Factors Influencing the Ability of Nuclear Localization Sequence

Dec 13, 2003 - Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS)...
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Bioconjugate Chem. 2004, 15, 152−161

Factors Influencing the Ability of Nuclear Localization Sequence Peptides To Enhance Nonviral Gene Delivery K. Helen Bremner,† Leonard W. Seymour,*,‡ Ann Logan,§ and Martin L. Read§ Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TH, UK, Department of Clinical Pharmacology, University of Oxford, Oxford, OX2 6HE, UK, and Department of Medicine, University of Birmingham, Birmingham, B15 2TH, UK. Received August 8, 2003; Revised Manuscript Received November 6, 2003

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin β-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.

INTRODUCTION

Gene therapy has tremendous potential as either a standalone treatment or adjuvant therapy for many forms of disease such as cancer (1). The most efficient methods of delivering genes are those based on viral vectors such as adenovirus and retroviruses. However, safety issues relating to inflammatory and immune responses to adenoviral proteins and malignant transformation due to insertional mutagenesis with retroviral delivery have raised concerns over the use of these vectors (2, 3). A safer alternative is to use nonviral (or synthetic) vectors such as polyplexes and lipoplexes for gene transfer. At present, however, the low levels of gene expression typically observed in vivo restrict the usefulness of nonviral vectors compared to viral vectors. Inefficient transfer of DNA from the cytoplasm to the nucleus has been identified as a major reason for this, particularly in postmitotic and quiescent cells where there is no periodic breakdown of the nuclear membrane (4, 5). By comparison, cellular and viral proteins enter the nucleus efficiently by means of nuclear localization sequences (NLS1), stretches of amino acids that bind to intracellular transport receptors such as importins and facilitate transfer through the nuclear pore. Early experiments showed that NLSs retain their activity when conjugated as a synthetic peptide to otherwise nonnuclear * To whom correspondence should be addressed. Phone: 441865-224986. Fax: 44-1865-224538. E-mail: Len.Seymour@ clinpharm.ox.ac.uk. † Cancer Research UK Institute for Cancer Studies. Present address: Department of Pathology, Columbia University, 630 W. 168th St. P&S 15-410, New York, NY 10032. ‡ Department of Clinical Pharmacology. § Department of Medicine.

proteins such as bovine serum albumin and transferrin (6). The application of NLS peptides for nonviral gene transfer has since been widely investigated (7) and results to date have indicated the potential of this approach to improve nuclear delivery of DNA. For example, coupling of streptavidin-NLS conjugates to biotinylated DNA enabled linear DNA fragments of between 310 and 1500 bp to enter the nuclei of digitonin permeabilized cells by an active transport process (8). Significant increases in gene expression were also observed following ligation of an oligonucleotide-NLS conjugate to one or both termini of a linear DNA molecule, although no conclusive localization or mechanistic data was given in this study (9). Results have been encouraging using linear DNA fragments but the type of chemistry used to conjugate peptides to circular plasmid DNA often results in loss of transcriptional capability. Two recent studies, for example, used either a photoactivation reaction (10) or a cyclopropapyrroloindole group (11) to couple NLSs at random locations on the DNA molecule. Sebestyen et al. demonstrated nuclear import in digitonin-permeabilized HeLa cells when >100 NLS peptides were attached to each plasmid, but transgene expression was completely 1 Abbreviations: DOTAP, 1,2-dioleoyl-3-(trimethylammonio)propane; DTT, dithiothreitol; EtBr, ethidium bromide; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HTLV, human T cell leukaemia virus type 1; ImpR1, importin R1; Impβ1, importin β1; NLS, nuclear localization sequences; PEI, polyethylenimine; PLL, polylysine; PNA, peptide nucleic acid; PNA-b, PNA-biotin; PNA-m, PNA-maleimide; RPC, reducible polycation; SV40p, SV40 promoter; Tn1, transportin 1; TXRed Mal, Texas Red maleimide.

10.1021/bc034140k CCC: $27.50 © 2004 American Chemical Society Published on Web 12/13/2003

Strategies To Enhance Nonviral Gene Delivery Using NLS Peptides

abolished (11). More encouragingly, Ciolina et al. found no decrease in expression until modification reached the level of 43 peptides per plasmid; however, despite binding of the conjugates to importin R, no increase in gene expression was observed (10). A strategy to prevent loss of gene expression is to attach NLS peptides to DNA at specific locations using peptide nucleic acid (PNA) molecules that have a repeating polyamide backbone rather than a repeating sugarphosphate backbone and hybridize to DNA in a highaffinity and sequence specific manner (12). Branden et al. recently demonstrated the utility of this approach with an 8-fold increase in transfection using PNA-NLS and 25-kDa PEI (13). The interaction between DNA and mono PNA is weakened under physiological conditions but has been recently overcome by the use of a homopyrimidine J-base containing bis PNA clamp that forms (PNA)2DNA triplexes in a highly stable, pH-independent manner (14). PNA clamps have so far been used to label DNA with fluorophores, but there is potential in this approach to attach a wide range of ligands to DNA to provide specific functions without compromising transcriptional capability (15). The exact conditions to successfully utilize the properties of NLS peptides to enhance nonviral gene transfer are, however, still not clear. It is unknown, for example, what type or length of NLS peptide is best suited for this application and NLS sequences that bind to a range of transport receptors, including importin β1 or transportin 1, have not been thoroughly investigated. The aim of the present study was to help define the requirements for successful NLS-enhanced transfection by comparing the ability of a panel of NLS peptides to enhance nonviral gene delivery. Two different strategies for incorporating NLS peptides in nonviral vectors were evaluated. In the first, NLS peptides were linked to DNA in a site-specific manner via an intracellularly stable thioether bond formed between a maleimide-labeled PNA clamp and a cysteine residue at the carboxy terminal of each peptide. The ability of NLS peptide-PNA/DNA conjugates to bind to their intracellular transport receptor and promote levels of gene expression was then examined. In the second strategy, the cationic nature of NLS peptides was utilized by investigating their ability to condense plasmid DNA into discrete particles and mediate transfection. EXPERIMENTAL PROCEDURES

