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Published as doi: 10.1096/fj.07-093765.
 (The FASEB Journal. 2008;22:2097-2107.)
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Nuclear-targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo

Matthieu D. Lavigne*,1, Laura Yates*, Peter Coxhead{dagger} and Dariusz C. Górecki*,2

* School of Pharmacy and Biomedical Sciences, and

{dagger} School of Biological Sciences, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, UK

2Correspondence: Molecular Medicine, Institute of Biomedical and Biomolecular Sciences, St. Michael’s Bldg., White Swan Rd., Portsmouth, PO1 2DT, UK. E-mail: darek.gorecki{at}port.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Poor nuclear entry, especially into nondividing cells, is a limiting factor in nonviral gene delivery. We have engineered a novel chimeric vector relying on the controlled assembly of a TAT-tagged multisubunit DNA binding protein (EcoR124I) with expression plasmids containing the EcoR124I recognition site. Molecular interactions of this molecular assembly were studied by electrophoretic mobility shift assay and atomic force microscopy. Maintenance of nanocomplexes in an appropriate stoichiometric ratio was both necessary and sufficient to produce a significant (>8-fold) increase in the activity of the therapeutic {alpha}-galactosidase A enzyme after intramuscular administration in the mouse model of Fabry disease. To our knowledge, this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle. Coinjection with pluronic SP1017 produced further enhancement of gene expression, demonstrating cumulative effects of the increased nuclear delivery by TAT chimeras and transcription activation by the pluronic. Cell penetration peptides (CPP), such as TAT, have been shown to improve delivery of macromolecules, when linked directly. However, in our system TAT-enhanced targeting took place even though it was linked to the plasmid DNA molecule indirectly via two noncovalent bonds. Therefore, this proof-of principle result indicates that TAT (and potentially other CPP) can be used for targeting modular chimeric vectors and therapeutic nanodevices.—Lavigne, M. D., Yates, L., Coxhead, P., Górecki, D. C. Nuclear-targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo.


Key Words: cell penetration peptides • gene therapy • TAT • pluronic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CLINICAL USEFULNESS OF NONVIRAL methods is still hindered by relatively low gene delivery and transgene expression efficiencies. For any gene supplementation approach (e.g., for treatment of inherited disorders), vectors must reach the nuclei of their target cells, only there the therapeutic gene can be expressed. It is a particularly difficult hurdle for nonviral vectors when used for targeting nondividing cells (neurons and myotubes). Since the discovery that skeletal muscle can be transfected in vivo by intramuscular injection of plasmid DNA, this organ system has attracted considerable attention as a potential source of secreted therapeutic proteins (1) . The safety, simplicity, and low costs of intramuscular injections and the fact that plasmid DNA (pDNA) can be maintained extrachromosomally in the nondividing muscle for months make it a very attractive alternative to other gene-targeting methods (2) . However, most studies so far have shown that targeting and expression in nondividing myotubes are not high enough for therapeutic purposes. Several studies (3) using different nonviral targeting methods showed that only 1% of pDNA internalized into the cells was actually delivered to the nucleus. Similarly, it was demonstrated that a minimum of 106 plasmid molecules have to be injected in order to observe gene expression in mouse myotubes in vivo (4) . Large nucleic acid molecules (the molecular mass of a therapeutic plasmid can vary between 2 and 10 MDa) cannot cross the intact nuclear envelope. In fact, macromolecules larger than 60 kDa require an active transport via interactions of nuclear localization sequences with specific nuclear import proteins (for example, importins; ref. 5 ). One of the main advantages of virus-derived vectors over nonviral systems stems directly from the fact that the viruses can divert the nuclear import machinery of the host cell to their own benefit. However, use of viral vectors is not without serious disadvantages. New physical methods, like pDNA electroporation or hydrodynamic injection (6) , can increase the number of expressing muscle fibers; however, these approaches also have limitations.

An alternative to viral vectors or physical targeting might be the rapidly developing assortment of polymeric vectors and nanoscale devices. We have previously shown that synthetic polymers can be engineered and used to improve nonviral delivery (7 8 9 10) . Furthermore, we have engineered a multisubunit and multifunctional DNA binding protein in order to regulate its activity in biological environments (11 , 12) . We based our system on a hybrid EcoR124I restriction-modification (R-M) enzyme (13) . The native nuclease is comprised of three subunits in a stoichiometric ratio of R2M2S (14 , 15) . The M2S subcomplex is responsible for DNA sequence recognition specificity (GAAnnnnnnRTCG) and DNA methylation (when recognizing hemimethylated DNA), while addition of the R subunit creates a restriction enzyme. This enzyme cuts target DNA at random locations, which can be >20 kbp from the recognition sequence. This is achieved by pulling DNA—a unique feature of this protein (most proteins traverse along DNA rather than pull it). This ATP-dependent translocation is being exploited to create nanoscale molecular motor devices (16) . In our laboratory, we attempt to use it for DNA compartmentalization and targeting. However, lack of efficient methods for targeting such nanoscale devices across cell membranes remains one of the obstacles for their practical application.

Engineering nonviral delivery systems with cell penetration peptides (CPP) or more specialized nuclear localization signals (NLS) could increase the level of entry of large biomacromolecules or nanosize assemblies into the cell nucleus (17) . Docking of NLS triggers conformational changes in the nuclear pore complex leading to the opening of a channel allowing molecules as large as 50 MDa to enter the nucleus. Thus, utilization of the CPP or NLS peptides could allow development of complex (and therefore large) nonviral vectors that would fulfil all the major requirements: target the tissue of interest, protect the genetic material, and produce sufficient level of expression.

