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doi:10.1006/mthe.2001.0472, available online at http://www.idealibrary.com on IDEAL

Adenovirus Hexon Protein Enhances Nuclear Delivery and Increases Transgene Expression ofPolyethylenimine/Plasmid DNA Vectors

Robert C. Carlisle, Thierry Bettinger, Manfred Ogris, Sarah Hale, Vivien Mautner, and Leonard W. Seymour*

CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, UK

*To whom correspondence and reprint requests should be addressed. Fax: +44-121-414-3263. E-mail: [email protected].

Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. Toincrease transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexonprotein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI andPEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expressionin HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake.Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showedreporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexonto promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclearlocalization sequences (NLS) by measuring transgene expression following intracytoplasmicmicroinjection of hexon–PEI/DNA complexes and NLS–albumin–PEI/DNA complexes in rat-1fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon–PEI/DNA> NLS–albumin–PEI/DNA > PEI/DNA > DNA alone > albumin–PEI/DNA. The activities of bothNLS–albumin–PEI and hexon–PEI were abolished by co-injection of wheat germ agglutinin, sug-gesting that both act by means of the nuclear pore complex (NPC); in contrast, excess freeNLS–albumin abolished transgene expression with NLS–albumin-PEI/DNA, but only partially inhib-ited hexon–PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependenton DNA concentration for all materials, although hexon conjugates showed much better activ-ity than NLS–albumin at low DNA doses (500–1000 plasmids/cell). Our data are consistent withhexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanismsthat are only partially dependent on the classical NLS import pathway. The hexon-mediatedmechanism of nuclear import enables substantially better transgene expression, particularly whenDNA concentrations in the cytoplasm are limiting.

Key Words: nuclear targeting, hexon, polyethylenimine, microinjection, transfection

ARTICLE

INTRODUCTION

The usefulness of many nonviral systems for gene deliv-ery is restricted by inefficient entry of plasmid DNA intothe nucleus of target cells, preventing access to cellulartranscription machinery. The nuclear membrane presentsa major barrier to entry, particularly in nondividing cells,and strategies are being developed to enable translocationof plasmid DNA through the nuclear pore complex (NPC).Consensus nuclear localization sequences (NLS) permitrapid delivery of nucleoproteins from the cytoplasm intothe nucleus [1], and attempts have been made to use NLSto promote nuclear entry of DNA in target cells. Successso far has been mixed, however, with some studies showing successful NLS-mediated nuclear delivery of

MOLECULAR THERAPY Vol. 4, No. 5, November 2001Copyright © The American Society of Gene Therapy1525-0016/01 $35.00

oligonucleotides or linearized plasmids [2,3], but less effi-cient transfer of intact plasmids [4].

The efficiency of nuclear delivery of plasmid DNA maybe enhanced by using mechanisms evolved by viruses. Forexample, adenovirus enters cells and delivers its DNA intothe nucleus using a highly efficient pathway. Binding of ade-novirus fiber protein to the coxsackie and adenovirus recep-tor (CAR) and interaction of the penton base with cell sur-face integrins [5,6] enables internalization into endosomes.Activation of the virus protease destabilizes the virus capsidand modified virus particles are released into the cytoplasm[7,8]. The residual virus capsid, containing the DNA core,then translocates efficiently to the NPC by shuttling alongmicrotubules [9,10]. The capsid has been shown to associate

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ARTICLE doi:10.1006/mthe.2001.0472, available online at http://www.idealibrary.com on IDEAL

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FIG 1. Intracytoplasmic microinjection in rat-1 fibroblasts of hexon–FITC (A), NLS–albumin–FITC(B), and albumin–FITC (C) as described in the text. Cells were photographed at the earliest achiev-able time after injection (5 min) and again after 30 and 60 min.

with tubulin, vimentin, and heat shock proteins [11]. Thedemonstration that the major capsid protein hexon associ-ates with heat shock protein-70 [12] has strengthened sug-gestions that hexon may have a key role in virus transloca-tion [9,13,14]. Upon reaching the nucleus, the capsid docksat the NPC [15], most of the hexon and other capsid pro-teins are shed into the cytoplasm, and the adenovirus core,consisting of DNA and associated proteins, is thought toenter directly into the nucleus [16].

The mechanism whereby hexon modulates transloca-
tion of the infecting virus capsidto the NPC seems to be differentfrom conventional NLS-medi-ated nuclear import pathways.NLS-bearing proteins bind cyto-plasmic importin proteins � and�, the importin–NLS–proteincomplex then binds compo-nents of the microtubular net-work, translocates to the NPC,and enables transfer of the NLSprotein through the NPC intothe nucleus [1]. In contrast,however, the amino acidsequence of hexon contains nodefinitive NLS, and the majority
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of hexon does not actually enter the nucleusduring the process of initial virus infection.Hence, the nuclear-homing activity of hexonseems different during initial infection andfollowing its de novo synthesis in the cyto-plasm, when it enters the nucleus efficientlyfor assembly of new virus capsids [17]. Nuclearimport of isolated hexon was demonstratedin a simple reconstituted cell system and anti-bodies raised against hsc70 inhibited thenuclear transfer of adenovirus DNA during theinitial stages of infection, but did not affectnuclear transfer of newly synthesized hexon atthe late stages [14].

Here we have examined whether hexonitself has nuclear import properties that can beused to improve the efficiency of nonviral sys-tems for gene delivery. To do this we have pre-pared covalent conjugates of hexon with thecationic polymer polyethylenimine (PEI) andexamined the biological activity of complexesformed by the association of conjugates withplasmid DNA encoding reporter genes.Transgene expression has been measured intransfection assays and following microinjec-tion of complexes into the cytoplasm and thenucleus. Results are compared with thoseobtained using simple PEI/DNA complexes andcomplexes formed using NLS-modified albu-min-PEI conjugates in order to compare the

activity of hexon with that of an NLS-bearing protein.