Materials. Peptides were synthesized by Severn Biotech Ltd (Kidderminster, UK) using standard fmoc chemistry with the following sequences: PKKKRKVEDPYC (SV40 T NLS) and SSDDEATADSQHSTPPKKKRKVEDPYC (Ext SV40 T NLS) containing the wild-type SV40 large T NLS, PKTKRKVEDPYC (mSV40 T NLS) and SSDDEATADSQHSTPPKTKRKVEDPYC (Ext mSV40 T NLS) containing a mutant SV40 large T NLS, MPKTRRRPRRSQRKRPPTPWAHFPGFGQSLC (HTLV) from the human T cell leukaemia virus type 1 Rex protein, GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGYC (M9) from heterogeneous ribonucleoprotein A1, and a scrambled version SPGNRMGAGYFKGSGNKSGFQGRNPGSGQGYGPNQGFYGC (Scr M9). Oligonucleotides containing either one, three, or five copies of the PNA binding site (AGAGAGAG) were purchased from Alta Bioscience (University of Birmingham, Birmingham, UK) and inserted into the KpnI-MluI cloning site of pGL3con (Promega). The sequence of oligonucleotides containing PNA-binding sites for pGL3con constructs were 5′-CGATATCCAGCTGTCTCTCTCTCA-3′ (one site),

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5′-CGATATCCTTAAGCTCTCTCTCTCTCTCTCTCTCTCTA-3′ (three sites), and 5′-CGATATCCTTAAGCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTA-3′ (five sites). A bis PNA clamp of sequence TCTCTCTCO-O-O-JTJTJTJT covalently attached at the amino terminus to biotin or maleimide was purchased from Gene Therapy Systems (San Diego, CA). J represents a pseudoisocytosine that is placed in the strand parallel to the DNA forming the “Hoogsteen strand”, and O represents an 8-amino-3,6-dioxaoctanoic acid linker. Preparation of Importin- and Transportin-GST Fusion Proteins. Importin R-GST and importin β-GST were grown from pGEX-2T plasmids (kindly provided by Y. Yoneda, Osaka University, Japan) and transportin 1 from a pGEX-5X plasmid (a kind gift from G. Dreyfuss, University of Pennsylvania) as previously described (16, 17). Proteins were expressed in BL21 (DE3) cells and induced with 1 mM IPTG for 18 h at 20 °C (importins) or 17 °C (transportin 1). Cells were harvested by centrifugation and disrupted by freeze thawing followed by sonication. Fusion proteins were recovered from cleared bacterial lysate using glutathione-sepharose (Pharmacia) according to the manufacturer’s instructions. Purified proteins were dialyzed against 20 mM HEPES-NaOH pH 7.3, 100 mM potassium acetate, 2 mM DTT containing 0.5 µg/mL protease inhibitor cocktail (Sigma). Purity of proteins was assessed by Coomassie staining following SDS-PAGE and Western blotting using an anti-GST antibody (Pharmacia). Interaction of NLS Peptides with Transport Receptors. Binding of NLS peptides to transport receptors was measured by ELISA similar to that previously described (17). Maxisorp 96-well plates were coated with peptide (41.9 pmol for SV40 peptide, 41.8 pmol for Ext SV40 peptide, 33.5 pmol for HTLV peptide, and 30.9 pmol for M9 peptide) in 50 mM NaHCO3 pH 9.8 at 4 °C overnight. After blocking with intracellular buffer (IB; 110 mM KCl, 5 mM NaHCO3, 5 mM MgCl2, 1 mM EGTA, 0.1 mM CaCl2, 20 mM HEPES-NaOH pH 7.4, 1 mM DTT, 5 µg/mL protease inhibitor cocktail) containing 5% BSA, plates were incubated at 4 °C overnight with 12.5 nM transport receptor-GST or GST alone in IB/1% BSA. After washing the plate three times, the primary antibody goat anti-GST was applied for 2 h at 1/1000 dilution in IB/1% BSA. The plate was again washed three times and incubated with HRP-labeled rabbit anti-goat antibody (1/1000 in IB/1% BSA) for 2 h. After a further three washes, the plates were incubated with OPD substrate for 20 min and the reaction stopped by addition of 50 µL of 4 M sulfuric acid. The absorbance at 490 nm was measured using a Victor multilabel plate reader and background readings subtracted. Preparation and Analysis of PNA/DNA Conjugates. PNA-biotin was vortexed for 2 min and heated at 55 °C for 15 min then added to plasmid DNA in PNA binding buffer (10 mM sodium phosphate buffer pH 7, 1 mM EDTA, 20 mM NaCl) and incubated at 37 °C for 24 h. Conjugate formation was assessed using streptavidinTexas Red (Molecular Probes) to probe for bound PNAbiotin. In brief, PNA-biotin/DNA conjugates were restricted with KpnI and HindIII, purified using Sephadex G-25 spin columns (Pharmacia) and incubated with a 10fold molar excess of streptavidin-Texas Red in the presence of 200 mM NaCl at room temperature for 30 min. Following gel electrophoresis on a 1.6% agarose gel in ethidium bromide-free conditions, the presence of Texas Red-labeled DNA was detected using a Typhoon gel scanner (Molecular Dynamics, λex 633 nm, λem 610 nm, 800 V). Gels were also stained in ethidium bromide

