Adenovirus Hexon Protein Enhances Nuclear Delivery and Increases Transgene Expression of Polyethylenimine/Plasmid DNA Vectors
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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: L.Seymour@bham.ac.uk.
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 hexonPEI/DNA complexes and NLSalbuminPEI/DNA complexes in rat-1fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexonPEI/DNA> NLSalbuminPEI/DNA > PEI/DNA > DNA alone > albuminPEI/DNA. The activities of bothNLSalbuminPEI and hexonPEI 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 freeNLSalbumin abolished transgene expression with NLSalbumin-PEI/DNA, but only partially inhib-ited hexonPEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependenton DNA concentration for all materials, although hexon conjugates showed much better activ-ity than NLSalbumin at low DNA doses (5001000 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
ARTICLEINTRODUCTIONThe 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 , 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 $35.00oligonucleotides or linearized plasmids [2,3], but less effi-cient transfer of intact plasmids .
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 associate473
ARTICLE doi:10.1006/mthe.2001.0472, available online at http://www.idealibrary.com on IDEALA
FIG 1. Intracytoplasmic microinjection in rat-1 fibroblasts of hexonFITC (A), NLSalbuminFITC(B), and albuminFITC (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 . Thedemonstration that the major capsid protein hexon associ-ates with heat shock protein-70  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 , 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 .
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 importinNLSproteincomplex then binds compo-nents of the microtubular net-work, translocates to the NPC,and enables transfer of the NLSprotein through the NPC intothe nucleus . In contrast,however, the amino acidsequence of hexon contains nodefinitive NLS, and the majority474of 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 . 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 .
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 theactivity of hexon with that of an NLS-bearing protein.
RESULTSNuclear Homing Activity of HexonFITC and NLSalbuminFITCWe assessed the ability of hexon, albumin, and NLSalbu-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 microscopyTABLE 1: Formation and characterization of proteinPEI conjugates
Reagent mixture Molar ratio of product Mass ratio of product
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-SPDPMOLECULAR 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 IDEALat 5, 30, and 60 minutes following injection. BothhexonFITC and NLSalbuminFITC 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, albuminFITCshowed no detectable nuclear accumulation, even 60 min-utes following microinjection. Nuclear localization usingFITC-labeled proteins was also examined using digitonin-permeabilized HepG2 cells . Again, NLSalbuminFITCand hexonFITC both showed substantial nuclear entry inpermeabilized cells, whereas albuminFITC remainedentirely extranuclear (data not shown). Similar data havepreviously been reported for hexonFITC .
Purification and Characterization of ProteinPEI 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
MOLECULAR THERAPY Vol. 4, No. 5, November 2001Copyright The American Society of Gene Therapyenable 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 PEIhexon and PEIalbumin conjugates(based on both types of linkage) using MonoS columnchromatography. Similar elution profiles were produced ineach case, and hexonthioetherPEI 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 PEIhexon 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 ProteinPEI Conjugates with DNAWe examined the ability of the proteinPEI 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 hexonPEI 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.475
ARTICLE doi:10.1006/mthe.2001.0472, available online at http://www.idealibrary.com on IDEALA
FIG. 3. Physicochemical characterization of the proteinPEI conjugates. (A) Agarose gel electrophoresis of proteinPEI/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 proteinPEI 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.
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 476complexes 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...