RESULTS

Nuclear Homing Activity of Hexon–FITC and NLS–albumin–FITCWe assessed the ability of hexon, albumin, and NLS–albu-min to translocate from the cytoplasm to the nucleus of rat-1 fibroblasts by injecting FITC-labeled proteins into the cyto-plasm. Cells were then examined by fluorescence microscopy

TABLE 1: Formation and characterization of protein–PEI conjugates

Reagent mixture Molar ratio of product Mass ratio of product

protein:PEI protein:PEI

PEI-s-hex 2.4 nmoles hexon-SVSB + 0.8 0.33

4.5 nmoles PEI-SPDP

PEI-ss-hex 2.5 nmoles hexon-SPDP + 1.0 0.4

4.5 nmoles PEI-SPDP

PEI-s-alb 21.6 nmoles albumin + 4.5 0.39

6.1 nmoles PEI-SPDP

PEI-ss-alb 24 nmoles albumin + 3.2 0.28

6.1 nmoles PEI-SPDP

MOLECULAR THERAPY Vol. 4, No. 5, November 2001Copyright © The American Society of Gene Therapy

ARTICLEdoi:10.1006/mthe.2001.0472, available online at http://www.idealibrary.com on IDEAL

at 5, 30, and 60 minutes following injection. Bothhexon–FITC and NLS–albumin–FITC showed rapid localiza-tion of fluorescence within the nucleus (Fig. 1). Transfer wasso rapid that nuclear uptake was already discernible by thetime of the earliest achievable examination (5 min), althoughthe intensity of nuclear fluorescence was further increasedin both cases by 30 minutes. In contrast, albumin–FITCshowed no detectable nuclear accumulation, even 60 min-utes following microinjection. Nuclear localization usingFITC-labeled proteins was also examined using digitonin-permeabilized HepG2 cells [18]. Again, NLS–albumin–FITCand hexon–FITC both showed substantial nuclear entry inpermeabilized cells, whereas albumin–FITC remainedentirely extranuclear (data not shown). Similar data havepreviously been reported for hexon–FITC [14].

Purification and Characterization of Protein–PEI ConjugatesWe set out to evaluate the possibility that adenovirushexon protein may improve the cytoplasm-to-nucleustransfer of polyelectrolyte complexes formed with plas-mid DNA, thereby increasing transgene expression. To

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enable covalent incorporation of hexon into the com-plexes, we synthesized conjugates between hexon and PEI,based on either stable thioether bonds or reducible disul-fide bonds. Conjugates were also formed between PEI andbovine serum albumin, as a negative control. We achievedpurification of PEI–hexon and PEI–albumin conjugates(based on both types of linkage) using MonoS columnchromatography. Similar elution profiles were produced ineach case, and hexon–thioether–PEI is shown as an exam-ple (Fig. 2). Elution was monitored by absorption at 280nm and 240 nm to enable simultaneous detection of pro-tein and PEI. The early peaks (fractions 2 and 4) were bufferpeaks resulting from the use of two loading injections.After application of the salt gradient, both spectrophoto-metric measurements showed two elution peaks (Fig. 2A),indicating the presence of protein in both peaks; this wasverified using bicinchoninic acid (BCA) analysis (Fig. 2C).Immunoblot analysis confirmed that hexon was presentin both peaks (Fig. 2B). In contrast, trinitrobenzenesul-fonic acid (TNBS) analysis, which determines free aminogroups, showed a signal in the second peak alone, demon-strating that PEI is only contained in this peak (Fig. 2D).These observations suggest that the earlier elution peakrelates to free hexon protein, whereas the later peak con-tains the PEI–hexon conjugate together with any free PEI.Parallel chromatography studies using free hexon and freePEI verified this interpretation. We obtained similar resultsfor the three other conjugates and in each case the secondelution peak was collected for further study. TNBS andBCA analysis were used to characterize the conjugates(Table 1).

Interaction of Protein–PEI Conjugates with DNAWe examined the ability of the protein–PEI conjugates tobind and condense plasmid DNA using an electrophore-sis band shift assay (Fig. 3A). Electrophoretic mobility ofDNA was abolished after binding to PEI (N/P ratio 7.5), andthe conjugates between PEI and hexon or albumin allmediated the same effect, suggesting the formation of par-ticulate structures under these conditions that cannotenter the gel. The interaction between DNA and the con-jugates was also assessed by monitoring the loss ofDNA/ethidium bromide fluorescence following sequentialaddition of PEI conjugates to free DNA, a measure of DNAcondensation. All conjugates showed a slightly decreasedability to condense DNA compared with free PEI, althoughcomplete condensation occurred in each case by N/P ratios

FIG. 2. Purification of thioether-based hexon–PEI conjugate. The conjugate waspurified by HPLC using a MonoS cation exchange column with elution usingan increasing gradient of NaCl, as described in the text. All four conjugatesgave similar results, and this conjugate is used as an example. (A) Spectrophotometric elution profile measured at wavelengths of 240 and280 nm, showing also the salt gradient (dashed line). (B) Immunoblot usingan anti-hexon antibody to identify the presence of hexon in fractions col-lected. (C) Determination of protein in fractions collected, using BCA assay.(D) Determination of primary amino groups using TNBS analysis.

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FIG. 3. Physicochemical characterization of the protein–PEI conjugates. (A) Agarose gel electrophoresis of protein–PEI/DNA complexes formed at N:P ratios of 7.5:1in 20 mM HEPES, pH 7.4, to achieve a final DNA concentration of 20 �g/ml. 1, DNA; 2, PEI/DNA; 3, PEI-s-hexon/DNA; 4, PEI-ss-hexon/DNA; 5, PEI-s-albumin/DNA;6, PEI-ss-albumin/DNA. (B) Determination of DNA condensation by measuring ethidium bromide fluorescence (�ex 510 nm and �em 590 nm) following serial addi-tion of aliquots of protein–PEI conjugates. Open circle, PEI; open diamond, PEI-s-hexon; filled diamond, PEI-ss-hexon; open triangle, PEI-s-albumin; filled triangle,PEI-ss-albumin; open square, PEI-albumin-NLS. (C) Transmission electron microscopy. TEM images of complexes formed between plasmid DNA and PEI (1), PEI-s-hexon (2), PEI-ss-hexon (3), PEI-s-albumin (4), or PEI-ss-albumin (5). Complexes formed at N:P ratio 7.5:1, in 20 mM HEPES pH 7.4, to achieve a final DNA con-centration of 20 �g/ml. (D) Photon correlation spectroscopy (PCS). Complexes were formed at N:P ratio 7.5:1, in 20 mM HEPES, pH 7.4, to achieve a final DNAconcentration of 20 �g/ml. We conducted three lots of 10 sub runs, with analysis using Contin software and monomodal distribution.

