
the incubator and illuminated for ~3 min. Alternatively cultures were
trypsinized and then illuminated in suspension prior to replating as
indicated under "Experimental Procedures." One concern on whether
the light treatment had any inherent cytotoxic effect was investigated
extensively. Fig. 5 shows results recording cell proliferation over 7 days after a 10-min illumination with the fiber optic lamp (in this
case in A549 human carcinoma cells but with identical results in other cell types). No cytoxicity nor any effect on cell growth was observed in this or a number of additional control experiments (Fig. 5 and data
not shown).
To examine the time course of uptake, complexes were applied to cells
for different periods, after which the cells were washed extensively
and images recorded immediately for particle uptake, or after
illumination for 10 s to record redistribution of the F-ODN 2 min
later (Fig. 6). Comparatively little uptake was observed within 1 h of application, and redistribution could be observed only
infrequently (Fig. 6a, 1 h). Between 2 and 4 h,
significantly increased numbers of particles were observed within the
cytoplasm and correspondingly increased release was observed, although
this was still suboptimal. By 6 h uptake was pronounced and
redistribution could now be observed in virtually all cells in the
field, although the efficiency of release continued to increase. While
we currently do not know the mechanism of uptake of the VP22.C1
particles, studies in live cells with labeled probes for endosomal
uptake or lysosomal fate failed to show significant co-localization or partitioning of the VP22 complexes to these compartments (data not
shown). However, uptake was sensitive to temperature. To examine this,
complexes were added to cells for 2 h at 37 or 4 °C, and localization then recorded immediately or after illumination (Fig. 6b). At 4 °C uptake was abolished, no intracellular
redistribution was observed, and the particles were found decorating
the surface of cells (Fig. 6b, 2 h 4 °C,
arrows). In contrast, at 37 °C the particles were found
within cells (Fig. 6b,
The efficiency of oligonucleotide delivery into cells following uptake
of the VP22.C1 particles and release was compared with Lipofectin-mediated delivery (Fig. 6c). To cater for the
requirement for efficient Lipofectin-mediated delivery, the
VP22.C1·F-ODN complexes and the Lipofectin·F-ODN complexes were
added to cells for 4 h in serum-free medium. The cells were then
incubated for a further 24 h in normal medium and then either
imaged directly for Lipofectin complexes or after illumination for
VP22.C1 complexes. A much brighter and uniform staining with nuclear
accumulation was observed for the F-ODN delivered by the VP22.C1
complexes than in the cells treated with Lipofectin·F-ODN complexes
(Fig. 6c, 24 h). Moreover after a further 48-h
incubation, cells treated with Lipofectin·F-ODN complexes exhibited
extremely weak staining. In contrast, in cells receiving the VP22.C1
complexes abundant redistributed F-ODN could still be observed,
virtually exclusively in the nuclei of cells (Fig. 6c,
48 h). The VP22.C1 particles remained within cells and additional
fields of cells could be illuminated to trigger oligonucleotide
redistribution several days after incubation with the VP22 complexes
(data not shown).
We also examined the fate of the protein, as opposed to the ODN, by
conjugating fluorescein to purified VP22.C1 prior to assembly of the
complexes. The conjugated protein (F-VP22.C1) was then further purified
and used to assemble the complexes with an unlabeled version of the ODN
used in earlier experiments. Complexes were then examined in living
cells. The conjugated protein retained the ability to assemble into
complexes that were taken up by cells (Fig.
7a). As when the complexes
were visualized by virtue of the tag on the ODN, we observed that the
protein was initially present exclusively in discrete cytoplasmic
particles (Fig. 7a), but upon illumination the protein could
be observed in a diffuse pattern in both the cytoplasm and nucleus
(Fig. 7b). While we have not quantitatively examined the
rate of distribution or nuclear entry by time lapse microscopy, a
qualitative evaluation indicated that the accumulation of the
fluorescent protein was slower and more was retained in the cytoplasm
than for the F-ODN. With the observation of uptake and release of both
protein and ODN, we have termed the complexes formed between VP22.C1
and short oligonucleotides as Vectosomes (derived from VP22 effected
complex transport bodies).