The shortest (11 amino acids: YGRKKRRQRRR) and probably the most efficient CPP described to date is the HIV-1 TAT-peptide (aa: 47–57 of tat protein). Since the early report of TAT-mediated delivery of β-galactosidase into mouse tissues (18) , this and also some other CPP have been shown to transport various macromolecules (proteins, liposomes, DNA, and polymers) into mammalian cell nuclei (19 20 21 22 23) . Nevertheless, as far as in vivo applications are concerned, the most successful demonstrations of efficient delivery involved monomeric TAT-engineered recombinant proteins (24 25 26) .

Here, we report the development of a prototypic chimeric device combining the nuclear targeting activity of TAT (expressed in fusion with the M subunit of EcoR124I) and the specific interactions between the multisubunit DNA binding protein (M2S) and a therapeutic plasmid encoding {alpha}-galactosidase A (AGA). The clinical model used here was that of Anderson-Fabry disease. Fabry is an X-linked lysosomal storage disorder (LSD) caused by AGA deficiency (27) . There is evidence from our laboratory and from others that LSDs could be corrected by gene transfer, expression, and enzyme secretion from a subset of cells or a specific tissue (28 , 29) . Our previous data suggested that plasmid vectors could be effective in targeting the AGA gene into skeletal muscle (28 , 30) . However, an insufficiently high level of AGA expression is still an apparent problem. The targeting system described here produced >8-fold increase in therapeutic AGA gene expression after a single injection into skeletal muscle in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of M2S-TAT expression vector
The pJS4M bacterial expression plasmid contains both the M and S sequences driven individually by T7 promoters, which results in coexpression of the two methylase subunits and the production of an active and stable M2S complex (31 , 32) . Polymerase chain reaction (PCR) amplification using a high-fidelity DNA polymerase (KOD-hifi, Novagen, San Diego, CA, USA) and primer set: Fw_NdeI-TAT-hsdM (5'-G GAA TTC CAT ATG TAC GGT CGT AAG AAA CGT CGT CAG CGT CGT CGT ATG AAA ATG ACA AGT ATT-3') and Rv_BamHI_hsdM (5'-ATG GAT CCT CAT TTC TGC ACC TCG C- 3') primers produced the M transcript with the inframe TAT sequence at its 5' end (restriction sites in italics). After extension in the presence of taq polymerase, the products were cloned into pGEMTeasy vector (Promega, Southampton, UK). The correct ~1.7kb insert was liberated using NdeI and BamHI and ligated into pJS4M3 vector digested with BamHI and NdeI (after limited digestion with NdeI). Positive clones were identified, and their accuracy was confirmed by DNA sequencing.

Purification of M2S and M2S-TAT proteins
The BL21(DE3) cells transformed with the pJS4M3 or pJSTATM plasmids were grown in Luria-Bertani (LB) supplemented with Ampicillin (150 µg/ml) until optical density (OD) reached 0.4–0.6. The cells were centrifuged (4500 rpm, 15 min) and washed in 10 mM Tris-HCl, l00 mM NaCl, pH 8.2, and the pellets were stored at –20°C. Cells were resuspended in the freshly prepared lysis buffer [50 mM Tris-HCl, pH 8.2; 25% sucrose; 5 mM EDTA; 3 mM dithiothreitol (DTT); 1 mM benzamidine; and 100 µM PMSF] and disrupted by sonication (with careful temperature monitoring). After centrifugation (4500 rpm, 15 min), the supernatant was adjusted to 250 mM NaCl and ultracentrifuged (50,000 rpm, 2 h). Supernatant fractions were mixed with 5x protamine sulfate buffer (PSB; 10 mg/ml protamine sulfate, 50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, and 0.25 M NaCl) adjusting volumes to obtain 1x PSB final, and the suspension was stirred gently for 30 min at 4°C. The nucleic acids were removed by centrifugation (10,000 rpm, 20 min), and the proteins in the supernatant were precipitated by saturating the solution with 44% (w/v) ammonium sulfate and stirring, followed by centrifugation (10,000 rpm, 20 min). The pellets were redissolved on ice in the freshly prepared (filtered and degassed) buffer A (10 mM Tris-HCl, pH 8.2; 1 mM EDTA; 100 mM NaCl; and 3 mM DTT). The sample was run on a desalting column (HiTrap Desalting, 1.5 ml, GE Healthcare, Little Chalfont, UK) equilibrated with buffer A using a protein purifier (ÁKTAprime Plus, GE Healthcare). Protein fractions were pooled and applied onto a diethylaminoethyl (DEAE) sepharose column (HiTrap DEAE FF, 5 ml, GE Healthcare) preequilibrated with buffer A. The bound proteins were eluted from the column using a linear gradient of NaCl (100–700 mM) by gradually replacing buffer A with buffer B (10 mM Tris-HCl, pH 8.2; 1 mM EDTA; 1 M NaCl; and 3 mM DTT). The M2S protein-containing fractions were identified by SDS-PAGE. Positive fractions were pooled and diluted in buffer A followed by purification on the HiTrap Heparin HP (GE Healthcare). The bound proteins were eluted with a linear gradient of NaCl in buffer B, and the purified samples were concentrated to ~1 mg/ml using a Centricon ultrafiltration device (30,000 molecular-weight cutoff, Millipore, Watford, UK). The proteins were stored for a short time at +4°C or for up to several months at –20°C in glycerol. All chemicals were from Sigma (Poole, UK) except when stated otherwise.