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of 5 and greater (Fig. 3B). These results were in contrast toresults we obtained using a lower molecular weight PEI (25kDa). This smaller PEI was also able to condense DNAeffectively when unmodified, but its protein conjugateswere unable to condense DNA (data not shown). This sug-gests that covalent modification of PEI with albumin orhexon decreases its ability to form complexes with DNA,although self-assembly of complexes still proceeds pro-vided the individual conjugate molecules contain suffi-cient positive charges.

Analysis by electron microscopy and photon correla-tion spectroscopy (PCS) (Figs. 3C and 3D) showed thatcomplexes formed by the disulfide-linked albumin withDNA (N:P ratio 7.5) were relatively heterogeneous, with anaverage diameter of 433 nm. In contrast, the other conju-gates all formed smaller and more monodisperse com-plexes at this N:P ratio (Figs. 3C and 3D), with averagediameters in the range of 120 to 210 nm. Studies we car-ried out at the N:P ratio of 5.0 produced larger and moreheterogeneous particles, particularly for the albumin-con-taining conjugates, with a tendency to aggregate (data notshown). Biological evaluation was carried out using

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complexes formed at the N:P ratio of 7.5 because this com-bined efficient DNA condensation with the most homog-enous and discrete nanoparticulate structure.

Uptake of Hexon-Containing PEI/DNA Complexesinto HepG2 CellsTo determine whether the presence of hexon or albuminwould promote binding or entry of complexes into cells,we measured cell accumulation using DNA trace-labeled bynick translation with 32P (Fig. 4A). At 4�C all the com-plexes showed similar amounts of cell association, 5–10times greater than free DNA, suggesting a component ofelectrostatic binding to the cell membrane. At 37�C allcomplexes again showed similar levels of cell association,approximately twice that measured at 4�C, still 5–10 timesgreater than free DNA. At both temperatures the amountof cell-association for simple PEI/DNA complexes was thesame as for the protein-containing complexes, confirmingno protein-mediated cell uptake of the complexes. Thegreater uptake of all substrates at 37�C than at 4�C suggeststhe involvement of an energy-dependent uptake mecha-nism at 37�C, probably endocytosis.



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FIG. 4. Influence of hexon on the uptake of complexes and transfection in HepG2 cells. (A) Uptake of protein–PEI/32P-DNA complexes into HepG2 cells. DNAwas radiolabeled and complexes formed as described in methods. At 48 h after seeding (24-well plates, 20,000 cells/well), HepG2 cells were incubated for 5 hwith complexes trace labeled with 32P-DNA, at either 4�C or 37�C, in serum-free medium. Incubation was conducted at 37�C (open square) or 4�C (filled square).Standard deviation shown, n = 4. (B) Transfection activity of protein–PEI/DNA complexes in HepG2 cells. Transfection studies were conducted as described inmethods using HepG2 cells plated at a density of 8000 cells/well in 96-well plates and grown for 48 h and then exposed for 5 h to DNA complexes (30 �l, plas-mid concentration 20 �g/ml, N:P ratio 7.5, 20 mM HEPES, pH 7.4). Luciferase expression was assayed 48 h post exposure. Standard deviation shown, n = 4.

Influence of Hexon on Transfection of HepG2 CellsWe examined the possibility that hexon can promotetransgene expression using a transfection assay.Hexon–PEI/DNA complexes were found to mediate sig-nificantly greater transgene expression than simplePEI/DNA complexes (Fig. 4B). Complexes formed usingthe disulfide-linked hexon–PEI conjugate gave the high-est transfection, 10–20 times greater than PEI/DNA. Thisactivity could be blocked by pretreating complexes withthe reducing agent dithiothreitol (DTT). Complexesformed using the non-reducible, thioether-linked,hexon–PEI conjugate demonstrated an enhancement oftransfection, 5–10 times that of PEI/DNA, that was resist-ant to pretreatment with DTT. Albumin–PEI conjugatesshowed no reproducible elevation of activity comparedwith PEI/DNA.

We evaluated the susceptibility of hexon–PEI/DNA andsimple PEI/DNA complexes to degradation by nucleases toassess the possibility that the enhanced transgene expres-sion of hexon–PEI/DNA reflects greater resistance to degra-dation by cytoplasmic nucleases. However, no differentialsusceptibility to degradation was found by micrococcalnuclease in a cell-free system (data not shown). In fact, pro-tection from cytoplasmic nucleases is unlikely to lead tosubstantial increases in transgene expression, as there is verylittle cytoplasmic degradation of DNA in the 24 hours fol-lowing microinjection of PEI/DNA complexes [19].

Microinjection Studies in Xenopus laevis OocytesWe examined the possibility that the transfection-enhanc-ing effect of hexon was mediated post-endosomally by


forming complexes using a luciferase-encoding plasmidand injecting them into the cytoplasm of Xenopus laevisoocytes. Transgene expression was determined after 24hours and showed a similar pattern to HepG2 transfec-tion experiments. The greatest luciferase expression wasachieved using the disulfide-linked hexon–PEI, reachinglevels of 4000 light units (l.u.)/oocyte (10–15 times greaterthan PEI/DNA complexes), which could be abolished bypretreatment with DTT (Fig. 5). Thioether-linkedhexon–PEI achieved up to 3000 l.u./oocyte and was resist-ant to DTT. In contrast, PEI–albumin/DNA complexesshowed no increased reporter gene expression comparedwith simple PEI/DNA complexes.