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solution (0.5 µg/mL) for 20 min and DNA fragments visualized using the Typhoon gel scanner (λex 532 nm, λem 526 nm, 550 V). The signal intensity of particular bands was quantified using ImageQuant software. PNAmaleimide was conjugated to plasmid DNA by incubating at 37 °C for 2 h in PNA binding buffer. Characterization of NLS Peptides. Cysteine-terminated NLS peptides were incubated with Texas-Red maleimide for 3 h in the dark, and reaction products analyzed on 16.5% Tris-Tricine SDS PAGE. Samples (0.5-20 µg of peptide) were loaded in sample buffer (2% SDS, 10% glycerol, 62.5 mM Tris.-HCl pH 6.8, 0.05% bromophenol blue) and were not heated prior to loading. Gels were scanned to detect Texas Red fluorescence using the Typhoon gel scanner (λex 633 nm, λem 610 nm, 800 V). Preparation of NLS Peptide-PNA/DNA Conjugates and Binding to Transport Receptors. PNAmaleimide/DNA conjugates were purified using Sephacryl S-500HR spin columns and incubated with a 20-fold molar excess of cysteine-terminated NLS peptides in 1 M NaCl for 1 h at room temperature followed by 2 h at 4 °C. The NLS peptide-PNA/DNA conjugates were then purified by ethanol precipitation and resuspended in water, and the amount of DNA was quantified by a fluorimetric assay using Hoechst dye (Sigma). Linear DNA conjugates were prepared by restriction digest using the enzymes KpnI and BamHI and purified by ethanol precipitation, and the amount of DNA was quantified using the Hoechst dye assay. Binding of NLS/DNA conjugates to transport receptorGST was measured using an in vitro binding assay similar to that described previously (10), whereby transport receptor-GST was immobilized on glutathionesepharose and the extent of NLS peptide-PNA/DNA binding measured. Briefly, 20 µL of glutathione-sepharose slurry was washed three times in 100 µL of binding buffer (20 mM HEPES-NaOH pH 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 5 mM MgCl2) containing 100 µg/mL BSA and incubated with 25 nmol transport receptor-GST in a total volume of 100 µL for 2 h at room temperature with end-over-end rotation. Beads were washed three times in binding buffer containing 1% BSA and then incubated with 0.5 µg of NLS peptide-PNA/DNA or DNA in binding buffer containing 1% BSA, again in a total volume of 100 µL for 2 h at room temperature with rotation. After washing three times, the beads in a volume of 100 µL of binding buffer/1% BSA were assayed for the presence of DNA by addition of 100 µL of PicoGreen (Molecular Probes, 1 in 200 dilution in TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA)) to samples in 96-well plates. After 2-5 min incubation in the dark, the fluorescence was measured using the Victor plate reader (λex 485 nm, λem 535 nm for 10 s). A DNA standard curve, also containing glutathione-sepharose beads, was included and the amount of DNA bound to the transport receptor was calculated for each sample. Formation of NLS Peptide/DNA Polyplexes. NLS peptide/DNA polyplexes were formed in water by the addition of the appropriate amount of NLS peptide to 20 µg/mL DNA, mixed and incubated at room temperature for 20 min prior to use. The ethidium bromide condensation and agarose gel retardation assays were used to assess polyplex formation. Briefly, fluorescence of a 10 µg/mL DNA solution with 400 ng/mL ethidium bromide added was measured at λex 532 nm, λem 610 nm using a fluorimeter (PerkinElmer, LS-50B). NLS peptides were added in small increments and the fluorescence meas-

Bremner et al.

ured. Background fluorescence from ethidium bromide alone was subtracted and the N:P ratio of NLS peptide to DNA was plotted as a percentage of the fluorescence in the absence of peptide. In the gel retardation assay DNA was incubated with NLS peptide or PLL in the range N:P of 0 to 10 prior to analysis by agarose gel electrophoresis in the presence of 50 µg/mL ethidium bromide. Maintenance and Passage of Mammalian Cells in Culture. The human cervical carcinoma cell line HeLa was grown in RPMI containing 1 mM glutamine, glucose (1 g/L) and 10% foetal calf serum (FCS). The PC-3 human prostatic carcinoma cell line was grown in DMEM supplemented with 1 mM glutamine, glucose (1 g/L) and 10% FCS. Transfection Studies. Cells were seeded at 30 000 cells per well in 48-well plates 24 h prior to transfection. In some experiments, NLS peptide/DNA polyplexes at a N:P ratio of between 1:1 and 10:1 were used to transfect cells and in others, a reducible polycation (RPC) was used in combination with the lipid DOTAP as the transfection agent as previously described (18). Briefly, the RPC was added to 50 µg/mL DNA solution at an N:P ratio of 2:1 and incubated at room temperature for 20 min. DOTAP was added at a (w/w) ratio of 5:1 to DNA in 7.5 mM HEPES-NaOH pH 7.4 and complexes incubated at room temperature for a further 30 min before use. Unless stated otherwise, 0.5 µg of DNA was added per well. Cells were incubated in serum-free medium at 37 °C for 2.5 h in the presence or absence of 100 µM chloroquine. The media was replaced with media containing 10% FCS, and the cells were cultured for a total of 8-24 h prior to analysis of reporter gene expression. Analysis of Reporter Genes. Luciferase expression following transfection was measured by a luminescence assay using cell lysates. The culture media was discarded, and cell lysates were harvested after freeze-thawing cells in 100 µL of lysis buffer. The lysate was gently vortexed, and 20 µL diluted into 100 µL of luciferase reaction buffer (20 mM glycylglycine, 1 mM MgCl2, 0.1 mM EDTA, 3.3 mM DTT, 0.5 mM ATP, 0.27 mM coenzyme A lithium salt). The luminescence was integrated over 10 s on a Lumat LB9507 (Berthold Instruments, UK), and the results were expressed as relative light units (RLU) per mg of cell protein, as determined using the Advanced Protein Assay (Totam Biologicals, Northampton, UK). MTS Assay for Cell Viability. Cells were seeded at 5000 per well in a flat-bottomed 96-well plate 24 h prior to the experiment. Following transfection (0.25 µg of DNA per well), the medium was changed and 20 µL of MTS reagent (Promega) added per well. Plates were incubated at 37 °C for 5 h prior to measuring the absorbance at 490 nm. Background absorbance was subtracted, and the results were plotted as percentage cell viability. RESULTS

NLS Peptides Used in Study. Three different types of NLS peptides were used in this study. Peptides SV40 and Ext SV40 were derived from the SV40 T antigen sequence that binds to importin R1, while control peptides mSV40 and Ext mSV40 contained the K128T mutation reported to abolish NLS function (19, 20). The HTLV peptide is a 31 amino acid sequence from the amino terminus of the HTLV Rex posttranscriptional regulatory protein that has previously been identified as mediating nuclear import by direct interaction with importin β1 (21). The 38 amino acid peptide M9 was

Strategies To Enhance Nonviral Gene Delivery Using NLS Peptides

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Table 1. NLS Peptides Used in Study

Figure 2. Schematic showing PNA binding sites used in study. (A) Relative location of oligonucleotides containing PNA binding sites inserted into the luciferase expression vector pGL3con (Promega). The relative positions of the SV40 promoter (SV40p), transcription start point and three restriction sites (Kpn1, HindIII, and BamH1) are indicated. (B) Sequences of oligonucleotides containing between one and five PNA binding sites used in study. The sequence of the PNA binding site within each oligonucleotide is highlighted and the name of the resultant plasmid DNA construct given (pPNA1, pPNA3 and pPNA5).

