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Gene Therapy (2005) 12, 1275–1282 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt RESEARCH ARTICLE Mechanism of efficient transfection of the nasal airway epithelium by hypotonic shock JL Lemoine1, R Farley2 and L Huang1 1 Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, PA, USA; and 2Department of Gene Therapy, Imperial College, National Heart and Lung Institute, London, UK The main barrier to gene transfer in the airway epithelium is the low rate of apical endocytosis limiting naked DNA uptake. Deionized water is known to stimulate the exocytosis of numerous intracellular vesicles during hypotonic cell swelling, in order to expand plasma membrane and prevent cell lysis. This is followed by the phase of regulatory volume decrease (RVD), during which the excess plasma membrane is retrieved by intensive endocytosis. Here we show that the more hypotonic the DNA solution, the higher the transfection of the nasal tissue. P2 receptors are known to be involved in RVD and we demonstrate that some P2 agonists and a P2 antagonist impair transfection in a time-dependent manner. Our study strongly suggests that the nasal airway epithelial cells take up plasmid DNA in deionized water during RVD, within approximately half an hour. Our simple gene delivery system may constitute a promising method for respiratory tract gene therapy. Gene Therapy (2005) 12, 1275–1282. doi:10.1038/ sj.gt.3302548; published online 12 May 2005 Keywords: airway; gene transfer; naked DNA; hypotonic shock; mechanism Introduction To date, transfection efficiencies in clinical trials of cystic fibrosis (CF) are too low to result in a notable correction of the chloride conductance defect.1,2 Two main extracellular barriers impede DNA uptake by airway epithelium. The first barrier is the mucus-gel layer involved in the mucociliary clearance of foreign bodies. The second is the low rate of endocytosis taking place on the apical side of airway epithelial cells.3 Moreover, the basolateral access is prevented by the presence of tight junctions. The respiratory epithelium contains these mechanisms of defense to limit viral and bacterial infections. We hypothesized that a hypotonic shock would force the apical membrane to undergo an intensive endocytosis, allowing plasmid DNA to access the intracellular compartments of the airway epithelial cells. Indeed, it is well known that a hypotonic shock comprises two phases: cell swelling and cell shrinkage or regulatory volume decrease (RVD). In the course of the first step, water molecules penetrate into the cells by osmosis. This initial cell swelling stimulates the exocytosis of numerous intracellular vesicles.4,5 By fusing with plasma membrane, they allow the membrane to expand and even to form some protrusions or blebs in fibroblasts and Ehrlich ascites tumor cells.6,7 Thus, cell swelling does not Correspondence: Professor L Huang, Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, 633 Salk Hall, 3501 Terrace Street, Pittsburg, PA 15213, USA Received 18 February 2005; accepted 26 March 2005; published online 12 May 2005 lead to cell lysis. Indeed, cultured cells were kept in a hypotonic medium for up to an hour without any loss of viability.8 The second step of the hypotonic stress is the internalization of the excess plasma membrane from both the apical and the basolateral sides to reform the lost intracellular vesicles. It has been extensively shown that P2 receptors are involved in this process. They are integral membrane proteins present on the apical and the basolateral surfaces and are divided into three subfamilies: X, Y and Z. The most important receptors, P2Y, are coupled to G proteins. P2Y1, P2Y2 and P2Y4 have been found on the apical side of the airway epithelial cells and P2Y6 on the basolateral side.9 Their natural agonist adenosine 50 -triphosphate (ATP) is present at high concentrations in secretory granules that fuse with the plasma membrane during cell swelling.10 Thus, ATP released in the extracellular medium can activate P2 receptors. A pathway transduced by P2Y receptors leads to the opening of potassium channels via a rise of intracellular calcium11 and another pathway results to the opening of chloride channels,12 including the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), via the activation of the protein kinase A.13 The release of potassium chloride reduces the ionic strength inside the cells but increases it in the extracellular environment, dragging water molecules out of the cells by osmosis. Those events progressively evict the excess intracellular water, allowing the retrieval of the excess plasma membrane by endocytosis. Here we demonstrate for the first time that in our in vivo model of transfection of the murine airway, the nasal airway epithelial cells take up plasmid DNA delivered in the deionized water during the step of cell Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al 1276 shrinkage of hypotonic shock, leading to its efficient transfection without damaging the cells. Results Effect of tonicity