Microinjection Studies in Rat-1 FibroblastsWe assessed the ability of hexon to mediate nucleartranslocation of DNA complexes by microinjection of plas-mid DNA encoding green fluorescent protein (GFP) intomammalian cells. TRITC-dextran was included to identifyinjected cells and exclude from the analysis those under-going cell division during the experimental period. Figure6A shows an example of rat-1 cells 18 hours subsequentto the injection of hexon-s-PEI/DNA (0.02 mg DNA/ml)into the cytoplasm. Figure 6B shows the results of a singlerepresentative experiment, evaluating transgene expres-sion activity following cytoplasmic and nuclear injectionof DNA and DNA complexes containing 2000plasmids/injection (0.02 mg DNA/ml). Results were scoredas the frequency (%) of GFP expression in injected cells forboth intracytoplasmic and intranuclear injection. Allmaterials showed a high frequency of transgene expression

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following direct intranuclear injection (60–80%; similarto the values found previoulsy [20]), except for albu-min–PEI/DNA complexes. Following intracytoplasmicinjection, PEI–hexon showed a strong stimulation of trans-fection efficiency, reaching over 50% efficiency of trans-fection. In contrast, free DNA showed little GFP expressionfollowing cytoplasmic injection (< 9%), whereas PEI/DNAcomplexes showed slightly more (< 12%), in accord withpublished observations [20]. Conjugates formed usingNLS–albumin–PEI showed greater activity (21%), but failedto achieve the levels shown by hexon.

Evaluation of the efficiency of cytoplasm-to-nucleustransfer by comparing frequencies of transgene expressionfollowing intracytoplasmic injection can be distorted bydifferential activity of the materials following arrivalwithin the nucleus. For this reason, we routinely expressedresults as a frequency ratio, defined as the ratio of trans-gene expression obtained for each material followingmatched intracytoplasmic and intranuclear injection,using the same needle and two halves of the same grid.This procedure had the additional benefit of controllingfor any variation between efficiencies obtained using dif-ferent grids, injection needles, and cell preparations,although any experiment achieving less than 20% fre-quency of transfection following intranuclear injectionwas considered unsuitable for analysis. The frequencyratios were calculated following cytoplasmic and nuclearinjection of substrates, with results from three completelyseparate experiments combined (Fig. 6). Whereas DNA

FIG. 5. Activity of protein–PEI/DNA complexes following microinjection intothe cytoplasm of X. laevis oocytes. Studies were conducted as described inmethods using oocytes at developmental stage IV, and a Drummond AutoOocyte Injector. The injection volume was 13.8 nl/oocyte, DNA concentration22 pg/nl (either noncondensed DNA or as polymer/DNA complex). Luciferaseexpression was assayed 48 h post injection. Standard deviation shown, n = 4.

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alone and PEI/DNA complexes show relatively low values(< 0.2), the frequency ratios observed for hexon-contain-ing complexes are greater than 0.7, with NLS–albumin–PEIshowing an intermediate value (~ 0.45). Furthermore, co-microinjection of free hexon (1000-fold excess; Fig. 6D)was shown to reduce the effect evident withhexon–PEI/DNA, but did not significantly effect PEI/DNAcomplexes.

We investigated the mechanism of hexon-mediatednuclear transfer by co-microinjection of wheat germ agglu-tinin (WGA) or excess NLS–albumin into rat-1 fibroblasts(Fig. 6C). WGA is a lectin that binds N-acetyl-b-D-glu-cosamine and inhibits nuclear pore function [21].Although it did not affect the frequency ratio of DNAalone or PEI/DNA complexes, it achieved over 50% inhi-bition of the activity mediated by hexon–PEI/DNA (boththioether and disulfide-based conjugates) and NLS–albu-min–PEI/DNA. This suggests the nuclear pore complex isintrinsically involved in the mechanism of hexon activity,similar to its involvement in the classical NLS pathway.The failure of WGA to inhibit PEI/DNA complexes suggeststhat a small amount of PEI/DNA may enter the nuclei ofcells through an NPC-independent mechanism. Althoughno conclusions can be made concerning this mechanism,the results fit well with those of Godbey, et al. [22].

Co-microinjection of excess competing NLS–albumininto the cytoplasm or nucleus also had no effect on theactivities of simple DNA or PEI/DNA (Fig. 6C). However,excess NLS–albumin decreased the transfection frequencyratios observed with hexon-containing complexes byabout 50%, confirming the involvement of componentsof the classical NLS import pathway in the mechanism ofactivity of hexon. However, co-microinjection of compet-ing NLS–albumin into the cytoplasm completely neutral-ized the activity of NLS–albumin–PEI/DNA complexes. Thedifferential competitive effects of NLS–albumin on theactivities of hexon–PEI/DNA and NLS–albumin–PEI/DNAsuggest either that hexon uses the classical NLS pathwaymore effectively than the NLS–albumin, or that additionalpathways are involved in mediating its nuclear-homingactivity.

We studied the influence of the amount of DNAadministered by cytoplasmic injection on transgeneexpression by varying DNA concentration in the injectatefrom 0.001 to 1 mg/ml while maintaining the N:P ratio at7.5. Microinjection of free plasmid DNA into the cyto-plasm showed very limited activity at low concentrations(0.001–0.08 mg/ml), whereas significant increases in thecytoplasm/nuclear frequency ratio (up to 0.4) wereachieved at higher DNA concentrations (Fig. 7). Thisincreasing ratio with rising DNA dose may reflect therequirement for a minimum number of plasmids to gainaccess to the nucleus to mediate detectable transgeneexpression, but could also reflect saturation of “sink”effects, such as degradation by nucleases, with increasingdoses of DNA.



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FIG. 6. Microinjection of rat-1 cells. (A) Example of microinjection results. Composite image of rat-1 cells 18 h post injection with hexon-s-PEI/DNA 0.02 mg/ml. 1, phase; 2, TRITC-dextran; 3, GFP; 4, overlay. (B) Efficiency of transgene expression following microinjection of DNA complexes into the cytoplasm or nucleus of rat-1 cells. Data are represented as the percentage of injected cells resulting in GFP expression following both intranuclear (open square) and intracytoplasmic (filled square)injection. Each pair of bars was performed using halves of the same grid, with the same needle for injection. Results shown are from a single representative experi-ment. (C) Influence of inhibitors of nuclear transport on transgene expression activity. Expression efficiency of complexes injected into the cytoplasm or nucleus of rat-1 cells, with data represented as transfection frequency ratio ((cytoplasmic % +) / (nuclear % +)). Filled square, no inhibitor; open square, NLS-albumin co-injected;filled square, WGA co-injected. Bar represents ± standard deviation; n = 3 separate experiments. (D) Influence of excess free hexon on nuclear targeting of hexon–PEI/DNA.Complexes and a 1000-fold excess of free hexon were co-microinjected into the cytoplasm or nucleus of rat-1 cells. Data represented as transfection frequency ratio((cytoplasmic % +) / (nuclear % +)), as described in the text.