Figure 1. Binding of NLS peptides to transport receptors. 96well plates were coated with 30-40 pmol/well of the (A) SV40 peptide, (B) Ext SV40 peptide, (C) HTLV peptide, or (D) M9 peptide in 50mM NaHCO3 at 4 °C overnight. After blocking with intracellular buffer containing 5% BSA, plates were incubated with 12.5 nM transport receptor-GST or with GST alone. The presence of the transport receptor was detected using goat antiGST antibody, HRP-labeled anti-goat and OPD substrate. The absorbance at 490 nm was measured and background subtracted. In panels A, B, and D binding of mutated (mSV40, Ext mSV40) and scrambled (Scr M9) peptides to transport receptors were also determined. Results are shown as a mean and standard deviation from at least three samples.

derived from heterogeneous ribonucleoprotein A1 reported to bind transportin 1, and a scrambled version of this peptide was used as a control peptide. The amino acid sequences of these peptides are shown in Table 1. For all peptides a cysteine residue was included at the carboxy terminus to enable attachment to PNA-maleimide/DNA conjugates. Binding of NLS Peptides to Intracellular Transport Receptors. The nature of the interaction of NLS peptides with a range of intracellular transport receptors was determined using an ELISA-based assay with purified GST fusion proteins and an anti-GST antibody. Significant binding to importin R1 was observed with the SV40 (Figure 1A) and Ext SV40 (Figure 1B) peptides but not the M9 peptide or the control peptides mSV40 and Ext mSV40. Unexpectedly, the HTLV peptide bound at similar levels to importin R1, importin β1 and transportin 1 (Figure 1C). However, the HTLV peptide bound only weakly to GST alone, indicating some degree of specificity for the interaction. The M9 peptide exhibited a strong interaction with transportin 1, its known receptor, whereas

the scrambled peptide control, Scr M9, did not bind (Figure 1D). A significant interaction was also observed between the M9 peptide and importin β1 (p < 0.05), although at 5-fold lower than with transportin-1. These data demonstrates that the NLS peptides are capable of binding to intracellular transport receptors in vitro with different degrees of interaction and specificity. The ability of these NLS peptides to enhance delivery of plasmid DNA was therefore next investigated. Characterization of PNA/DNA Conjugates. In the first strategy to utilize NLS peptides for gene delivery, PNA/DNA conjugates were prepared and characterized prior to attachment of NLS peptides. The requirements for efficient binding of the PNA to DNA were examined using constructs based on the plasmid pGL3con containing one, three, or five copies of the PNA binding sequence AGAGAGAG inserted immediately upstream of the SV40 promoter. Figure 2 shows the relative location and sequence of PNA binding sites inserted into pGL3con. These constructs were incubated with PNA-biotin and the presence of bound PNA-biotin probed using TexasRed labeled streptavidin. Comigration of streptavidin Texas-Red with DNA following electrophoresis demonstrated significant binding of PNA-biotin to constructs containing three or five copies of the PNA binding site (Figure 3A). By comparison, there was no significant binding of PNA-biotin to DNA without any binding sites (Figure 3A) or to constructs containing only one binding site (data not shown). The interaction between PNA and plasmid DNA was investigated in more detail by restriction of PNA-biotin/DNA conjugates with KpnI and HindIII, generating DNA fragments of either 277 bps (containing three PNA sites) or 293 bps (containing five PNA sites). Probing for PNA-biotin with streptavidin Texas-Red showed that PNA-biotin bound specifically to the PNA binding site, as only short DNA fragments (5 kb) without any known PNAbinding site (data not shown). Binding of streptavidin Texas-Red to PNA-biotin also resulted in retardation of the shorter DNA fragments producing discrete bands. For example, addition of streptavidin Texas-Red to the DNA

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Bremner et al. Table 2. Efficiency of PNA Binding to DNA construct

PNA-ba

%DNA boundb

pPNA1 pPNA3 pPNA3 pPNA5 pPNA5

37.5 3.13 12.5 1.88 7.5

1.7 12.1 18.0 15.7 79.4

a Molar excess of PNA-biotin added to DNA. b Percentage of DNA bound by PNA-b determined by measuring ethidium bromide fluorescence of free DNA.

Figure 3. Characterization of PNA/DNA conjugates. (A) PNAbiotin (PNA-b) was added to pGL3con (Lane 1), pPNA3 (Lane 2), and pPNA5 (Lane 3) at a 7.5-12.5 molar excess, incubated at 37 °C for 24 h, and a 10-fold molar excess of streptavidin Texas-Red added in 200 mM NaCl for 30 min. Samples were run on a 0.8% ethidium bromide (EtBr)-free agarose gel and conjugate formation assessed by scanning for (i) Texas Red fluorescence on a Typhoon fluorimager. The gel was then stained in (ii) 50 µg/mL EtBr and scanned for the presence of DNA. Lane 4 (-) contains streptavidin-Texas Red alone with no DNA present. (B) The PNA-b/pPNA5 conjugate was prepared as in (A) using 1.9 to 7.5 molar excess of PNA-b and restricted with KpnI and HindIII prior to addition of streptavidin-Texas Red. Samples were run on a 1.6% agarose gel for 3 h and visualized for (i) Texas Red and (ii) EtBr fluorescence as in (A). Restriction fragment of 293 bp is shown only. (C) Analysis of agarose gel from (B) using ImageQuant software with image density plotted as a function of distance migrated. The peaks numbered 1 to 5 are indicated, and the lines represent using PNA-b at a molar excess of 1.9 (intact) and 7.5 (dashed).