of the DNA solution on transfection of the nasal tissue We evaluated gene transfer in the mouse nose, a commonly used model of the airway epithelium because of the easy access,14 with plasmid DNA (pDNA) in the deionized water, sodium chloride and phosphatebuffered saline (PBS). Luciferase activity was reduced in a dose-dependent manner by sodium chloride and maximal transfection efficiency was produced in the deionized water, with values 92- and 102-fold higher than those obtained in 150 mM NaCl (saline) and PBS, respectively (Figure 1). It was recently shown that PBS and saline are more potent vehicle for pDNA than deionized water in the skin15 and the muscle.16 This suggests that a different mechanism takes place in the airway than in the skin or the muscle. Either the tonicity or the ionic strength of the pDNA solution prevents the transfection of the nasal epithelium. To determine whether transfection efficiency depends on the tonicity of the pDNA solution, we perfused the nasal epithelium with plasmid DNA in sucrose solutions of increasing tonicity, which also decreased transfection in a dose-dependent manner. The most hypotonic pDNA solution is therefore the most potent condition for transfecting the nasal tissue. Localization of transgene expression in the nasal tissue by b-galactosidase immunostaining To localize transgene expression within the nasal tissue, 100 mg of b-galactosidase expression plasmid pSVb was delivered in deionized water or in saline. No specific staining was found in the nasal tissue of untreated mice (Figure 2a), the nasal tissue perfused with pNGVL3-luc in deionized water (Figure 2b) or with pSVb in saline (Figure 2c). On the other hand, a positive and uniform staining was observed in the airway epithelial cells following their perfusion with pSVb in deionized water (Figure 2d). In all areas examined, more than 90% of the airway epithelial cells were significantly transfected (arrowheads). Compared to the untreated tissue (Figure 2a) and the tissue perfused with pSVb in saline (Figure 2c), the nasal tissues that were perfused with pNGVL3-luc in deionized water (Figure 2b) or with pSVb in deionized water (Figure 2d) were not significantly changed. This result indicates that hypotonic shock treatment did not damage the nasal epithelial tissue. Effect of the preperfusion of deionized water on transfection of the nasal airway epithelium with pDNA We then sequentially perfused mice with deionized water for different periods of time, followed by pDNA in deionized water. There was a time-dependent decrease of the luciferase activity. The administration of deionized water for 20 min followed by the perfusion of the nose with pDNA resulted in a maximal reduction of the luciferase expression (seven-fold). Transfection efficiency returned to the level achieved with the simple perfusion of mice with pDNA, when deionized water was predelivered for as long as 30 min. Neither the perfusion of the nasal tissue with deionized water after pDNA, nor the delivery of saline prior to the one of pDNA affected transfection of the nasal epithelium (Figure 3). These data suggest that the simple perfusion of deionized water improves gene transfer within a 30min period, after which the system has recovered to its original state and could be restimulated. We hypothesized that the perfusion of the nose for a period shorter than 30 min with deionized water prior to the delivery of pDNA would start a hypotonic shock before the nasal tissue sees the pDNA, thus the endocytosis of DNA could not take the full benefit of the hypotonic shock. This interpretation is in agreement with the report that cells incubated in a hypotonic medium swell and reach their maximal size in 2–3 min and shrink back to their initial volume in approximately 30 min.17 Our data suggest that DNA uptake by the nasal respiratory tissue occurs during the step of cell shrinkage (RVD). When a Figure 1 Effect of tonicity of the pDNA solution on transfection of the nasal tissue. Comparison of luciferase expression following the delivery of plasmid DNA in deionized water, sodium chloride, phosphate-buffered saline (PBS 1 ) and sucrose (n ¼ 3–4 mice per group); **Po0.005 and ***Po0.0005 versus deionized water. Gene Therapy Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al 1277 Figure 2 b-Galactosidase immunostaining of the nasal airway epithelium. Nasal tissues harvested from mice that received 100 mg of plasmid DNA were fixed, sectioned and immunohistochemically stained for the b-galactosidase 24 h following administration. Representative sections of the airway epithelium of untreated animals (a), animals that received the luciferase expression plasmid pNGVL3-luc in deionized water (b), animals that received pSVb in saline (c) and animals that received pSVb in deionized water (d) are shown. Hematoxylin stain (blue) marks nuclei and AEC stain (red) marks b-galactosidase expression. The arrowheads point to some of the b-galactosidase-expressing nasal epithelial cells. 