Examples of other classes of small oligonucleotides were also examined
for Vectosome formation, cellular uptake, and release. In each case we
altered the amount of protein used to maintain overall neutral charge
of the mixture. Short double-stranded DNAs, hammerhead ribozymes, and
RNA/DNA hybrids of the type reported to promote correction of mutations
within cells, were all recruited into particles which entered cells and
were induced to release the different ODNs after illumination. The
results for a 36-base hammerhead ribozyme, tagged at its 5' end with
fluorescein, are shown in Fig. 7, c and d. As for
each of the ODN cargoes, immediately upon examination the ribozyme was
present in discrete cytoplasmic particles (Fig. 7c), while
following illumination the ribozyme was released and distributed to
cytoplasm and nucleus, with pronounced nuclear accumulation (Fig.
7d).
Finally, we examined whether Vectosomes could be used for the delivery
of a third moiety that could be added to the complexes. To show in
principle that this could be achieved we tested whether a biotinylated
oligonucleotide could be used to assemble Vectosomes and if so, whether
streptavidin could then be recruited into the complexes and into cells.
In this analysis the same ODN tagged with fluorescein at its 5' end as
before was synthesized with biotin coupled to its 3' end. Streptavidin
was conjugated to a different fluorochrome (Alexa 594), allowing ready
discrimination between the two cargoes. We showed first that the
addition of biotin to the 3' end of the ODN had no effect on assembly
of the complexes, on cellular uptake, or on release of the ODN after illumination (data not shown but see also Fig. 7, e and
f). We then added streptavidin-Alexa 594, either to the
biotinylated F-ODN prior to incubation with VP22.C1, or after assembly
of the complexes. In both cases recruitment of the streptavidin-Alexa 594 into particles was observed. As before, the complexes were then
diluted in medium containing 10% serum and added to the cells. Import
was then examined initially or after light induced release, using
separate channels for simultaneous recording of the distribution of the
F-ODN or the streptavidin in the same fields. (Control experiments
demonstrated that there was no cross-channel leak between fluorescein
and Alexa 594, data not shown, see also Fig. 7.) Initially upon
examination the distribution of the F-ODN in the discrete particles in
the cytoplasm was mirrored by the distribution of the streptavidin
(Fig. 7, compare panels e and g). After
illumination, the F-ODN was observed to redistribute as before (Fig.
7f), and in the same cells it could be seen that the
streptavidin now also redistributed into a diffuse cytoplasmic pattern
(Fig. 7h). Strikingly it could also be seen that while the
F-ODN exhibited significant accumulation in the nucleus, the
streptavidin was observed virtually exclusively in the cytoplasm (see
arrowed cells). We interpret these results to indicate that the
streptavidin protein was able to be "piggy-backed" into cells by
virtue of the biotin on the ODN used to assemble the Vectosomes, and
that it could be released into the cytoplasm upon activation. It is
reasonable to propose that its lack of accumulation in the nucleus is
simply due to the fact that it lacks a nuclear localization signal and
is above the size limitation for passive diffusion into the nucleus.
While it is impossible to determine from this level of analysis whether the cytoplasmic streptavidin remains complexed to the biotinylated ODN,
the results demonstrate that in principle, Vectosomes may be designed
with different ligands on the ODN, or protein, to import additional cargoes.
Biological Activity of an Antisense Oligonucleotide--
The
efficacy of VP22 protein in delivering functional molecules was
assessed using an antisense oligonucleotide. We selected an antisense
ODN against c-raf, a kinase which is involved in the
mitogen-activated signaling pathway. Previous systematic evaluation identified an antisense ODN targeted to the 3'-untranslated region of
the c-raf RNA which inhibited proliferation of a number of cell types including the human carcinoma line A549 (15, 16). We
therefore used an anti-c-raf 20-mer phosphorothioate
corresponding to that previously identified (15). As with other
standard 20-mers the anti-c-raf ODN was incorporated into
Vectosome particles and release of the ODN was observed after
illumination (data not shown). We next tested the effects of these
Vectosomes on A549 cell proliferation, including in parallel Vectosomes
assembled with a control mismatched ODN (ODNm), and also free
anti-c-raf ODN as an additional control (Fig.
8). Each sample was tested in duplicate.