fragment containing five copies of the PNA binding site produced five distinct bands with reduced electrophoretic mobility (Figure 3B and 3C). Similarly, plasmids containing three copies of the PNA binding site gave three retarded bands in the presence of streptavidin TexasRed (data not shown). These retarded bands most likely indicate that an increasing number of PNA-biotin molecules are bound to each plasmid. The amount of PNA-biotin used correlated with the intensity of Texas-Red fluorescence in retarded bands, reflecting the level of PNA-biotin bound to the DNA. For example, two major retarded bands were observed using the DNA fragment containing five PNA-binding sites with PNA-biotin at a molar excess of 1.9, whereas five distinct bands of reduced intensity were observed using 4-fold more PNA-biotin (Figures 3B and 3C). The proportion of DNA bound by PNA-biotin was determined by quantifying the ethidium bromide fluorescence of free

DNA (Table 2). These results indicated that approximately 80% of plasmid DNA containing five PNA binding sites was bound by PNA-biotin when 7.5 molar excess was used. In contrast, only 25% of plasmid DNA containing three PNA binding sites was bound to PNA-biotin at a molar excess of 12.5. Altogether these results indicate that while PNA bound specifically to plasmids containing three copies of the PNA binding site, at least five binding sites were required to achieve an appropriate level of PNA/DNA conjugation that might prove useful for gene delivery applications. Preparation and Characterization of NLS Peptide-PNA/DNA Conjugates. To determine the reactivity of terminal cysteine residues the SV40 and Ext mSV40 peptides were incubated with Texas-Red maleimide and reaction products analyzed by Tris-Tricine SDS-PAGE. Two major bands were observed corresponding to the fluorescently labeled NLS peptide and free Texas-Red maleimide (Figure 4A, lane 2i,ii). Increasing the amount of peptide used from 1 to 20-fold molar excess (Figure 4A, lanes 2-4i,ii) resulted in complete loss of free Texas-Red maleimide and more labeled NLS peptide. Similar results were obtained with all of the cysteine terminated NLS peptides (data not shown). On the basis of these results a 20-fold molar excess of NLS peptide to PNA-maleimide was subsequently used in conjugation reactions. The next step was to attach NLS peptides directly to the PNA/DNA conjugates. However, the NLS peptides used in this study are cationic, containing lysine and arginine residues, and are capable of binding nonspecifically to DNA by electrostatic interaction. To overcome this problem NLS peptide-PNA-maleimide/DNA conjugates were prepared by incubating PNA-maleimide/DNA conjugates with a 20-fold molar excess of each NLS peptide in 1M NaCl. Unreacted peptide was then removed by ethanol precipitation. To verify conjugate formation and removal of free peptide, PNA-maleimide/ DNA conjugates were labeled with an N-terminal FITClabeled HTLV peptide and restricted to release a 293 base pair fragment. Analysis of the restriction fragments by gel electrophoresis demonstrated that fluorescence of the NLS peptide was only associated with DNA fragments containing the PNA binding sites and not with the rest of the plasmid (Figure 4B). Using an agarose gel-based assay to generate a peptide standard curve with FITClabeled HTLV peptide, it was estimated that on average 1.26 NLS peptides were bound to each plasmid (data not shown). However, as the PNA was bound to 80% of the DNA, this figure was recalculated to an average of 1.6 copies of the NLS peptide bound to 80% of the DNA. These results demonstrate that site-specific conjugation between NLS peptides and the PNA-maleimide/DNA conjugates was achieved. In Vitro Biological Activity of NLS Peptide-PNA/ DNA Conjugates. The biological activity of NLS peptidePNA/DNA conjugates was evaluated by their ability to bind to intracellular receptors and transfection activity

Strategies To Enhance Nonviral Gene Delivery Using NLS Peptides

Figure 4. Conjugation of NLS peptides to PNA/DNA. (A) NLS peptides (i) SV40 and (ii) Ext mSV40 were incubated with Texas-Red maleimide (TX-Red mal) for 3 h, analyzed on a 16.5% Tris-Tricine SDS-PAGE gel and scanned using a Typhoon fluorimager at λex 633 nm, λem 610 nm. NLS peptides were added at 1-, 10-, and 20-fold molar excess. Positions of labeled peptides and free TX-Red mal are indicated. (B) NLS peptide-PNA/DNA conjugates were prepared as previously described using either PNA-maleimide (lane 1), PNA-biotin (lane 2) or no PNA (lane 3), and a FITC-tagged HTLV peptide. The conjugates were then restricted with KpnI and HindIII and samples run on a 1.6% ethidium bromide (EtBr)-free agarose gel. The peptide was detected by scanning for (i) FITC using a Typhoon fluorimager at λex 532 nm, λem 526 nm. The gel was stained in (ii) 50 µg/mL EtBr and scanned for the presence of DNA at λex 532 nm, λem 610 nm. Specific conjugate formation was indicated by comigration of the peptide with the DNA fragment containing PNAbinding sites.

in HeLa cells. Using an in vitro binding assay based on GST-receptor fusion proteins bound to glutathionesepharose beads, approximately 5 ng of SV40 and Ext SV40-PNA/DNA conjugates (p < 0.05) and 3.5 ng of the HTLV-PNA/DNA conjugate (p ) 0.0123) bound to importin R1-GST, compared to 2 ng with DNA alone (Figure 5A). In contrast, there was no significant binding observed with the HTLV-PNA/DNA conjugate to importin β1 or the M9-PNA/DNA conjugate to transportin 1-GST. Previous studies have indicated that NLS peptides attached to linear DNA molecules enhance nuclear delivery (9). Therefore, NLS peptide-PNA/DNA conjugates were restricted with KpnI and BamHI to generate linear DNA molecules of 2491 base pairs prior and their transfection ability assessed. Transfection of HeLa cells using 0.25 µg of DNA with a reducible polycation/DOTAP delivery system (18) showed that reporter gene expression of unmodified linear DNA was 10-fold less than intact circular DNA after 24 h (Figure 5B). Attachment of the Ext SV40 peptide to the PNA/DNA conjugate mediated a significant 7-fold increase in transgene expression (p ) 0.035) compared to linear DNA and PNA/DNA controls. There were no significant improvements in gene expression observed using any of the other NLS peptides. These results indicate that biological activity of linear DNA can be enhanced by linkage of NLS peptides using PNA