40, original magnification. Figure 3 Sequential perfusion of deionized water and plasmid DNA in water. Deionized water was delivered for 10–30 min shortly followed by pDNA in deionized water for 15 min. pDNA in deionized water was delivered for 15 min shortly followed by deionized water for 20 min (DNA-20 min water) and saline (150 mM NaCl) was delivered for 20 min shortly followed by plasmid DNA in deionized water for 15 min (20 min saline-DNA). **Po0.05, ++ Po0.01 and ***Po0.0005 versus pDNA alone (n ¼ 3–4 mice per group). cycle of cell swelling and cell shrinkage is over (in about 30 min), a second cycle can begin and luciferase expression returns to the level achieved with a simple perfusion of pDNA (Figure 4). Effect of ATP on transfection of the nasal epithelium The crucial role played by P2 receptors in RVD has been abundantly illustrated.18,19 Their overstimulation by exogenous ATP accelerates RVD,20 whereas their blockade by the antagonist suramin slows it down.21 To assess whether P2 receptors are involved in deionized watermediated transfection, we delivered pDNA along with ATP in the deionized water. There was a dose-dependent decrease in luciferase expression in the nasal tissue, with values 10-fold lower at 400 nmol of ATP (Figure 5). We hypothesized that exogenous ATP accelerates the phase of cell shrinkage, during which nasal cells take up pDNA. Since the DNA solution was delivered within 15 min, in the presence of exogenous ATP, RVD would nearly be over while most of the pDNA were not yet delivered (Figure 4). We tested this hypothesis by shortening the duration of delivery of the DNA/ATP solution. The faster the perfusion of mice with the plasmid DNA/ATP solution, the higher the luciferase activity, and the perfusion of the nose lasting as short as 7 min gave rise to gene expressions as high as pDNA alone delivered in deionized water in 7 min (Figure 5). Gene Therapy Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al 1278 Effect of suramin on transfection of the nasal respiratory tissue Similarly, we perfused the nasal epithelium with pDNA and a P2 antagonist, suramin. There was also a dosedependent reduction of the luciferase activity, as high as 15-fold at 50 nmol of suramin. Interestingly, the delivery of pDNA along with 400 nmol of ATP and 50 nmol of suramin resulted in a similar transfection efficiency as that of the pDNA alone (Figure 6). This demonstrates that neither exogenous ATP nor suramin are toxic to the nasal tissue. In the presence of suramin, RVD would last longer and after the 15-min perfusion most of pDNA would have been degraded by extracellular DNases prior being taken up by cells22 (Figure 4). To test whether suramin affects transfection by slowing down RVD, we slowed down the delivery of the DNA/suramin solution. The slower the perfusion of Figure 4 Schematic of the changes of the cell volume in the course of a hypotonic shock. Cell volume in the absence of any drug (—), in the presence of ATP (- - -) and in the presence of suramin (– – –). mice with the pDNA/suramin solution, the higher the gene expression, and the delivery lasting 45 min completely abolished the deleterious effect of this drug on gene transfer (Figure 6). Interestingly, it has been shown that RVD is also slowed down in CF because of a limited release of endogenous ATP during cell swelling.23,24 This last experiment suggests that pDNA in deionized water should preferentially be administered over a long period of time in the airway of CF patients. Impairment of transfection of the nasal tissue by other P2 agonists One may argue that ATP may interact with ATP-binding proteins other than the P2 receptors. To determine whether P2 receptors are the real target of ATP, pDNA was delivered along with other well-known P2 agonists. ADP, dATP and UTP are potent ligands of P2 receptors, whereas adenosine and AMP are not.25 At the same dose as ATP (400 nmol), ADP, dATP and UTP affected luciferase activity as much as ATP and adenosine had no effect (Figure 7). Gene expression achieved with AMP is intermediate between adenosine and P2 agonists. This result can be explained by the presence of ectoadenylate kinase on the apical surface of the airway.26 The enzyme converts one molecule each of AMP and ATP into two molecules of ADP. We hypothesize that ecto-adenylate kinase would convert a part of exogenous AMP into ADP, which is an agonist. Since the P1 agonist adenosine does not impair gene transfer, the data demonstrate that P1 receptors are not involved in the transfection and it is known that they are not involved in RVD either.27 Similarly to ATP, the perfusions of the nasal epithelium with plasmid DNA along with 400 nmol of P2 agonists and 50 nmol of suramin resulted in luciferase activities as high as the ones achieved with the plasmid DNA alone. This strongly suggests that the target of the nucleotides and suramin is the P2 receptor and not other ATP-binding proteins. Figure 5 Effect of ATP on transfection of the nasal epithelium. Luciferase activities achieved following a 15-min perfusion of the nose with plasmid DNA and up to 400 nmol of ATP; +++Po0.001 and ***Po0.0005 versus pDNA alone. Transfection