Complexes were assembled and introduced into monolayer cultures of A549
cells. One day later the cells were trypsinized, and while in
suspension one set was subjected to illumination to induce ODN release
from Vectosomes while another set was held in suspension without light release. For each test sample 2 × 105 cells were then
plated out and incubated for 2 days, after which time cell counts were
recorded. The results (Fig. 8) show that compared with the control
cells with no treatment (open bars), no effect on
proliferation was observed for any of the test samples in the absence
of illumination (compare 1d to 3 days, We previously reported upon the unusual trafficking properties of
the HSV protein VP22 including the observation that when soluble
cell-free extracts containing VP22 were applied to the medium of tissue
culture cells, the protein could subsequently be detected within cells,
accumulating in the nuclei (4). Based upon these observations we
proposed that VP22 protein itself could be exploited for the delivery
of different types of cargoes. In this work, we develop a system for
the expression and purification of the core conserved region of VP22
(VP22.C1, residues 159-301) and examine its ability to deliver small
nucleic acids into cells. The salient points of our observations are
discussed as follows. First using gel retardation assays we show that
VP22.C1 binds small nucleic acids, e.g. antisense
oligonucleotides or ribozymes. We believe this binding is likely to be
due to a nonspecific electrostatic interaction and we have not observed
any significant sequence specificity for binding or complex formation.
Second, at ratios of protein to ODN designed to yield a neutral
mixture, particles were formed which did not enter the native gels but
which were not simply nonspecific aggregates. The VP22.C1/ODN particles
were observed by phase-contrast microscopy and electron microscopy to
be single regular spheres with diameters ranging from 50 nm to 2 µm.
The majority of particles ( Third, the VP22.C1·ODN complexes, termed Vectosomes are stable in
serum, and are taken up by cells after which they remain in a stable
form in the cytoplasm, with little detectable disassembly or dispersal
over a period of days. The mechanism of uptake of the Vectosome
particles is currently unknown. Indeed, even with regard to uptake of
free ODNs, although there appears to be agreement that some form of
endocytosis is involved, a detailed understanding of the mechanism and,
e.g. whether specific receptors are required, is still
lacking. However, it would seem unlikely that uptake of Vectosomes is
via classical receptor-mediated (or receptor independent) endocytosis.
Incubation at low temperature (+4 °C) did inhibit uptake indicating
that an energy dependent process was involved but the particles are
considerably larger than endocytic vesicles and initial results
indicate that they did not co-localize with markers for endosomal entry
or lysosomal fate (data not shown). It is possible that uptake could be
mediated by some form of macropinocytosis, a process which is known to
operate constitutively in many transformed cell types and which
accommodates intracellular vesicles up to several microns in size (19).
Future work including whether, e.g. components of the
extracellular matrix such as glycosaminoglycans are involved, and
studies with selective inhibitors and cell lines defective in specific
pathways should help identify routes of entry. One striking feature of
the Vectosomes is the observation that they remain stable within cells,
yet are somehow primed for dynamic release of the ODN and protein cargo
upon light activation. It would seem reasonable to propose that some
component of the complexes responded in some way to light, inducing a
reorganization or disruption and release of both protein and ODN. One
possibility, since a fluorescent tag was used to track the fate of the
ODN, is that when concentrated in the assembled particles, light
excitation of the fluorochrome induces reactive species which may then
disrupt the particle and/or any membranous component in which it
resides. While we have observed release when the fluorescent tag was on the protein rather than the ODN, following the fate of the Vectosomes in the absence of any tag cannot be readily performed in living cells.
However, we have observed by phase-contrast microscopy that Vectosome
complexes assembled without a fluorescent tag on either the protein or
ODN, frequently undergo some distortion or disappear, after light
activation, although this effect appears with a slower time course.
Further work is in progress to examine the requirement or otherwise of
a tag on the ODN, and for example, the efficiency of release in
relation to wavelength and absorption maxima of the fluorochromes used.
Interestingly some form of photochemically induced internalization of
DNA complexes has been previously reported (20, 21). In that work the
authors reported that application of photosensitizers, such as aluminum
phthalocyanine could render the release of internalized macromolecules
sensitive to light, possibly by disrupting the endosomal/lysosomal
compartments, and they achieved significant increase in delivery of a
test cargo in surviving cells. The photosensitizers used in that work
normally exhibit some degree of cellular toxicity upon light
activation. The fluorescent tags we have used on the other hand are
thought to be relatively poor photosensitizers and we have observed no intrinsic cytotoxic effect, either of the complexes or of the light-induced release. We have further shown that proteins can be
piggy-backed onto these complexes, indicating also that
additional types of linkers may be used to incorporate the cargoes.
Such linkers may be manipulated by means other than light.