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Figure 5. Biological activity of NLS peptide-PNA/DNA conjugates. (A) Binding to intracellular transport receptors. Glutathione-sepharose beads were incubated with 25 nmol of transport receptor-GST (ImpR1-importin R1, Impβ1-importin β1, and Tn1-transportin 1) as indicated for 2 h, washed in NLS buffer/ 1% buffer, and incubated with 0.5 µg of NLS peptide-PNAmaleimide/pPNA5 for 2 h at room temperature. The picogreen assay was used to determine the amount of DNA bound to beads. In control experiments the transport receptor-GST was omitted (-) as indicated. Significant difference was observed between Ext SV40-PNA/DNA conjugates and DNA alone (*p < 0.05). (B) Transfection activity of linear NLS peptide-PNA/ pPNA5 conjugates. HeLa cells were transfected with 0.25 µg of 2491 bp linear DNA fragments generated from NLS peptidePNA/DNA conjugates using RPC/DOTAP as delivery vector. Luciferase expression was assayed after 24 h and normalized to protein concentration. Significant difference was observed between Ext SV40-PNA/DNA conjugates and DNA alone (*p < 0.035). Results are shown as a mean and standard deviation from at least three samples.

technology but it is dependent on the type of NLS peptide used. Polyplex Formation Using NLS Peptides. A second strategy to enhance gene delivery was investigated using NLS peptides to condense plasmid DNA into discrete particles and mediate transfection in mammalian cells. The ability of NLS peptides to condense DNA was examined using the ethidium bromide condensation and agarose gel retardation assays. Two different controls were used, an oligolysine peptide consisting of 18 lysine residues (K18) and a 7.5 kDa PLL polymer (PLL). K18 was of similar molecular weight to the smaller NLS peptides such as the SV40 peptide, whereas PLL was used as a control for larger peptides resulting from any dimerization of NLS peptides via disulfide bridges between the terminal cysteine residues. Plasmid DNA was efficiently condensed by PLL (Figure 6A) and K18 (data not shown) with an almost complete loss of fluorescence (∼90%) at an N:P ratio of between 1 and 2. The HTLV

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Figure 6. Polyplex formation using NLS peptides. (A) EtBr condensation assay with the following peptides: M9 (filled squares), Ext SV40 (filled circles), SV40 (filled triangles), HTLV (crosses), and PLL 7.5kDa (empty squares). Results are shown as a mean and standard deviation from at least three samples. (B) Agarose gel electrophoresis of plasmid DNA complexed with the indicated NLS peptide at N:P ratios of 1.0, 2.0, 5.0, and 10.0. The control lane (-) contains plasmid DNA alone. The relative locations of wells and free DNA are indicated.

peptide also produced a similar reduction in fluorescence at an N:P ratio of 2 to 3. In contrast, the SV40, Ext SV40 and M9 peptides were relatively poor at condensing DNA with only a 15-40% reduction in fluorescence at an N:P ratio of between 5 and 10. Similar trends were observed using the agarose gel retardation assay where DNA was retained in the wells at an N:P ratio of 1 to 2 with PLL (Figure 6B), K18 (data not shown), and HTLV peptides (Figure 6B), whereas free DNA was still observed with the SV40, Ext SV40, and M9 peptides even at an N:P ratio of 10. These results indicate that the HTLV peptide was the only NLS peptide capable of fully condensing DNA. Photon correlation spectroscopic analysis provided additional evidence that the HTLV peptide efficiently condensed plasmid DNA with polyplexes observed less than 90 nm in diameter at an N:P ratio of 10 (data not shown). Transfection Activity of NLS Peptide/DNA Polyplexes. PC-3 cells were transfected with NLS peptide/ DNA polyplexes formed at an N:P ratio of 10 and luciferase gene expression assayed after 24 h to determine transfection activity (Figure 7A). No significant gene expression was observed using polyplexes formed with the SV40, Ext SV40, or M9 peptides in the presence of 100 µM chloroquine compared to cells alone (Figure 7A). This result was most likely due to the inability of these peptides to condense DNA. In contrast, HTLV/DNA polyplexes mediated levels of gene expression (1.4 × 106 RLU/mg protein) that were approximately 186-fold higher

Figure 7. Transfection activity of NLS peptide-based polyplexes. (A) PC-3 cells were transfected with 0.5 µg pGL3con complexed with M9, SV40, Ext SV40, and HTLV peptides at an N:P ratio of 10, unless otherwise indicated, in the absence (light gray bars) or presence (dark gray bars) of 100 µM chloroquine. DOTAP/DNA complexes (black bar) were used as the positive control. (B) PC-3 cells were transfected with either 0, 0.1, 0.25, or 0.5 µg of pGL3con complexed with HTLV peptide (filled diamonds), PLL 7.5 kDa (filled triangles), and K18 (filled squares) at an N:P ratio of 10 in the presence of 100 µM chloroquine. The fold differences in levels of expression achieved using the HTLV peptide compared to PLL (grey bars) or K18 (empty bars) are shown in the inset. Luciferase expression was assayed 24 h after transfection and normalized to protein concentration as in (A). (C) Cytotoxicity of NLS peptide-based polyplexes. PC-3 cells were incubated for 5 h with 0.5 µg of pGL3con complexed with the HTLV peptide, K18, and PLL 7.5 kDa at an N:P ratio of 10 in the presence of 100 µM chloroquine. MTS reagent was then added to the cells and the absorbance at 490 nm measured. Results are shown as a mean and standard deviation from at least three samples.

than cells alone. In the absence of chloroquine, significant levels of reporter gene expression (2.5 × 105 RLU/mg protein) were still observed but were 5.6-fold lower indicating the requirement for the endoosmolytic reagent.