Construction of reporter plasmids pX61TH and pCS2TH
The plasmid pX61 (AGA expression cassette driven by human CMV IE promoter; refs. 28 , 30 ) has been modified to include the EcoR124I enzyme recognition site. The pBendEcoR124I (gift from Darren Mernagh, University of Portsmouth, Portsmouth, UK) plasmid was digested with MluI, and the resulting fragment containing the EcoR124I site (GAATTCGAGGTCG) was cloned into pX61 digested with MluI. The pCS2 vector [green fluorescent protein (GFP) expression cassette] was modified to include the EcoR124I site by PCR amplification of pX61TH using a primer set F1055-NsiI 5'-AGGTAAATGCATTAAGCTACAACAAGGGGCT-3' and R1513-NsiI 5'-GCAGTTATGCATCGTCAATGGAAAGCTCCTATTG-3', followed by digestion and cloning of the resulting product into the NsiI site in the reporter vector backbone. All modifications were confirmed by DNA sequencing.

Nondenaturing electromobility shift assay (EMSA)
M2S-TAT (stock solution at 0.67 µM) and M2S (stock solution at 1 µM) were mixed in various amounts adjusted to obtain final molar protein-to-DNA ratios ranging from 0.2 to 2.5 with the 5x EMSA binding buffer solution (50% glycerol; 125 mM NaCl; 250 mM Tris-HCl, pH 8.0; 50 mM MgCl2; and 5mM DTT). Samples were incubated for 10 min at room temperature, before addition of the end-labeled fluorescent oligomer duplexes (final concentration of 2 µM) containing the EcoR124I recognition sequence (Invitrogen, Paisley, UK). It should be noted that he fluorophore (FITC) was included only on the forward primer (FITC-5'-CCG TGC AGA ATT CGA GGT CGA CGG ATC CGG-3'). Samples were incubated another 10 min at room temperature, loaded onto 6% acrylamide/bisacrylamide (19:1) gel, and run at 100 V at +4°C. The gel was rinsed with H2O before being analyzed in a phosphorimager (Fuji, Bedford, UK) at 550 V at 473 nm with an LPG filter.

Atomic force microscopy (AFM) studies
Samples of M2S-TAT/pDNA and M2S/pDNA complexes were prepared freshly in deposition buffer (containing HEPES and MgCl2), and suspensions were incubated on mica for 10 min to allow maximal immobilization of complexes before analysis (33) . A NanoScope IV/MultiMode AFM (Veeco Instruments, Santa Barbara, CA) with a J-scanner (max. x, y, z-translation=200, 200, 16 µm3) was used throughout this study. TappingMode imaging was performed in air using silicon cantilevers (Type NSG01, resonant frequency=150–190 kHz, spring contant, K=8–10 N/m; NDT-MDT, Moscow, Russia) with integrated tips at a resolution of 512 x 512 pixels and a scan rate of 3.05 Hz. Postcapture image analysis was conducted using NanoScope software (Version 6.12r1, Veeco Instruments, Santa Barbara, CA, USA).

In vitro cell transfection assays
COS-7 monkey kidney fibroblasts were maintained at 37°C in a humidified atmosphere containing 5% CO2, in 75 cm2 tissue culture flasks (Nunc, Loughborough, UK) in Dulbecco’s modified Eagle’s medium supplemented with L-glutamine (8 mM), penicillin (200 U/ml), and streptomycin (0.2 mg/ml; all Sigma), and 10% (v/v) fetal calf serum (Invitrogen). Cells (2x105) were plated in 6-well plates 24 h before the transfection.

For studies in growth-arrested cells, COS-7 (~2.5x105) were cultured in medium with 5 µg/ml Aphidicolin (Sigma). After transfections, cells were maintained in Aphidicolin-containing medium until analyzed, as described below.

Four micrograms of endotoxin-free plasmid DNA pCS2TH containing a GFP eukaryotic expression cassette and 1.2 µl M2S-TAT were mixed to cover the 1:1-to-1:2 M ratios, incubated briefly, and diluted in Optimem (Invitrogen); subsequently, some samples were complexed, if required, with Lipofectamine (Invitrogen) at room temperature for 20–30 min.

The cells were washed with sterile PBS, overlaid with the transfection mixtures (diluted to 1 ml/well in OPTIMEM), and incubated for 4 h at 37°C with 5% CO2. After that period, it was replaced with complete medium. To evaluate the efficiency of gene expression, cells were analyzed by flow cytometry 48 h post-transfection (FACSCalibur, Becton-Dickinson Biosciences, San Jose, CA, USA). The monitoring of GFP expression and cell death was performed in two different channels: GFP ({lambda}ex=488 nm/{lambda}em=520 nm) and propidium iodide ({lambda}ex=530 nm/{lambda}em=620 nm), respectively. We analyzed 20,000–100,000 events per sample. Flow cytometry data were expressed as mean ± SE, where n > 4. For tracing studies, the expressions in live cells were analyzed ({lambda}ex=488 nm/{lambda}em=520 nm) and images were obtained using confocal fluorescence microscopy (LSM 510 Meta, Zeiss, Oberkochen, Germany) with an x40 water immersion objective.