Current work also indicates that Vectosome assembly, delivery, and
release are all observed using second generation VP22 variants modified
to incorporate a proapoptotic peptide. Complexes assembled with this
modified VP22 result in regulated, light-dependent
apoptosis. Thus, while the precise mechanism of light activation
remains to be established, the ability to assemble stable,
macromolecular complexes between ODNs and a readily manipulated
protein, and to deliver and regulate release of the cargo offers the
prospect that these complexes may provide a very useful versatile
delivery agent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-herpesviruses (5, 6). We
suggested that this property of VP22 might be useful in the field of
gene therapy, to amplify delivery of candidate gene products by
expressing them as VP22 fusion products. The augmented delivery and
activity of functional proteins potentially useful in gene therapy
applications have now been demonstrated for p53 and thymidine kinase
(7, 8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (1 mM) and incubation continued for a further 3-5 h. The
cultures were then cooled on ice, the bacteria pelleted, and suspended
in lysis buffer (70 ml/1.6 liters culture) containing 50 mM
sodium phosphate, pH 8.0; 300 mM NaCl, 5 mM
imidazole, pH 8.0; 5 mM
-mercaptoethanol; 0.5 mM phenylmethylsulfonyl fluoride; 1 µg/ml leupeptin and
pepstatin. Lysosyme (1 mg/ml) was added and the samples incubated on
ice for 30 min followed by sonication (3 × 10 s on ice).
Nonidet P-40 (0.1%), DNase (5 µg/ml), and RNase (5 µg/ml) were
then added and incubation continued for a further 20 min. Viscosity was
reduced by drawing the sample through a 21-gauge needle 3 times before removal of insoluble material by centrifugation (14,000 rpm for 15 min
at 4 °C).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-herpesviruses that the C-terminal half of the protein was the only
region exhibiting a high degree of conservation (Fig.
1a). Moreover studies of deletion mutants
in transient transfection assays have shown that this C-terminal region
retained most of the activity of the intact protein.2 We therefore
developed a system for the bacterial expression of the C-terminal half
of the protein (residues 159-301) including a 6xHis tag at the C
terminus to facilitate purification. Expression from this construct
(pVP24, Fig. 1b) resulted in high yields (5-10 mg/liter) of
soluble protein (termed VP22.C1) which could be readily purified by a
one-step purification procedure using Ni-NTA affinity chromatography
(Fig. 1c). Generally throughout the course of this work, as
judged by total protein staining of polyacrylamide electrophoresis gel,
preparations of
98% purity were achieved. The identity of the
protein was also confirmed by N-terminal amino acid sequencing and mass
spectrometry of the purified protein indicated a mass of 18,415 ± 11, acceptably within experimental error from the predicted mass of
18,424. Thus we established a relatively straightforward scheme for the
production and purification of high yields of intact soluble
protein.
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Fig. 1.
Purification of VP22.C1. a,
schematic summary of the conservation of the C-terminal half of VP22.
Computer alignment of VP22 species as indicated (HSV-1,
herpes simplex virus type 1; VZV, varicella zoster virus;
BHV, bovine herpesvirus; EHV, equine herpesvirus;
MDV, Marek's disease virus) showed that while there is
relatively little sequence conservation within the N-terminal region,
the C-terminal region of ~100 amino acids exhibits significant
homology across the species. b, summary of the pET24 based
construct for expression of VP22. C1. c, VP22.C1
purification. VP22.C1 (indicated by the arrow) was purified
by Ni-NTA chromatography as described under "Experimental
Procedures" and the resulting fractions were analyzed by
SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.
Lanes: 1, unbound supernatant containing VP22.C1 from
DEAE-Sepharose clarification; 2, flow-through from Ni-NTA
chromatography; 3, early wash from Ni-NTA chromatography;
4-20, bound material eluted in a gradient of imidazole from
0 to 500 mM. Fractions pooled from this particular
purification for further analysis are indicated by the
bracket.
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Fig. 2.
VP22.C1 binding to F-ODN and particle
formation. a, electrophoretic mobility shift assay
showing complex formation between VP22.C1 and F-ODNs. A 20-mer
phosphorothioate F-ODN (0.6 nmol) was incubated alone (lane
1) or in the presence of increasing amounts (0.06, 0.12, and 1.2 nmol) of VP22.C1 (lanes 2-4) and the complexes separated in
a 6% nondenaturing agarose gel. Complexes (C1, C2, and
C3) were visualized by shortwave illumination on a
standard photoilluminator box. b, the VP22.C1·F-ODN
complexes formed at a molar ratio of protein/ODN of 2:1 (as in
lane 4) were applied to a microscope slide in PBS 10 min
after preparation and examined by fluorescence microscopy (top
panel). c, similar mixtures were applied to a
copper-coated EM grid, negatively stained, and examined by electron
microscopy. Left panel shows a low magnification
conventional light microscope image of the grid itself with the
particles just visible; right panels, high magnification
electron micrographs showing the regular spherical nature of the
assembled particles.