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We observed that PEI/DNA complexes showed a morepronounced dependence on concentration, with the fre-quency ratio rising to 0.7 at a DNA concentration of 0.2mg/ml. This is very similar to the effects reported byPollard, et al. [20], who noted improved activity ofPEI/DNA complexes compared with DNA alone over thesame dose range. Complexes formed using NLS–albu-min–PEI showed a very low frequency ratio at small DNAconcentrations, but showed better activity than PEI/DNAwith the frequency ratio rising to 0.7 at a DNA concen-tration of 0.08 mg/ml. Hexon-bearing complexes showedthe most powerful action of all, however, with the


frequency ratio rising to a maximum of 0.7 at a DNA con-centration of only 0.02 mg/ml, equivalent to 2000 plas-mid copies per cell. These data suggest that hexon has amore powerful nuclear homing activity than theNLS–albumin, mediating efficient nuclear-transfer activityat relatively low plasmid concentrations.

DISCUSSION

The adenovirus virion has a diameter of about 90 nm, andis thought to translocate to the nuclear pore complex inassociation with chaperone proteins and microtubules

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[13,23], probably mediated through hexon, the major cap-sid protein. The ability of hexon to target cell nuclei hasbeen demonstrated before, both as a constituent of thecapsid [12] and as a free protein in digitonin-permeabilizedcells [14]. Microinjection studies performed here using freehexon-FITC confirmed its ability to transfer rapidly fromthe cytoplasm into the nucleus in viable cells.

Polyelectrolyte DNA complexes have sizes comparableto adenovirus [24,25], hence it is reasonable to considerthat hexon may also be able to enhance their efficiency ofnuclear delivery. Here we have conjugated hexon proteincovalently to the cationic polymer PEI using eitherthioether or disulfide bonds. The conjugates were able toform discrete complexes with plasmid DNA, similar tothose produced by PEI–albumin conjugates or PEI alone.We found the cellular uptake of all complexes to be sim-ilar, compatible with the slight negative charge on bothhexon and albumin, suggesting there was no change inelectrostatic cell binding or internalization of any com-plex. However, despite the same amount of material enter-ing cells, the hexon-containing complexes mediated muchgreater levels of transgene expression than the other com-plexes, implying that hexon was mediating an intracellu-lar effect to promote transfection.

Microinjection into the cytoplasm of X. laevis oocytesshowed superior reporter gene expression forhexon–PEI/DNA complexes compared with PEI/DNA,indicating that the activity of hexon was mediated afterits entry into the cytoplasm. Despite their different class

FIG. 7. Microinjection/dose escalation study in rat-1 cells. Expression efficiencyof complexes injected into the cytoplasm or nucleus of rat-1 cells, with datarepresented as transfection frequency ratio. Complexes were formed at anN:P ratio of 7.5:1 in 20 mM HEPES, pH 7.4, to achieve a final DNA concen-tration ranging between 0.001 and 0.5 mg/ml DNA. Filled square, DNA; opencircle, PEI/DNA; open diamond, PEI-s-hexon/DNA; filled diamond, PEI-ss-hexon/DNA; �, PEI-albumin-NLS/DNA.

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origins, X. laevis oocytes provide a simple and useful modelfor assessment of nuclear-homing activity than can haveimplications for mammalian cells. The large size of oocytespermits reporter gene activity to be quantified directly inhomogenates and their lack of cell division restrictsnuclear entry to trans-membrane mechanisms, precludingnonspecific uptake during periods of membrane disinte-gration. This system showed that the presence of hexonreproducibly increased reporter gene expression 15- to 20-fold, consistent with a nuclear-homing function thatallows the polyelectrolyte complexes to gain access to cel-lular transcription machinery.

We observed similar activity of hexon following directcytoplasmic injection in mammalian cells, with approxi-mately 10-fold greater transgene expression for hexon-containing complexes compared with PEI/DNA controls.The proposed mechanism of action, namely increasedcytoplasm-to-nuclear transfer, was illustrated by express-ing results as a cytoplasmic/nuclear transfection frequencyratio to permit direct comparison of the nuclear transferof different materials. Hexon displayed very effective abil-ity to promote nuclear transfer of DNA complexes, achiev-ing transfection frequency ratios of 0.7, much greater thanthose observed for free DNA or PEI/DNA complexes. Theactivity of complexes containing NLS–albumin–PEI con-jugate was also studied to enable comparison of the activ-ity of hexon with classical NLS pathways. These complexesalso showed greater nuclear transfer activity than simplePEI/DNA complexes, though were not as powerful ashexon-containing complexes. The reasons for the poortransgene expression mediated by albumin–PEI/DNA com-plexes (even following direct intranuclear injection) areunknown, but may relate to their tendency to aggregate,observed by electron microscopy (EM), leading to interac-tions with intranuclear components and preventing trans-gene expression.

We observed an interesting difference between hexon-mediated and NLS-mediated cytoplasm-to-nucleus trans-fer when the influence of DNA concentration was exam-ined in mammalian cell microinjection. Although bothNLS–albumin and hexon achieved a maximum cyto-plasm/nucleus transfection frequency ratio of about 0.7 atDNA concentrations above 0.08 mg/ml, hexon showedmuch better nuclear transfer activity at lower DNA con-centrations. Indeed, even at concentrations of 0.005 mgDNA/ml, hexon-containing complexes showed transfec-tion frequency ratios over 0.4, whereas complexes con-taining NLS–albumin showed no activity greater than freeDNA. These data suggest that hexon may be active at lowerconcentrations than classical NLS sequences, perhaps indi-cating a higher affinity for a cytoplasmic receptor or evenan alternative mechanism of action.