Analysis of the therapeutic gene expression in vivo
Fabry (AGA knockout) mice (34) or C57Bl6 were used in accordance with the UK Home Office Guidelines and with the approval of the Institutional Ethical Review Board. High-purity, endotoxin-free plasmid preparations were carried out using an Endofree Maxi Kit (Qiagen, Crawley, UK). M2S-TAT/pDNA and M2S/pDNA complexes were prepared as described above at the required protein-to-DNA ratios (1:1 and 1:2.5).

Anesthetized 4-wk-old mice were injected with the naked plasmid DNA solution or with plasmids complexed with protein (M2S-TAT, or M2S, or iM2S-TAT) at the required protein-to-DNA ratios (1:1 and 1:2.5) in sterile saline solution. A 27G1/2 needle fitted with a plastic collar to maintain depth of injection constant was used. The standard injection had a volume of 25 µl (20 µg of pDNA); it was administered aseptically into both tibialis anterior muscles.

Pluronic SP1017 solution (lot number SP1017–2; Supratek Pharma, Laval, QC, Canada) was combined with M2S-TAT/pDNA and gently mixed to get the required final concentrations of DNA (20 µg) and 0.01% w/v of SP1017 in 25 µl saline. The formulations were used immediately for injection. In some experiments, the same amount of SP1017 was injected 24 h postinjection of M2S-TAT/pDNA. Cyclophosphamide (Baxter Healthcare, Thetford, UK) was administered intraperitonealy at 150 mg/kg on days –1, +2 postinjection of constructs. At specific times postinjection, muscles were removed, flash-frozen in liquid nitrogen, and ground, and the tissue powder was resuspended in the reporter lysis buffer (RLB) (Promega, Southampton, UK). After brief centrifugation, supernatants were used for further analyses: the protein concentrations were measured using the bicinchoninic acid kit (Sigma), and the AGA enzymatic activity was measured fluorimetrically as described before (28 , 30) . The data are presented as means ± SE. All results were analyzed using ANOVA with Bonferroni’s multiple comparison test, and P < 0.05 was considered as statistically significant.

Serum levels of anti-hAGA antibodies were measured using ELISA, as described previously (30) . Briefly, Maxisorp plates (Nunc) were coated with recombinant human AGA (0.5 µg/well). Each sample was measured in duplicate. Bound anti-hAGA antibodies were detected using horseradish peroxidase-conjugated sheep anti-mouse IgG (1/500) and a substrate mix containing 3,3',5,5'-tetramethylbenzadine (TMB) and H2O2 (Sigma). Titers were defined as the reciprocal of the highest dilution of serum that produced an OD450 equal to 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In an attempt to engineer a nanoscale DNA targeting and compartmentalization device, we were trying to exploit the specific characteristics of the recombinant EcoR124I DNA binding protein combined with advantages offered by "smart" synthetic polymers responsive to biological stimuli. We have previously modified this multisubunit and multifunctional protein in order to regulate its activity in biological environments (11 , 12) and demonstrated that smart polymers can be designed and used to improve nonviral gene targeting (7 8 9 10) . Here, as a proof-of-principle we show that CPP can be used to target complex, hybrid macromolecular systems into nondividing muscle fibers in vivo.

Engineering, expression, and molecular characterization of the chimeric vector
To assess the potential of TAT-mediated targeting of complex macromolecular assemblies, we first engineered the M subunit of the EcoR124I protein. The TAT-encoding sequence has been cloned in frame with the M cDNA sequence at its 5' end using PCR amplification with modified primers followed by ligation of the resulting ~1.7 kb fragment between the second NdeI and BamHI sites of the pJS4M3 M2S expression vector (Fig. 1 A).


Figure 1
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  		Figure 1. A) Schematic representation of the pJSTATM, M2S-TAT expression vector and the major purification steps producing M2S-TAT protein (for details see Materials and Methods). Inset: sequence alignment between pJSTATM and its parent pJS4M3 plasmid demonstrating the insertion of the TAT cDNA in frame with the 5'-end of the M subunit. B) SDS-PAGE analysis of the wild-type (Mtase) and the TAT-tagged version (TAT-Mtase). Coomassie detection of various amounts of protein, with two clear S and M bands (~59 and ~46 kDa respectively) present. Note a small shift in the electrophoretic mobility of the TAT-M subunit consistent with ~1.5 kDa mass difference due to the TAT-tag.

The recombinant fusion protein M2S-TAT and control M2S (Mtase) have been expressed and purified. A modified version of the protocol described previously (32) was adopted with sequential purification on DEAE-Sepharose followed by heparin column chromatography. The two subunits of M2S eluted together as a complex as the columns were run under nondenaturing conditions. However, M2S and TAT- M2S eluted at different salt concentrations, probably due to the affinity of TAT peptide for heparin (data not shown).

Analysis of protein extracts by SDS-PAGE revealed two strong bands corresponding to the TAT-M and the S subunits of molecular mass ~59,000 and ~46,000 Da, respectively (Fig. 1B ). A small shift in the electrophoretic mobility of the TAT-M subunit was observed, consistent with an ~1.5 kDa mass difference for the TAT-tagged version.