80%) were within a size range from 0.3 to 1 µm as judged by light scattering analysis (data not shown). Although
several artificial cationic polymers, such as polylysine have been
shown to form aggregates with negatively charged nucleic acids, this is
the first example of which we are aware of an encoded polypeptide such
as VP22.C1 promoting the formation of regular discrete particles of the
type observed here. The formation of regular spherical VP22.C1.F-ODN
particles prompted us to examine their uptake and delivery into cells.
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Fig. 3.
Cellular uptake of particles. Particles
assembled as described in the text were added to cells and incubated
overnight at 37 °C in medium containing 10% serum. a and
c, free F-ODN in COS cells; b and d,
VP22.C1·F-ODN complexes in COS cells; e and f,
VP22.C1·F-ODN complexes in HeLa cells. Panels a, b, and
e represent initial localization, while panels c,
d, and f show localization 2 min after 10-s
epifluorescence illumination. Arrowheads in panel
b indicate those cells in which the ODN was observed in a diffuse
pattern immediately upon examination. Intact arrows indicate
those cells with the typical pattern of localization before
(b and e) and after (d and
f) illumination (g). Time course of light-induced
redistribution of internalized ODNs. HeLa cells were incubated
overnight in medium containing 10% serum with VP22.C1·F-ODN
complexes. The cells were then focused by phase-contrast microscopy and
localization captured immediately (0 s). Oligonucleotide redistribution
was initiated by a 10-s illumination with a Hg lamp and live images
were captured at times indicated by confocal microscopy.
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Fig. 4.
Animation showing light-triggered
redistribution of F-ODN delivered by VP22. COS cells were
incubated overnight at 37 °C in medium containing 10% serum with
VP22.C1·F-ODN particles. The cells were washed in fresh medium and
illuminated 10 s with the Hg lamp. A time series of 30 sequences,
one every 10 s was recorded by confocal microscopy with phase
contrast recorded in the red channel and fluorescence recorded in the
green channel. The images were compiled into a movie, located at
http://www.jbc.org.
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Fig. 5.
Cell growth is not affected by
illumination. A549 cells were plated at 104 cells per
well in 24-well plates on day 0. The following day (day 1) the cells
were illuminated for 10 min with a fiber optic halogen lamp (Schott
KL2500LCD). Following illumination the cells were incubated for the
times indicated, then trypsinized and counted.
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Fig. 6.
Characterization of VP22.C1·F-ODN particle
uptake. a, time course of uptake of VP22.C1·F-ODN
particles into cells. Particles were added to cells (T24 bladder
carcinoma cells) as usual and the cells incubated at 37 °C in medium
containing 10% serum. At various times (1, 2, 4, or 6 h) after
particle addition, the cells were washed extensively and uptake
recorded immediately ( ), or 2 min after a 10-s illumination with the
Hg lamp (+), to record ODN redistribution. Particle uptake and ODN
redistribution could be observed between 2 and 4 h after addition,
but continued to increase. b, temperature dependence of
particle uptake into cells. T24 cells were incubated with VP22.C1/F-ODN
particles for 2 h at 37 or 4 °C in medium containing 10%
serum. The cells were washed and uptake recorded immediately (
), or 2 min after a 10-s illumination with the Hg lamp (+), to record ODN
redistribution. At 4 °C complexes were seen to decorate the outside
of cells (indicated by arrows), and no ODN redistribution
was observed. c, comparison between VP22 and Lipofectin
delivery of fluorescent oligonucleotides. HeLa cells were incubated for
4 h in serum-free medium with VP22.C1·F-ODN complexes or
Lipofectin/F-ODN as indicated. The final amount of ODN in the medium
(500 nM) was the same in both cases. The medium was then
replaced with fresh medium containing 10% serum and the cells were
further incubated overnight. One day later, cells incubated with VP22
complexes were subject to a 10-s illumination and the distribution of
the ODN examined 2 min later, while cells incubated with Lipofectin
complexes were examined directly (24 h). Two days after illumination (3 days after original incubation with the complexes) distribution of the
ODN was compared again, without any further illumination (72 h). ODN
distribution from the VP22 complexes was still readily observed.