To compare the mechanism of action of hexon and clas-sical NLS sequences, we included wheat germ agglutinin(WGA), an inhibitor of NPC function, in mammalian cellcytoplasmic injections. When WGA was co-injected we



observed a decrease in transgene expression efficiency ofhexon–PEI/DNA down to levels obtained with PEI/DNA.This effect was concordant with previous observations thatWGA inhibits both the attachment of virus to the nuclearmembrane [26] and the nuclear import of free hexon [14],suggesting involvement of the NPC in both mechanisms.As expected, WGA exerted a similar inhibition of transgeneexpression gained using NLS–albumin–PEI/DNA complexes.

We also observed that co-injection of excess NLS–albu-min into the cytoplasm decreased transgene expressionwith hexon–PEI/DNA by about 50%, suggesting sharedcomponents in the NLS pathway and the pathway used byhexon–PEI/DNA complexes. Indeed, nuclear localizationof both free hexon and adenovirus nucleocapsids has beenreported to be inhibited by NLS–albumin in permeabilizedcells [14]. Co-injection of NLS–albumin also decreasedtransgene expression activity of NLS–albumin–PEI/DNAcomplexes, although this inhibition was more effectiveand levels were diminished to those observed for PEI/DNA.This differential inhibition by excess free NLS–albuminsuggests that either hexon–PEI/DNA uses classical NLS-mediated mechanisms more efficiently than NLS–albu-min–PEI/DNA or it uses an additional alternative mecha-nism. The former possibility cannot be dismissed as thenumber of NLS per albumin molecule was not optimizedin this study. Conversely, the second possibility gains sup-port from observations showing that although excessalbumin–NLS strongly diminishes the nuclear localizationof FITC–hexon, it does not ablate it completely [14].

The high efficiency of transgene expression obtainedthroughout these studies using disulfide-based hexon–PEIconjugates was unexpected, because several reports havedemonstrated intracellular reducing activity that can beused to activate disulfide-stabilized gene delivery systems[27,28]. Consequently the disulfide bond might beexpected to be reduced in the endosome or cytoplasm,releasing the hexon and leaving the PEI/DNA complexwithout any nuclear-delivery mechanism. It is possiblethat the time scale is too short during hexon-mediatednuclear targeting for this to occur, compared with vector-activation systems that may remain in the cytoplasm forperiods of days. Alternatively, there are indications thatthe reducing conditions in the nucleus are significantlystronger than in the cytoplasm [29], raising the possibil-ity that some of the intracellular reducing activitiesobserved in other studies may have actually involved pro-cessing in the nucleus. In this case, it is possible that thedisulfide-based hexon–PEI conjugate may be relatively sta-ble within the cytoplasm.

Although inefficient entry of DNA into the nucleus isa crucial factor limiting the success of nonviral genedelivery, attention has only recently begun to focus onthis challenge. Nuclear delivery using NLS has been eval-uated with the NLS linked directly to DNA [2,4,30] oralternatively conjugated to DNA-condensing agents [31].We consider, however, that polyelectrolyte complexes


have more physical properties in common with infectingviruses than with simple proteins, hence viral mecha-nisms for nuclear delivery of DNA may provide usefulmechanisms for enhanced delivery of plasmid DNA. Wehave shown here that adenovirus hexon imparts nuclearhoming activity, leading to increased rates of transgeneexpression, in a variety of experimental systems. It is par-ticularly interesting that the mechanism seems to beeffective at low cytoplasmic concentrations of DNA, asachievable concentrations are likely to be low in mosttherapeutic applications.

Although it will be possible to incorporate hexon pro-tein into targeted gene delivery systems as a whole protein,further characterization of its activity is seen as a priorityin order to assess whether it can synergize with extracel-lular targeting ligands or endosomolytic molecules.Peptide sequences mediating the same functions wouldbe more easily incorporated into most gene delivery sys-tems, providing simpler chemistry and better definition ofthe vectors formed, and with fewer implications forimmunogenicity. Similarly, identification of the key cyto-plasmic proteins through which hexon mediates itsnuclear homing activity might enable design of simpler oreven superior gene delivery strategies.

MATERIALS AND METHODS

Chemicals. Polyethylenimine (PEI) 800 kDa (Mr) and trinitrobenzenesul-fonic acid (TNBS) were obtained from Fluka (UK). We obtained 3-(-2-pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP), N-acetyl-L-cysteine, 5,5�-dithiobis(2-nitrobenzoic acid) (DTNB), dithiothreitol (DTT),TRITC-dextran, Bis Tris Propane, TRITC-labeled wheat germ agglutinin(WGA), and DEAE Sepharose from Sigma (Poole, UK). MonoQ and MonoScolumns were from Amersham Pharmacia Biotech (St. Albans, UK) and N-succinimidyl-(4-vinylsulfonyl)benzoate (SVSB) was purchased from Pierce(Rockford, IL).

Production and radiolabeling of plasmid DNA. A luciferase expressionvector (pGL3 Promega, USA) was used in transfection, uptake, and X. lae-vis oocyte studies and a plasmid containing the GFP reporter gene (C16084-1, Clontech USA) was used in microinjection studies. The plasmidswere grown in Escherichia coli HB101 and purified using Qiagen GigaprepKits (Crawley, W. Sussex, UK). For radiolabeling the expression vector waslinearized using HindIII restriction enzyme and was labeled by nick trans-lation using 32P-dCTP and the Ready-to-Go DNA labeling beads fromAmersham Pharmacia Biotech. Unincorporated nucleotides were removedusing MicroSpin S-300 Sephacryl columns (Amersham Pharmacia Biotech).

Isolation and purification of adenovirus hexon protein. Hexon proteinwas isolated and purified to homogeneity from the lysate of adenovirustype 5 infected human 293 cells, using an established procedure [32].Briefly, following removal of intact virus by CsCl density gradient cen-trifugation, the soluble antigen was dialyzed into Bis Tris Propane (10 mM,pH 7.4) and then purified using a DEAE fast-flow Sepharose anion exchangecolumn and an increasing gradient of salt. Fractions containing hexonwere identified by western blot, using a guinea pig anti-hexon antiserumraised in-house. Hexon was further purified using Mono-Q ion exchangecolumn chromatography and purity was demonstrated using silver stain-ing and western blot analysis with anti-adenovirus and anti-fiber antibod-ies.