The wild-type Mtase protein is constituted of one S and two M subunits associated as a trimer (M2S). This complex is able to bind a specific DNA recognition site (GAAN6RTCG) and transfer a methyl group onto the second adenosine residue of this sequence. Without the presence of a methyl donor molecule (such as S-adenosyl-methionine), the Mtase complex is acting only as a DNA binding protein. In the native enzyme in vivo, the presence of a third component, the R subunit, renders the M2S a fully functional Type I restriction and modification system (11 , 35) . The functional interaction of the TAT-modified M2S with DNA was assessed by EMSA. The mobility of the fluorescently labeled oligonucleotide was affected by the presence of increasing concentrations of both proteins. Figure 2 B shows that binding of TAT-M2S to DNA is very close to stoichiometric, as all the DNA is retarded above a protein-to-DNA ratio of 1. This was very similar to the interaction of the native M2S. Addition of the R subunit created a supershift of the retarded band on the gel (Fig. 2B ; R/M2S-TAT). This showed that addition of the TAT peptide at the N terminus of the M subunit did not interfere with the proper functional complex assembly. Heat inactivation (Fig. 2B ; iM2S-TAT) prevented DNA binding, confirming that this specific property of this protein depends on the presence of intact M2S complexes.


Figure 2
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  		Figure 2. Analyses of DNA binding by M2S-TAT and the native M2S. A) Schematic representation of the complex between one molecule of pX61TH plasmid vector and the M2S-TAT complex via protein-DNA interactions. B) EMSA. Note the shift of the specific fluorescent DNA probe containing the EcoR124I recognition sequence by both M2S-TAT and M2S at protein/DNA ratios >1:1. No displacement occurred when M2S-TAT was inactivated (lane iTAT-Mtase). Addition of the R subunit caused supershift (lane R/M2S-TAT) confirming proper interactions between the TAT-tagged M2S and the restriction subunit. C, D) Tapping mode AFM images showing M2S-TAT/pDNA complexes prepared at 1:1 ratio (C) or pDNA only (D). Note the presence of a single M2S-TAT complex associated with one pDNA molecule. Comparison of height profiles confirms the specificity of the complex (4:1) in comparison with single string of DNA (1:1) and the supercoiled double string of DNA (2:1).

Further characterization of the interactions of M2S-TAT with DNA and specifically with the therapeutic plasmid pX61TH (encoding AGA) was performed using AFM. Figure 2C, D shows interactions between M2S-TAT and pX61TH. This "bead on a string" structure was observed for the complexes made at 1:1 ratio. The height of the peak corresponding to the "bead" (~4 nm) is more than twice that of a single DNA molecule (~1 nm), while two overlapping "strings" of a supercoiled pDNA give a predicted height of ~2 nm. Freshly prepared complexes showed structures ranging in size from 2–5 nm when supercoiled and complexed with M2S-TAT and were ~500 nm in length if completely stretched. When the R2M2S complex was used, it increased the size of the bead structure significantly in agreement with the larger molecular size of this complex (data not shown). Moreover, in keeping with the EMSA results, there was no evidence of nonspecific binding of M2S-TAT to pX61TH, as only one protein molecule per pDNA molecule could be observed, even at higher M2S-to-pDNA ratios (data not shown).

Enhanced gene expression in cells transfected with M2S-TAT/pDNA complexes
Having demonstrated that the M2S and M2S-TAT proteins were fully functional in terms of their abilities to form complexes with pDNA, we decided to carry out transfection assays at an optimal protein-to-pDNA ratio (1:1) in order to evaluate the effects on gene expression. In vitro transfection experiments were performed using M2S-TAT complexed to pCS2TH plasmid DNA encoding the GFP reporter gene. The initial analysis using fluorescent microscopy showed early GFP expression in a subset of COS-7 cells 24 h post-transfection. This signal was enhanced 48 h post-transfection (data not shown).

For multiple quantitative assays, flow cytometry was adopted, which enabled accurate quantification of the number of GFP-expressing cells and the mean intensity of expression and also provided assessment of the toxicity of the various complexes used, expressed as the number of dead (propidium iodide stained) cells.

There was higher overall transfection efficiency for pCS2TH with M2S-TAT (Fig. 3 A, B) with about 50% more cells expressing GFP (P<0.01) and a >35% (P<0.01) increase in mean GFP intensity when compared with Lipofectamine-pDNA complexes. Moreover, M2S-TAT complexes did not produce any increase in cell death when compared with controls. Interestingly, however, the pCS2TH-M2S-TAT complex alone was unable to transfect cells, as GFP expression occurred only when it was used in combination with Lipofectamine (Fig. 3B ).


Figure 3
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  		Figure 3. Analysis of GFP gene expression using M2S-TAT system in vitro. A) Examples of flow cytometry plots demonstrating GFP expression and cell death in control cultures and cells transfected with plasmid/Lipofectamine and combined with M2S-TAT (as described in Materials and Methods). B) Summary graph of the flow cytometry data. Complexation with M2S-TAT significantly enhanced GFP expression only when used with Lipofectamine. P < 0.01. Values represent averages of three individual experiments.

Enhanced gene expression after skeletal muscle targeting in vivo
The safety, simplicity, and low cost of intramuscular injection and the fact that plasmid DNA can be maintained for a long time in the nondividing muscle make targeting and expression of secreted therapeutic proteins in this organ system a very attractive proposition. We are developing this method in order to produce therapeutic levels of AGA, the enzyme missing in the Anderson-Fabry disease.