) and redistribution by light had
begun to be observed (Fig. 6b, +).
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Fig. 7.
Light-induced release of fluorescein-labeled
VP22.C1 from internalized F-VP22.C1·ODN complexes. COS cells
were incubated overnight in medium containing 10% serum with
F-VP22.C1·ODN complexes assembled with the untagged version of the
ODN previously used. The cells were focused by phase-contrast
microscopy and localization recorded immediately (a), then
subjected to illumination (10 s, Hg lamp), returned to the incubator
and examined again 20 min later (b). The same field is
recorded in a and b, but is offset slightly in
the second recording. c and d, delivery and
release of a ribozyme. COS cells were incubated overnight in medium
with VP22.C1·F-ribozyme complexes. The following day the cells were
focused by phase-contrast microscopy and localization recorded
immediately (c) or 2 min after illumination (d).
The F-ribozyme can be seen to be in complexes similar to those seen
with the F-ODN and to redistribute after light activation.
e-h, piggy backing of streptavidin into cells. COS cells
were incubated overnight in medium with
VP22.C1·F-ODN-biotin·streptavidin-Alexa 594 complexes, assembled
with a biotinylated version of the F-ODN previously used. The following
day the cells were washed and the distribution of the ODN or
streptavidin as indicated recorded prior to illumination (e
and g) or 2 min after illumination (f and
h). The streptavidin can be seen to be in complexes and
co-localizing with the ODN prior to release, and to redistribute after
light activation, but accumulating in the cytoplasm. Examples of cells
to indicate the different distribution of the F-ODN and streptavidin
after release are indicated with arrows.
light). Illumination itself had no effect on cell proliferation (control, open bar, + light) nor did illumination in the presence of
the free anti-c-raf ODN (hashed bar). However, a
significant reduction in proliferation after illumination was observed
for the Vectosome complexes assembled with the anti-c-raf,
while only a minor effect was observed for the Vectosomes assembled
with the control ODN. The effect with the
Vectosome·anti-c-raf complexes represents a pronounced
effect on cell proliferation, consistent with previous reports, and
indicates that release of the ODN from the complexes to the cytosol
(and/or nucleus) was required for inhibition. The absence of any effect
of illumination by itself, together with the lack of any effect of the
Vectosome complexes incorporating the anti-c-raf ODN in the
absence of light, indicate that Vectosome particles may offer a
regulatable system for the delivery and release of the cargo ODNs or
proteins.
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Fig. 8.
Growth inhibition of cells by
anti-c-raf ODN requires light-induced release.
A549 cells were incubated overnight at 37 °C in medium containing
10% serum with VP22.C1 particles prepared with an
anti-c-raf F-ODN or a mismatched F-ODNm. The
following day (1d) the cells were trypsinized, illuminated
for 3 min in suspension, and the cells (2 × 105)
replated for further incubation in fresh medium. Approximately 2 days
later (3 days) the cells were counted. Results are expressed as the
mean of two independent experiments each performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80%) were within a narrower size range of
300 nm to 1 µm. We do not yet understand the basis for the assembly
of these regular particles but some speculation is warranted.
Preliminary results from ongoing work indicate that the native form of
VP22.C1 is a dimer with a native size of ~36 kDa. Thus it could be
that each molecule of the dimer could bind the ODN and that there could
be two or more binding sites per ODN, resulting in a cross-linking that
may underpin assembly of the complex. Also it is possible, since VP22
is assembled as a major component of HSV virions and L-particles
(1-3), that properties important for the complex assembly we observe
are somehow involved in virion assembly. Further analysis will be
directed toward understanding requirements for complex assembly itself.
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ACKNOWLEDGEMENTS |
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We thank Len Packman and Dan Hill for mass spectrometry and electron microscopy services, Lisa Kueltzo for light scattering data, and Jackie Ferguson for plasmid construction.
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FOOTNOTES |
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* 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 a supplementary movie.
To whom correspondence should be addressed. Tel.: 44-1883-715028;
Fax: 44-1883-714375; E-mail: P.OHare@mcri.ac.uk.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M010294200
2 J. Webb, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: ODN, oligonucleotides; HPLC, high performance liquid chromatograpy; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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