SPDP modification of PEI. PEI (800 kDa, 21.5 nmoles) was dissolved in0.5 ml boric acid/borax buffer (0.2 M, pH 8.4) and 43 nmoles or 215 nmolesof SPDP in 100% ethanol added. After 3 h at room temperature the PEI-

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dithiopyridyl product was purified by Sephadex G25 gel filtration. PEI con-centration was measured by determination of amino groups using a TNBSassay [33], and incorporation of dithiopyridyl was calculated by measur-ing the change in absorbance at 343 nm following the addition of DTT [34].Yields of 18 nmoles PEI modified with 24 nmoles dithiopyridyl and 12.3nmoles PEI modified with 70.6 nmoles dithiopyridyl were obtained.

Modification of proteins using SVSB and SPDP. Hexon (2.7 nmoles) andalbumin (28.5 nmoles), each dissolved in boric acid/borax buffer (pH 8.4,0.2 M, 1 ml), were added separately to 6 nmoles SVSB or 57 nmoles SPDPin 100% ethanol, and allowed to react for 3 h at room temperature.Products were purified using Sephadex G25 and protein concentrationdetermined using the BCA assay. For SVSB modification, vinyl sulfone con-tent was calculated using �-mercaptoethanol and Ellmans reagent [35].Yields of 2.4 nmoles hexon modified with 2.5 nmoles vinyl sulfone and21.6 nmoles albumin modified with 30 nmoles vinyl sulfone were obtained.For SPDP modification, dithiopyridyl content was calculated by measuringthe change in absorbance at 343 nm following the addition of DTT. Yieldsof 2.5 nmoles hexon modified with 3.7 nmoles dithiopyridyl and 24nmoles albumin modified with 32 nmoles dithiopyridyl were obtained.

Synthesis and purification of PEI–hexon conjugates. DTT was added toPEI-SPDP to reduce precisely 90 mol% of the SPDP to free thiol groups. Theresulting PEI-SH was reacted with molar equivalents of hexon-dithiopyridyland hexon-vinyl sulfone, and with the same masses of albumin-dithiopy-ridyl and albumin-vinyl sulfone, to produce conjugates with similar com-position (Table 1). The conjugations were allowed to proceed for 12 h atroom temperature. PEI–protein conjugates were purified using a MonoSHR/5 cation exchange column and a Bio-Tek 360 HPLC system with UVdetection at 240 and 280 nm (Kontron). The column was equilibrated with20 mM HEPES, pH 7.4, containing 0.5 M NaCl, samples loaded in the samebuffer and eluted using an increasing gradient of NaCl and a flow rate of0.5 ml/min. Fractions (1.0 ml) were assessed for hexon content byimmunoblotting, protein content using the BCA assay and amine contentusing the TNBS assay. Fractions containing conjugates were pooled andconcentrated using large volume concentrators with a molecular weight cutoff of 10 kDa (Amicon, Millipore, Bedford, MA) and the amine and pro-tein content determined.

Synthesis of NLS-modified bovine serum albumin. SVSB (in 100%ethanol) was added to albumin (dissolved in 0.2 M boric acid/borax buffer,pH 8.4) in a fivefold molar excess, allowed to react for 3 h at room tem-perature and then purified using Sephadex G25. The albumin-SVSB pro-duced contained an average of 2.9 vinyl sulfone groups per albumin. NLSpeptide (PKKKRKVEDPYC, Alta Bioscience, Birmingham, UK) was thenadded in 30-fold molar excess to albumin-SVSB to ensure complete modi-fication of the vinyl sulfone. The resulting thioether-based NLS-albuminconjugate was purified after 3 h using Sephadex G25 and a non-denatur-ing PAGE gel was run to confirm that an NLS-albumin conjugate had beenformed.

Synthesis of NLS-modified bovine serum albumin–PEI (NLS–albumin–PEI).PEI was modified with SPDP and albumin–NLS with SVSB as described above.PEI–SH was generated using DTT and allowed to react with NLS–albu-min–SVSB. After 18 h at room temperature the product was purified asdescribed above for PEI–hexon conjugates. A yield of 0.74 nmoles PEI con-jugated to 0.23 nmoles albumin-NLS was obtained.

Formation of complexes between plasmid DNA and PEI or protein–PEIconjugates. Complexes were routinely formed by adding a small volumeof PEI or PEI–protein conjugate to a solution of plasmid DNA in 20 mMHepes, pH 7.4, to achieve a final DNA concentration of 20 �g/ml anddefined N:P ratio (the molar ratio of amino groups in PEI to phosphates inDNA).

Measurement of complex formation by loss of ethidium bromide/DNAfluorescence. Self-assembly of PEI/DNA complexes was assessed by moni-toring loss of ethidium bromide fluorescence. Ethidium bromide and DNAwere mixed in 20 mM Hepes, pH 7.4, to achieve final concentrations of400 ng/ml and 20 �g/ml, respectively. Fluorescence was measured (�ex 510nm and �em 590 nm) using a Perkin Elmer LS50B spectrofluorimeter, set to100%, and aliquots of cationic polymer were added sequentially to thecuvette, mixed by inversion, and residual fluorescence measured.

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Agarose gel electrophoresis. Electrophoresis of complexes was performedusing a Horizon 58 gel electrophoresis kit (Gibco, UK), with 0.8 % (w/v)agarose gels containing ethidium bromide (500 ng/ml). Following 20 minelectrophoresis at 90 V gels were examined using a UV transilluminator.

Transmission electron microscopy. Carbon films were prepared by subli-mation on freshly cleaved mica and recovered by flotation on Cu2+/Rh+

grids. The grids were dried, stored on filter paper and glow discharged (110mV, 25 s) before use. A sample of complex (5 �l) was pipetted onto a gridand left for 1 min. Negative staining was achieved by the addition of uranylacetate (20 �l, 1 % w/w) to the grid, with the excess liquid removed usingfilter paper after 20 seconds. Samples were analyzed using a JEOL JEM100xcxII with acceleration 80 kV.