Here we have used the Fabry mice model to test the targeting efficacy of the novel hybrid vector in muscles in vivo. The hAGA expression plasmids: pX61TH (with M2S binding site, Fig. 2A ) and the control pX61 were prepared with or without complexation with active (M2S-TAT, M2S) or inactivated (iM2S-TAT) proteins. The pX61 plasmid series has been shown before to produce significant levels of biologically active AGA after its intramuscular injection (28 , 30) . Here, the pDNA ± (M2S±TAT) complexes were prepared in saline (20 µg pDNA/25 µl saline) using 1:1 and 2.5:1 protein-to-DNA molar ratios, as established before by EMSA and AFM, and injected into Fabry mice muscles (tibialis anterior).

One week postinjection, AGA activity levels were assessed (Fig. 4 ). First, it was confirmed that the insertion of EcoR124I sequence in the pX61TH plasmid backbone did not affect the temporal patterns or the levels of expression, as the AGA activity 1 wk postinjection was comparable for both constructs (data not shown).


Figure 4
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  		Figure 4. AGA enzymatic activity in Fabry mice after skeletal muscle targeting in vivo. The AGA activity in muscle extracts (nmol/h/mg of protein) 1 wk postinjection with 20 µg of pX61TH combined with M2S-TAT or M2S at two different molar ratios. Statistical analysis of the data showed a very significant overall difference between M2S-TAT/pX61TH at the optimal (1:1) ratio and all the other treatments (#P<0.001). All the other treatments were comparable one to another (P<0.001), demonstrating that the effect depends on the optimal M2S-TAT to plasmid ratio or the presence of active M2S-TAT and therefore the enhancement is triggered by the specific complexes. All values are presented as mean ± SE for n samples in each experimental group.

Importantly, complexation of pX61TH with M2S-TAT had a critical effect on AGA expression levels. The AGA activity was increased ~8 times with complexes made at a 1:1 protein-to-plasmid ratio. Interestingly, at a 2.5:1 ratio, the enhancing effect was significantly lower, as it only produced 2-fold increases in the AGA activity levels in comparison to the naked plasmid.

To further characterize the mechanism of such an increase in gene expression, the plasmid pX61TH was complexed with the wild-type M2S (at both optimal 1:1 and suboptimal 2.5:1 ratios). In this case, the levels of AGA activity remained unchanged with respect to those obtained with pDNA only (Fig. 4) . Moreover, if M2S-TAT was inactivated (as confirmed by its inability to bind DNA in EMSA, see lane iTAT-Mtase in Fig. 2B ) before being complexed with pDNA (either pX61TH or pX61), the AGA activity levels did not increase significantly, as compared with plasmid alone. This confirmed that the increase in gene expression was not caused by the presence of the TAT peptide alone. M2S-TAT with pX61 (without M2S binding site) was also ineffective (data not shown). Overall, the highly significant increase in gene expression in this experimental paradigm was observed only for the complexes formed between fully functional M2S-TAT and the pX61TH (M2S binding) plasmid at the molar ratio of 1:1. Interestingly, although human AGA is a potent antigen in the knockout mouse (30) , the presence of M2S-TAT complexes did not cause any increase in the immune response to AGA (see Supplemental Data).

Pluronic block copolymer enhances expression of the M2S-TAT-targeted gene
To test whether the enhanced AGA expression in muscle is caused by increased plasmid targeting into the nucleus, we used pluronic SP1017. Pluronic block copolymers increase expression of plasmids already located within the nucleus of muscle cells. Their mechanism of action depends on promoter transactivation, particularly of promoters containing the nuclear factor-{kappa}B (NF-{kappa}B) binding site (e.g., hCMV; refs. 30 , 36 ). The pX61 is driven by CMV promoter and is known to be responsive to pluronic up-regulation (30) .

We have therefore compared AGA expression levels when combining pX61TH ± M2S-TAT with pluronic SP1017–2 at (0.01%). It was fist confirmed that pX61TH was sensitive to pluronic enhancement, as AGA activities were increased ~5-fold (Fig. 5 A), and this effect was comparable to the values reported before for pX61 (30) . Crucially, we observed an additional significant increase (~9-fold) in AGA activity levels when M2S-TAT/pX61TH complexes were coinjected with this polymer (Fig. 5A ).


Figure 5
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  		Figure 5. Pluronic block copolymer enhances expression of the M2S-TAT targeted gene. The relative AGA activity in muscle extracts (represented as the percentage of the value obtained for the naked pX61TH plasmid). A) 1 wk postinjection of plasmid combined (1:1) with M2S-TAT there was a significantly higher enzymatic activity when compared with plasmid alone or to that of plasmid combined with SP1017 pluronic polymer. Combination of M2S-TAT and SP1017 produced a further very significant increase of AGA activity. B) The enhancement by pluronics was also observed when SP1017 had been injected with 24 h delay but coadministration of cyclophosphamide (Cy) abolished the NF-kB dependent enhancing effect of SP1017. All values are presented as mean ± SE.