of the nose with pDNA and 400 nmol of ATP for as short as 7 min; *Po0.05 versus 400 nmol of ATP for 15 min (n ¼ 3–4 mice per group). Gene Therapy Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al 1279 Figure 6 Effect of suramin on transfection of the nasal respiratory tissue. Transfection of the nasal tissue with pDNA and up to 50 nmol of suramin for 15 min. Luciferase expression following delivery of plasmid DNA along with 400 nmol of ATP and 50 nmol of suramin for 15 min (ATP+suramin-15 min); ++ Po0.01 and ***Po0.0005 versus pDNA alone. Perfusions of the nasal passages with plasmid DNA and 50 nmol of suramin for up to 45 min; *Po0.05 and **Po0.005 versus 50 nmol of suramin for 15 min (n ¼ 3–4 mice per group). Figure 7 Effects of other P2 agonists on transfection of the nasal epithelium. Transfections efficiencies achieved with pDNA, 400 nmol of nucleotide(side)s and 50 nmol of suramin; +++Po0.001 and ***Po0.0005 versus pDNA alone (n ¼ 3–4 mice per group). Discussion Our data are in agreement with the model of transfection by hypotonic shock of the nasal tissue. Indeed, we have shown that the more hypotonic the DNA solution was, the more potent was the transfection of the nasal respiratory epithelium. pDNA must be delivered within a 30-min period to take the full benefit of the hypotonic treatment. We have demonstrated that P2 agonists, which are known to accelerate the phase of cell shrinkage in the process of hypotonic shock, accelerated the transfection as well. Similarly, we have shown that a P2 antagonist, suramin, which slows down the RVD, also slowed down the transfection. Although the delivery of pDNA in water was first reported by others,28–30 we herein provided some evidence that deionized water is not solely a vehicle for pDNA, but actively participates in the transfection process. Naked pDNA in deionized water specifically transfected the airway epithelial cells, as demonstrated by immunohistochemistry for b-galactosidase, whereas pDNA in saline did not. The major concern about the delivery of water in the airway is the potential damage caused by the hypotonic DNA solution to the tissue. However, the nasal tissues that received pDNA in deionized water were not detectably different from the untreated tissue (Figure 2), indicating that the hypotonic shock procedure was well tolerated by the tissue. One may argue that a hypotonic shock might increase the paracellular permeability of the airway epithelium to plasmid DNA, allowing this latter to gain access to the basolateral side of the cells. The epithelial cells would take pDNA up from a larger surface area of plasma Gene Therapy Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al 1280 membrane. However, we were unable to detect any specific staining of the mesenchymal cells underlying the airway epithelium (Figure 2d). If pDNA was able to cross the airway epithelium, those underlying mesenchymal cells would have been transfected. Moreover, from Wang et al,31 upon a hypotonic shock, the transepithelial resistance of human airway epithelial cells drops in a short period of time, plateaus for about 3 h and slowly increases back to the base line in about 24 h. A similar time course was obtained with human tracheal cells by Widdicombe et al.32 The model of transfection by hypotonic shock involving the opening of the tight junctions fails to explain the data shown in Figure 3. Indeed, the preperfusion of water for 10–25 min should not significantly impair the transfection of the airway epithelium. On the other hand, the model of transfection involving an exacerbated rate of uptake of pDNA is consistent with the results of Figure 3. Widdicombe et al32 have shown that human tracheal cells exposed to RITC-labeled dextran (2000 kDa) in deionized water efficiently take up this fluorescent macromolecule. Koberna et al33 have also demonstrated that various fluorescent low-molecular weight compounds, such as nucleotides, peptides and dyes, are rapidly taken up by cell lines, a human primary cell culture, drosophila and xenopus cells, as well as slices of the rat liver by hypotonic shock. In contrast, the same molecules were not significantly internalized when the cells were exposed to an isotonic medium. Finally, van der Wijk et al34 have more recently shown that the hypotonic swelling of intestine 407 cells induces the endocytosis of TRITC-labeled dextran (10 kDa). Numerous fluorescent vesicles appeared in the hypo-osmotically stimulated cells but not in the cells exposed to an isotonic solution of TRITC-labeled dextran. The rate of endocytosis increased after a lag phase of 2–3 min and lasted for about 15 min. It is highly likely that pDNA is also endocytosed by the nasal epithelial cells in our study, although the direct evidence will have to be obtained in future studies. So far, the shortcoming of nonviral vectors was their insufficient potency to deliver genes in the normal airway.35 The luciferase expression achieved by our simple nonviral system approaches that of recombinant Sendai virus in the nose36 and is comparable to that achieved with pDNA/PEI