Photon correlation spectroscopy (PCS). A Zetasizer 1000 (Malvern, UK)was used for size determination of complexes (500 �l samples). Resultswere analyzed using Contin software with monomodal deconvolution.Polydispersity is defined as the peak width at half height divided by peakheight.

In vitro studies of protein nuclear homing activity. Proteins were labeledwith FITC according to the manufacturer’s protocol (Pierce) to achieve alabeling ratio of 10 FITC per protein molecule and then purified usingSephadex G15. Rat-1 cells (20,000 cells/50 �l) were plated onto glassCellocate grids (BDH/Merck) and grown in DME with 10% serum (24 h,37�C). FITC-labeled protein (0.35 mg/ml in HEPES buffer, pH 7.4) was back-loaded into Femptotip microinjection needles (BDH/Merck) and cellsmicroinjected with an Eppendorf transjector 5246 and Eppendorf micro-manipulator 5171 (set to injection pressure 50 psi, back pressure 45 psi, andinjection duration 0.3 s). Images were captured using an inverted fluores-cence microscope (Zeiss Axiovert 100) and analyzed using Metamorph soft-ware.

Studies of cellular uptake and transfection. Uptake studies were con-ducted using HepG2 cells 48 h after seeding them in 24-well plates (20,000cells/well). Cells were incubated for 5 h with complexes trace labeled with32P-DNA, at either 4�C or 37�C, in serum-free medium. Medium was sam-pled and cells washed in PBS and dissolved in lysis solution (Promega) formeasurement of radioactivity using a scintillation counter (Packard).

In transfection studies, HepG2 cells were plated at a density of 8000cells/well in 96-well plates and grown for 48 h with DMEM containing 10%fetal calf serum. Fresh DMEM was applied (200 ml/well; without serumexcept where indicated) and DNA complexes added (30 �l, plasmid con-centration 20 �g/ml, N:P ratio 7.5, 20 mM HEPES, pH 7.4), giving a finalDNA concentration of 2.7 �g/ml. Cells were incubated at 37�C and after 5h medium was replaced with fresh DMEM containing 10% FCS (200�l/well). Luciferase expression was assayed after 48 h using a standard lumi-nescence protocol.

Microinjection of DNA complexes into isolated X. laevis oocytes. Maturefemale frogs were anesthetized by immersion in benzocaine, killed by rapiddestruction of the brain and oocytes were dissected and individually exam-ined in a petri dish containing modified Barth’s solution (MBS, pH 7.6).Only oocytes at developmental stage IV were used, and complexes weremicroinjected using a Drummond Auto Oocyte Injector (DrummondScientific Company). The injection volume was 13.8 nl/oocyte, 22 pg/nlDNA concentration. Oocytes were maintained in MBS for a further 48 hin small petri dishes at 18–20�C (individually grouped for specific studies),at which time viable oocytes were pooled (10 oocytes/ml) in lysis solution(100 mM potassium phosphate, 0.2% vol/vol Triton X-100, 1 mM dithio-threitol, pH 7.8) pipetted up and down, and samples centrifuged at 6000g(MicroCentaur, 10 min). The clear supernatant (20 �l) was then assayedfor luciferase activity using a standard luminescence protocol.

Microinjection of DNA complexes into mammalian cells. Microinjectionstudies were conducted in rat-1 fibroblasts using an Eppendorf transjector5246, an Eppendorf micromanipulator 5171, and an inverted fluorescencemicroscope (Zeiss Axiovert 100). Rat-1 cells (20,000 cells/50 �l) were platedonto glass Cellocate grids (BDH/Merck) and grown in DME with 10% serum(24 h, 37�C). The grids were then washed with PBS and transferred intoserum-free DME buffered with HEPES. PEI/DNA complexes were formed atan N:P ratio of 7.5:1 in 20 mM Hepes, pH 7.4, to achieve a final DNA con-centration of 20–500 �g/ml and a final volume of 50 �l. To enable



visualization of the technical success of injections, 2 �l TRITC-dextran (150kDa, 50 mg/ml) was added to each sample. Before injection the sampleswere centrifuged at 6000g for 5 min (MSE Microcentaur) to remove largeaggregates, and analyzed by electrophoresis before and after centrifugationto check that the DNA concentration was not substantially decreased.Samples were back-loaded into Femptotip microinjection needles(BDH/Merck) and cells injected using an injection pressure of 40–70 psi, aback-pressure of 60–70 psi, and an injection duration of 0.3 s. Half of thegrid was used for injections into the cytoplasm and the other half for injec-tions into the nucleus, with 100–150 cells injected into each compartmentin each determination. To ensure reproducibility, the same needle wasused for both cytoplasmic and nuclear injections. Observation using UVillumination and appropriate filter sets enabled determination of the num-ber of successful injections. The glass grid was then washed with PBS, trans-ferred back into DME containing 10% serum, and incubated at 37�C. After18 h the number of GFP-positive cells was counted. Cells were only countedif they were still displaying TRITC-dextran staining restricted to the com-partment injected, showing they had not been through cell division. Thepercentage of cells expressing GFP following cytoplasmic injection was cal-culated, as was the percentage of cells expressing GFP following intranu-clear injection. In some studies WGA (final concentration 0.2 mg/ml) orNLS-albumin (0.06 mg/ml), potential inhibitors of nuclear transport, wasmixed with the test agent immediately before microinjection into cells. Inaddition studies whereby 1000-fold excess of free hexon was co-injectedwere also conducted, aggregation of complexes caused by such high con-centrations of hexon was overcome by the pre-addition of albumin (5mg/ml).

ACKNOWLEDGMENTSWe thank Libuska Oupická (University of Birmingham) for production of plas-mid DNA, Carsten Kneuer (Faustus Translational Cancer Research GmbH) forinitial inspiration to evaluate nuclear homing activity of hexon, and many col-leagues for useful discussions. This work was supported by the Cystic FibrosisTrust, Association pour la Recherche sur le Cancer, Medical Research Council,and the Cancer Research Campaign.

RECEIVED FOR PUBLICATION APRIL 17; ACCEPTED AUGUST 20, 2001.

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