Importantly, in order to rule out the possibility that pluronics act as a pDNA cocarrier rather than as biological response modifiers, we confirmed that SP1017 enhanced AGA expression even when it was injected separately (24 h after plasmid administration) at the time when plasmid would have already reached the nucleus (Fig. 5B ). It should be noted that the difference between this delayed injection and coinjection with pDNA in Fig. 5B was statistically insignificant (P=0.28). Moreover, the enhancement by pluronics was prevented by administration of cyclophosphamide (+Cy; Fig. 5B ), a specific NF-{kappa}B pathway inhibitor confirming the effect of SP1017 being mediated via NF-{kappa}B responsive elements on the hCMV promoter. This result indicates that M2S-TAT increases the nuclear targeting of pX61TH plasmid DNA, which is then expressed more efficiently in the transactivating presence of pluronics.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gene delivery into the nucleus of eukaryotic cells is inefficient, largely because of the significant barriers within the target cell including the plasma membrane and nuclear envelope. A group of basic proteins, including the HIV-1 Tat protein, have been shown to facilitate the entry of attached molecules into cells through a novel energy- and receptor-independent manner (23) . This property of cell-penetrating peptides has been used here to target a multimeric, noncovalent assemblage of DNA binding protein complex and a specifically modified therapeutic plasmid.

We have engineered this novel gene delivery vector exploiting a TAT-tagged multisubunit DNA binding protein derived from EcoR124I and expression plasmids with the EcoR124I recognition site. This vector produced >8-fold increase in the activity of the therapeutic AGA enzyme 1 wk after intramuscular administration in the mouse model of Fabry disease. While intramuscular injection of plasmid DNA is widely used to target gene expression, the efficiency of this method is low and attempts to increase muscle targeting of plasmids by vector modification were largely unsuccessful. To our knowledge this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle in vivo.

CPP, such as TAT, have been shown to improve delivery of macromolecules, when linked directly (37) . However, in our system TAT-evoked targeting of the therapeutic plasmid took place even though it was linked to the pDNA molecule indirectly via two noncovalent bonds (one between the M and S subunits, forming the M2S complex and the second between the DNA-binding S subunit and the DNA).

This finding is important from the application point of view because some proteins that would require targeting using CPP cannot be engineered directly, as their termini are functionally critical. Therefore, the possibility of combining those with a carrier molecule harboring a CPP is a promising approach. Moreover, in some other proteins addition of the highly charged and hydrophobic peptide could affect their function, irrespective of CPP localization. Our result demonstrates that CPP might aid the targeting of complex assemblies of macromolecules where only some of the components (and distant from the active site) contain the targeting peptide.

Molecular interactions of this chimeric assembly were confirmed by EMSA and AFM and were found essential. Maintenance of the nanocomplex in an appropriate stoichiometric ratio was both prerequisite and sufficient to boost expression levels. The EcoR124I is known to have a strong DNA binding affinity (108 M–1; ref. 32 ), and this might be an essential factor for maintaining the stability of this targeting complex, particularly in vivo.

Increased nuclear localization of pDNA in vivo was confirmed by the enhancement of expression by pluronic SP1017. Specifically, promoters containing the NF-{kappa}B binding site (e.g., hCMV) are stimulated by pluronics (30 , 36) and pX61 and pX61TH used here respond strongly to such stimulation (30) . The observed enhancement by pluronics administrated with 24 h delay and abolition of the enhancing effect by cyclophosphamide, a drug preventing NF-{kappa}B binding to DNA (38 , 39) , demonstrate that our AGA plasmid was already targeted into the muscle cell nuclei. There it interacted with SP1017. These data also confirmed that pluronics act as an exogenous synthetic biological response modifier. In this context, the recent study by Chang et al. (40) is of significant interest. These authors demonstrated that nonionic triblock copolymer of PEG-PLGA-PEG enhanced gene delivery efficiency into skeletal muscle. However, unlike the pluronic block copolymers, consisting of ethylene oxide and propylene oxide blocks (EO-PO-EO), this polymer appears to be acting via physical modification of pDNA and increased diffusivity of complexes in injected muscle. This result poses some critical questions regarding structure-function correlation of block copolymer vectors but also opens up new possibilities, as novel fascinating chemistries for these materials are now accessible.

Various approaches were used to attach CPP or NLS to the DNA directly via peptide nucleic acid (PNA clamps, hairpins, or electrostatic interactions; refs. 19 , 41 ) or by grafting on various polymers and liposomes (20 , 42 , 43) . The drawbacks of many of these methods were application of poorly controllable processes, the cost of production, and the time needed for the preparation of the vector. Our approach took advantage of a well-characterized engineering of TAT-fusion proteins and the controlled in vitro assembly of the CPP-containing protein and pDNA. A tight control of the nature and numbers of interactions between TAT-functionalized molecules and target pDNA is important for the maximum nuclear import efficiency (stoichiometry issues). This approach also limits the nonspecific electrostatic interactions of TAT peptides or TAT-containing polymers (positively charged) with negatively charged DNA. This targeted interaction of specific DNA sequences within the DNA molecule with the targeting protein complex restricts the possibility of multiple steric hindrances resulting from unspecific charge-charge interactions between TAT-containing molecules and random DNA sequences. This lowers the probability of subsequent transcriptional blockade of targeted expression plasmids.

Recombinant proteins combining DNA-binding and the membrane transduction domains were also used recently by Vaysse et al. (44) and Rajagopalan et al., (45) . Such systems transfected various cells in vitro and the complexes were found in the nucleus. As in our case, also in these studies the addition of cationic lipids enhanced the uptake and expression (45 , 46) showing that TAT alone is not a very efficient transfection tool in vitro. In an additional experiment, we found (as was expected) a significantly lower transfection efficiency in the COS-7 cells arrested using Aphidicolin when compared with dividing cells. However, the use of TAT-M2S complexes only slightly increased the level of transfection (P>0.05; data not shown). In contrast, in our in vivo system the presence of TAT was sufficient to increase gene expression >8-fold, without any additional transfection aids (e.g., lipofection).