and pDNA/CK30PEG10K.37 Unlike viral vectors, pDNA alone is not proinflammatory, especially when the CpG dinucleotides are eliminated.38 It could therefore be repeatedly administered in the airway. Furthermore, several currently used antiasthmatic drug solutions, such as cromoglycate and salbutamol, were found to be hypo-osmolar.39,40 Consequently, it is clinically conceivable to deliver a hypotonic pDNA solution in the airway of patients suffering from inherited or acquired respiratory disorders. The nebulization of a naked DNA solution results in a nearly complete fragmentation of the DNA molecule, which becomes therapeutically inactive.41 The patient does not inhale most of the misted solution, which falls back in the reservoir of the nebulizer and is misted again. The recirculation of the solution leads to the shear damage of the DNA molecules. However, Trudell Medical International (London, Ontario, Canada) has recently developed the AeroProbet, which is an intra- Gene Therapy corporeal nebulizing catheter suitable for the delivery of shear sensitive drugs (McLachlan, G et al, abstract # 496, ASGT’s 7th annual meeting). The drug solution and a gas flow through different capillaries composing the catheter, which converge at the distal tip as minuscule orifices. The pressurized gas mists the drug solution into fine droplets. The drug solution is directly aerosolized in the lung and does not recirculate in the catheter, which might be used in conjunction with a bronchoscope. It is expected that a single passage of the naked DNA solution in this type of nebulizing catheter would not extensively break the DNA molecules. The device may be connected to a pump to slowly deliver a DNA solution into the airway of CF patients, in order to take into account of the slow rate of cell shrinkage of CF epithelial cells after a hypotonic treatment (limited release of ATP during the cell swelling). Finally, this simple technique of transfection might be used to deliver other nucleic acids in the airway, such as siRNAs to treat viral infections (influenza), as well as genes in other mucosa, such as the uro-genital and the gastro-intestinal tracts. Material and methods Materials All nucleotides, adenosine and suramin were purchased from Sigma-Aldrich (St Louis, MO, USA). Plasmid DNA pNGVL3-luc carries the firefly luciferase gene driven by the cytomegalovirus (CMV) promoter. It was custom prepared as endotoxin-free DNA by Bayou Biolabs (Harahan, LA, USA). The pSVb plasmid comprises the b-galactosidase gene under the control of the SV40 enhancer/promoter. Gene transfer to the murine nasal epithelium All experiments were carried out in female CD1 mice (6–8 weeks old, Harlan) anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (Sigma-Aldrich). They were handled according to the instructional guidelines for animal care and use. As previously described by Griesenbach et al,42 a catheter was placed 2.5 mm within the left nostril and the diverse solutions of naked plasmid DNA (100 mg/mouse in 75 ml of deionized water) were perfused at a rate of 5 ml/min using a peristaltic pump (pump P-1, Amersham Biosciences). During the perfusion, the mice were kept in an inverted position in order to maximize the time contact between plasmid DNA and the nasal tissue. At 24 h after perfusion, nasal turbinates were harvested, homogenized, frozen/thawed three times and assayed for luciferase activity (luciferase assay, Promega, Madison, WI, USA). The luciferase activity of untreated mice was nullified. b-Galactosidase immunostaining CD1 mice were perfused with the b-galactosidase expression plasmid pSVb, in deionized water or saline, and with pNGVL3-luc in deionized water, as described above. After 24 h, the nasal passages were harvested, fixed in formalin, embedded in paraffin and sectioned. The sections were incubated with a biotinylated anti-b- Mechanism of transfection of the airway epithelium with naked DNA in water JL Lemoine et al galactosidase antibody (Sigma-Aldrich) and then a complex avidin/biotinylated peroxidase (Vectastain, Vector laboratories). Finally, the sections were incubated with the peroxidase substrate AEC (3-amino-9-ethylcarbazol, Sigma-Aldrich) and counterstained with hematoxylin (Sigma-Aldrich). In the group of mice perfused with pSVb in water, the determination of the percentage of transfected airway epithelial cells was carried out by counting the number of b-galactosidase-positive and -negative cells in the airway epithelium in several sections of the nasal tissue of three animals. Acknowledgements We thank Xinsheng Nan (University of Edinburgh, UK) for the gift of the plasmid pSVb and Uta Griesenbach (Imperial College, UK) for helpful discussions. This study was supported by the Cystic Fibrosis Research Trust (UK). References 1 Alton EWFW et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a doubleblind placebo-controlled trial. Lancet 1999; 353: 947–954. 2 Hay JG et al. Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther 1995; 6: 1487–1496. 3 Pickles RJ et al. 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