Strategies To Enhance Nonviral Gene Delivery Using NLS Peptides

Similar levels of expression were observed using HTLV/ DNA polyplexes formed at an N:P ratio of 5. However, no transgene expression was observed at an N:P ratio of 1, reflecting the incomplete condensation of DNA at this N:P ratio. The transfection activity of HTLV/DNA polyplexes was also compared against those formed with K18 and PLL, which are both capable of condensing DNA efficiently. Measurement of luciferase expression 24 h after transfection demonstrated that the HTLV peptide mediated levels of transgene expression approximately 4.7-14.7fold greater than PLL and 9.6-32.3-fold greater than K18 depending on the amount of DNA used (Figure 7B). This difference was not due to cellular toxicity as viability assays showed that there was no toxicity observed using any of the NLS peptides (Figure 7C). Altogether these results indicate that HTLV-based polyplexes mediate efficient transfection of cells at levels that are significantly higher than those achieved with polyplexes based on PLL of similar molecular weight. DISCUSSION

A number of cellular barriers restrict transgene expression but inefficient transfer of DNA from the cytoplasm to the nucleus has been identified as the major contributory factor with only 0.1% of naked DNA or 1% of polyplex DNA reaching the nucleus following microinjection into the cytoplasm (22). A common strategy to enhance nuclear delivery of DNA has been to incorporate synthetic NLS peptides into gene delivery vectors that bind intracellular transport receptors, such as importin R, and utilize the highly efficient physiological protein nuclear import pathways. Many of these approaches have however met with limited success (10, 11, 23, 24), although one study reported a 10-1000-fold increase in gene expression (9). The aim of the present study was to facilitate the development of NLS-based vectors by helping to identify the requirements for NLS-enhanced transfection, including the type of NLS peptide, morphology of DNA and method of incorporating the NLS peptide. Two different strategies using a range of NLS peptides were evaluated in order to enhance nonviral gene delivery. In the first approach we inserted PNA-binding site oligonucleotides into the plasmid pGL3con and demonstrated site-specific conjugation of NLS peptides via a PNA-maleimide clamp. A major advantage of this approach was that, unlike a number of previous studies (10, 11), NLS peptide-modified DNA did not have diminished transcriptional capacity. In addition, we were able to show in vitro binding of the Ext SV40-PNA/DNA conjugate to an importin R1-GST fusion protein and a corresponding 7-fold increase in reporter gene expression using linear DNA. This increase in gene expression was comparable to the 2-8-fold increases observed in the majority of related studies (8, 13). Whereas, there was no significant improvement in gene expression observed using other NLS peptides such as HTLV or M9. The number of NLS peptides required to enhance nuclear delivery of DNA, whether circular or linear, is the subject of controversy with some researchers favoring a single NLS and others preferring as great a number of peptides as possible. It has been proposed that a single or cluster of NLS peptides attached to one end of linear DNA is sufficient for nuclear import and that more may actually hinder the process (8, 9). This hindrance could in principle occur through multiple NLSs causing docking of the DNA to several nuclear pore complexes and has been calculated to be physically possible with DNA over

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1 kb in size (25). In this study, a mixed population of NLS peptide-PNA/DNA conjugates was produced containing a cluster of different numbers of the NLS peptide, and it was estimated using a fluorescently tagged HTLV peptide that on average 1.26-1.6 NLS peptides were bound to each plasmid. Enhanced transfection was only observed, however, when the Ext SV40 peptide was conjugated to DNA, indicating that the type and length as well as the number of NLS peptides play an important role in promoting nuclear delivery. Initial binding studies demonstrated a strong interaction between the NLS peptides with their corresponding intracellular receptors. In the case of the SV40 and Ext SV40 peptides, for example, this interaction was highly specific as a single mutation from a K to T residue abolished binding. Surprisingly, the HTLV peptide bound with a similar affinity to all of the receptors examined, which may have been a nonspecific interaction as this peptide is highly positively charged. Transportin 1, however, has 24% sequence homology with importin β1 (26) and possesses two distinct, nonoverlapping cargobinding sites, one for M9-like sequences, and one for more basic sequences (27). The HTLV peptide may have therefore bound to the second cargo site on transportin 1. A likely explanation for the lack of NLS activity observed with the HTLV and M9 peptides was their apparent loss in ability to interact with transport receptors following conjugation to DNA. There was also relatively weak binding observed between the Ext SV40PNA/DNA conjugate and importin R1-GST fusion protein. This lack of binding was most likely due to electrostatic interaction between the negatively charged phosphate groups of DNA with the cationic NLS peptides. The positive charges of the lysine and arginine residues of many NLS peptides are critical for interaction with transport receptors, and their neutralization would likely hinder cargo recognition. Possible ways to overcome this problem include increasing the spacer length separating the NLS peptide from the DNA or to increase the number of NLS peptides attached to the DNA. Ciolina et al., for example, showed that increasing the number of SV40 NLS peptides attached to DNA from 45 to 100 also resulted in enhanced binding of DNA to importin R1-GST (10). An advantage of the PNA technology used in this study is the possibility to increase the number of NLS peptides attached to the DNA by insertion of additional PNA-binding sites. However, it is likely that to achieve maximum NLS-mediated transfection a balance will be required between improving the interaction of NLS peptide/DNA conjugates to transport receptors and the proposed hindrance of binding to multiple nuclear pore complexes (9). An important consideration throughout this study was to fully characterize and purify NLS peptide-PNA/DNA conjugates prior to biological evaluation. The presence of unconjugated NLS peptides, for instance, may compete for binding to transport receptors. NLS peptides were therefore attached to PNA/DNA conjugates in the presence of high salt conditions to avoid nonspecific interactions and unreacted peptides removed by ethanol precipitation of the DNA. A fluorescence-based assay was also developed to analyze binding of PNA to DNA indicating that at least five binding sites were required for efficient PNA binding (>80% of DNA bound). No significant PNA binding was observed with plasmids containing just one site or to linear DNA (data not shown). Although some short PNAs can interact stably with their complementary sequence (28), weaker binding of PNA to linear DNA compared to circular DNA has been