It is clear that CPPs are capable of introducing a wide range of cargoes into living cells. However, the mechanism of internalisation is not fully elucidated. Even for the most extensively studied TAT peptide the suggested mechanisms can vary widely. These involve a range of processes from the energy-independent penetration of membranes to endocytotic uptake. This uncertainty is clearly related to a number of factors, including the type of complexes, size and properties of the cargo protein, the extracellular or intracellular delivery (the latter concerning nuclear targeting only), and many others. For example, TAT fusion proteins can be taken up largely into cytoplasmic vesicles, whereas peptides fused to TAT entered the cell rapidly by a mechanism dependent on membrane potential (47) . The analysis of various size cargoes expressed inside the cell indicated that TAT-tagged peptides cross the nuclear envelope by passive diffusion (25) . On the other hand, the study of splice-correcting peptide nucleic acids tethered to a variety of CPP indicated that their mechanism of uptake was mainly via endocytosis (48) and the synthetic peptide TAT-RGD targeting of pDNA also involved caveoli-mediated endocytosis (46) . In another recent study (49) , the TAT-RGD functionalization was found to increase the vector escape from endosomes and as TAT peptide has also been shown to improve gene transfer via enhanced cell surface adherence, it is possible that TAT may improve gene delivery by more than one mechanism (50 , 51) . TAT-facilitated endosomal escape might be of great significance for the targeting of nanoassemblies as the mechanism of uptake of such complexes is likely to involve endocytosis.

The clinical model used in this study was that of Anderson-Fabry disease (27) . There is evidence that this (and other) LSDs could be corrected by gene transfer, expression, and enzyme secretion from a subset of cells or from a specific tissue (29 , 30 , 52 53 54 55) . Gene therapy has several advantages over an alternative enzyme replacement approach, as it offers a long-term therapeutic effect, eliminates risks associated with repeated parenteral administrations and is less expensive (56) . However, the insufficiently high level and short duration of AGA expression are still apparent problems.

We show here that our novel, chimeric nonviral AGA gene delivery system provides significantly increased expression levels after skeletal muscle targeting in AGA knockout (Fabry) mice. Although in this preliminary study we were not looking for therapeutic effects, based on our previous data (30) it is expected that such expression levels would result in AGA serum activity of at least 30% of that found in normal individuals.

Importantly, using this method, we have found that we could significantly decrease the plasmid dose without affecting expression levels. It is important for clinical applications, as most current experimental protocols use unrealistically high plasmid copy numbers.

In our proof-of-principle experiment, we used EcoR124I. This choice of protein was dictated by the specific properties of the modified EcoR124I, namely functioning as the molecular DNA motor. Our long-term aim is to develop a nanomolecular DNA compartmentalization and targeting device (12) . DNA itself finds numerous new applications. For example, in the development of the autonomous nanoscale diagnosis and treatment devices: the recent and the most spectacular being as a biomolecular computer (57 , 58) . Therefore, DNA targeting and vector development are very important areas of research and are also beyond the classical therapeutic applications. The potential exploitation of single molecules and their nanoscale assemblies as functional machines is expected to create a significant technological breakthrough in biotechnology and medicine. This strategy could be also applicable to target genomic context vectors (e.g., a transgene and its relevant regulatory regions) and ultimately as complex systems as artificial chromosomes.

M2S is a bacterial protein that, in the longer term, might be immunogenic in vivo. Importantly, the presence of TAT fusion protein did not increase the short-term immune responses after muscle injection: The AGA antibody titers were not significantly different when compared with those triggered by plasmid alone and the decrease in enzyme levels was not accelerated (see Supplemental Data). However, for a technically less-demanding way to deliver DNA to the cell nucleus, less immunogenic moieties could be used. Interestingly, some mammalian proteins possess such properties. For example, engineered histones are able to mediate the efficient delivery of DNA into cells through the process of histone-mediated transduction, with further enhancement achieved by the addition of NLS (59) . Similarly, chimeric fusion proteins of the human high-mobility group protein HMGB2 with various NLS facilitated efficient gene delivery in vitro (60) . Use of similar autologous proteins offers a distinct advantage for gene therapy purposes; however, their efficacy in vivo remains to be tested.

On the other hand, vectors that trigger strong immune responses against transgene-encoded protein after intramuscular injection are highly advantageous for DNA-based vaccination. Thus, the TAT-M2S targeting system, as it stands, may not be suitable for gene therapies where long-term transgene expression is required. However, it may be useful for DNA vaccination.

In summary, the results presented here demonstrate that TAT can be used effectively for transduction of complex, multisubunit nanoscale devices into nondividing cells in vivo. We believe that this strategy can be of interest to those who wish to exploit CPP for molecular targeting.


   ACKNOWLEDGMENTS
 
We thank the Wellcome Trust (grant 067484) and the Institute of Biomedical and Biomolecular Sciences (University of Portsmouth) for financial support. We also thank F. S. Ahmadian for input into pCS2TH cloning, J. Beveridge for technical assistance with confocal microscopy, J. Rice for cell culture, J. Whitaker for FACS, J. Smith for help with AFM analyses, and P. Cox and K. Firman for critical suggestions on the manuscript.


   FOOTNOTES
 
1 Current address: Molecular Biology Laboratory, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

Received for publication August 2, 2007. Accepted for publication December 13, 2007.


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