Particle Formation by a Conserved Domain of the Herpes Simplex Virus Protein VP22 Facilitating Protein and Nucleic Acid Delivery*,

Nadia Normand, Hans van Leeuwen, and Peter O'HareDagger

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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 beta -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).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

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.


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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.

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 (>= 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.

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.


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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.

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.


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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.

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, -) and redistribution by light had begun to be observed (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).


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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.

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- 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

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 (>= 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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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

The on-line version of this article (available at http://www.jbc.org) contains a supplementary movie.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: ODN, oligonucleotides; HPLC, high performance liquid chromatograpy; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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