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previously observed by up to two to 3 orders of magnitude (29). In addition, secondary structure of the plasmid may hinder binding of PNA to DNA which is dependent on the exact sequence of the plasmid DNA used (30). The morphology of the DNA used was also observed to be an important factor in this study to influence NLSenhanced transfection. Initial transfection results showed a modest 2-fold increase in gene expression using the SV40 peptide attached to PNA/circular DNA (data not shown). However, the level of gene expression was enhanced to 7-fold compared to unmodified DNA when the NLS peptide-PNA/DNA conjugate was restricted with the enzymes KpnI and BamHI to produce a linear DNA molecule with the cluster of NLS peptides at one end. This is in agreement with previous studies that have shown enhanced gene expression using linear DNA with NLS peptides following ligation of an oligonucleotideNLS conjugate (9) or incorporation of a modified nucleotide for chemical linkage of the NLS peptide (8). It is also still not clear what delivery vectors should be used with NLS peptide/DNA conjugates to properly exploit the NLS-transport pathways. Previous studies have observed enhanced gene expression with NLS peptide/DNA conjugates using cationic lipids and PEI (9). However, the intracellular trafficking and location of DNA release from these types of vectors are thought to differ greatly, with cationic lipids releasing DNA into the cytoplasm and PEI/ DNA polyplexes remaining largely intact (31, 32). PEI might even be expected to hinder NLS activity by masking the peptide and preventing interaction with transport receptors. In this study we used a reducible polycation/lipid combination that has recently been shown to facilitate release of nucleic acids into the cytoplasm and is therefore likely to promote availability of NLS peptides for interaction with transport receptors (18). In the second approach we examined the ability of NLS peptides to condense DNA and mediate transfection. Of all the NLS peptides examined, the HTLV peptide was the most efficient at condensing DNA and mediated transfection at levels 5-32-fold higher than control polyplexes formed with PLL of similar molecular weight. Chloroquine potentiated the transfection activity of HTLV/DNA polyplexes by 5-10-fold, an expected result, as the HTLV peptide does not contain any lytic sequences known to mediate endosomal escape. This is the first study to the best of our knowledge that has demonstrated efficient transfection mediated by a peptide based on the HTLV sequence. The inability of the SV40 and M9 peptides to mediate transfection is probably due to the lack of positively charged amino acids in their sequence since a minimum length of six to eight cationic amino acids are known to be required to compact DNA into structures active in gene delivery (33). It still remains to be investigated whether the increased level of transfection observed using the HTLV peptide is NLS-mediated or due to some other mechanism. The arginine-rich sequence of the HTLV peptide is similar to many protein transduction domains (PTDs), such as that in HIV TAT (34), which mediate cross-membrane transfer and are also thought to have nuclear homing capacity (35). Therefore, it is possible that the HTLV peptide used in this study is functioning as a nuclear homing PTD. CONCLUSION

Based on these results, a number of factors were identified that directly influence the ability of NLS peptides to enhance nonviral gene transfer. These included the type of NLS peptide, morphology of DNA, and

Bremner et al.

method used to incorporate NLS peptides into nonviral vectors. Highly cationic NLS peptides such as the HTLV sequence were most effective at condensing DNA and enhanced levels of gene expression compared to polylysine controls. Peptides based on the SV40 but not the M9 and HTLV sequences were able to promote gene expression when attached to the ends of linear DNA. Proper characterization of NLS peptide/DNA conjugates was also an important consideration in evaluating the transfection capability of NLS-modified vectors. Further development of NLS-modified vectors is required before they can be used routinely; however, these results help to define the requirements for NLS-modified vector design and construction. It may also prove necessary to include additional components used by viruses to achieve effective nuclear delivery, such as the triple-stranded DNA flap for HIV-1 genome nuclear import (36), before more efficient nonviral delivery systems can be developed. ACKNOWLEDGMENT

We express our gratitude to Dr. Y. Yoneda (Osaka University, Japan) for the pGEX-2T plasmids, Dr. G. Dreyfuss (University of Pennsylvania) for the pGEX-5X plasmid and Dr. L. Haynes (University of Birmingham, UK) for the GST protein. We also thank Dr. R. Grand (University of Birmingham, UK), Dr. G. McNab (University of Birmingham, UK), and Dr. D. Jans (Monash University, Australia) for useful discussions. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and Cancer Research UK. Supporting Information Available: Western blot analysis of receptor GST fusion proteins. Sequence data demonstrating insertion of oligonucleotides into pGL3con. Tris-Tricine SDS-PAGE analysis demonstrating reactivity of NLS peptides with maleimide-Texas Red. Characterization of NLS peptides by HPLC analysis and mass spectroscopy. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Read, M. L., and Bremner, K. H. (2002) Antisense strategies and nonviral gene therapy for cancer. Expert Opin. Ther. Pat. 12, 379-391. (2) Buckley, R. H. (2002) Gene therapy for SCID-a complication after remarkable progress. Lancet 360, 1185-6. (3) (2003) French gene therapy group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID). J. Gene Med. 5, 8284. (4) Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A., and Welsh, M. J. (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270, 189979007. (5) Brunner, S., Sauer, T., Carotta, S., Cotten, M., Saltik, M., and Wagner, E. (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7, 401-7. (6) Lanford, R. E., Kanda, P., and Kennedy, R. C. (1986) Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell 46, 575-82. (7) Bremner, K. H., Seymour, L. W., and Pouton, C. W. (2001) Harnessing nuclear localization pathways for transgene delivery. Curr. Opin. Mol. Ther. 3, 170-7. (8) Ludtke, J. J., Zhang, G., Sebestyen, M. G., and Wolff, J. A. (1999) A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA. J. Cell Sci. 112 (Pt 12), 2033-41.

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