Use of ultrasound to enhance nonviral lung gene transfer in vivo - Nature

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Feb 15, 2007 - We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung. Gene Therapy (2007) 14, ...
Gene Therapy (2007) 14, 768–774 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

Use of ultrasound to enhance nonviral lung gene transfer in vivo S Xenariou1,2, U Griesenbach1,2, H-D Liang3,4, J Zhu1, R Farley1,2, L Somerton1,2, C Singh1,2, PK Jeffery1, S Ferrari1,2, RK Scheule5, SH Cheng5, DM Geddes1,2, M Blomley3 and EWFW Alton1,2 1

Department of Gene Therapy, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, UK; 2UK Cystic Fibrosis Gene Therapy Consortium, London, UK; 3Ultrasound Group, Imaging Sciences Department, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, London, UK; 4School of Engineering, University of Cardiff, Cardiff, UK and 5Genzyme Corporation, Framingham, MA, USA

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/ pDNA or PEI/pDNA gene transfer compared to Optisontreated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound

being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung. Gene Therapy (2007) 14, 768–774. doi:10.1038/sj.gt.3302922; published online 15 February 2007

Keywords: sonoporation; ultrasound; gene transfer; nonviral vectors; lung; cystic fibrosis

Introduction Our aim is to establish a clinically relevant treatment for cystic fibrosis (CF), a monogenic, lethal disease, in which pulmonary failure is the major cause of mortality. We are, thus, interested in optimizing lung gene transfer, and more specifically, gene transfer to the respiratory airway epithelium. The lung offers the advantage of allowing for topical administration of therapeutic agents, usually via nebulization. In spite of this, it has proved to be a formidable target for gene delivery, with a series of extracellular barriers such as mucociliary clearance and mucus, and in the case of CF, a layer of infected sputum. In addition, there are several intracellular obstacles, including clearance by uptake into endosomes, and the nuclear membrane that must be breached.1 Efforts to overcome these barriers with the use of mucolytic agents,2 viscoelastic gels,3 or agents that disrupt tight junctions, thus allowing access to receptors present on the basolateral membrane,4 have enhanced transfection efficiency to airway epithelium, but further improvements would always be welcome. Correspondence: Dr U Griesenbach, Department of Gene Therapy, National Heart and Lung Institute, Imperial College, 1B Manresa Road, London SW3 6LR, UK. E-mail: [email protected] Received 5 September 2006; revised 21 December 2006; accepted 21 December 2006; published online 15 February 2007

Both viral and nonviral vectors have been used in CF clinical trials, delivered either to the nasal epithelium or the lung of CF patients.5,6 Our particular focus is on nonviral vectors because of their capacity to be repeatedly administered,7 a critical need in the treatment of a lifelong condition. Proof of principle for the delivery and expression of cystic fibrosis transmembrane conductance regulator (CFTR), the gene mutated in CF, with nonviral vectors has been established, in preclinical models8 as well as in clinical trials.5,9 However, in general, nonviral gene transfer agents are not as efficient as viruses, since they lack the natural mechanisms to invade cells, and CFTR expression in clinical trials resulted in partial correction of the bioelectrical defect.10 Attempts to improve the efficiency of nonviral vectors are being made, including the addition of peptide ligands to target the vector to receptors localized on the apical membrane of airway epithelial cells,11 and the development of nanoparticles to improve nuclear entry.12 Here we have assessed, if sonoporation, the use of high-frequency ultrasound (US) increases nonviral gene transfer to the lung. US is a versatile technique, and has several medical applications. High-frequency (41 MHz), low-intensity US is commonly used for imaging, for example to detect foetal abnormalities. In contrast, US shock waves, at lower frequencies but higher intensities, are used to destroy gallbladder or kidney stones (lithotripsy), whereas a similar type known as high-intensity focused

Use of ultrasound to enhance nonviral lung gene transfer in vivo S Xenariou et al

Results Optison reduces nonviral gene transfer in the mouse lung Initial studies were carried out to investigate the effect of Optison on nonviral vectors. Naked pDNA, PEI/pDNA and GL67/pDNA, mixed with Optison at 1:1 (v/v) ratio, were delivered to the mouse lung. In all cases, the addition of Optison significantly reduced reporter gene expression (Figure 1). Naked pDNA was also mixed with Optison at a ratio of 1:0.25 (v/v, DNA/Optison), but this formulation also significantly reduced transfection efficiency (data not shown). Sonoporation does not increase GL67/pDNA or PEI/ pDNA transfection efficiency when mixed with Optison To determine suitable US conditions, mice were instilled with phosphate-buffered saline (PBS; 100 ml, n ¼ 3) and exposed to 1 MHz US for 10 or 20 min. As animals

769 100 Luciferase (RLU/mg protein)

ultrasound (HIFU) is currently being evaluated in preclinical models as a method to ablate tumours.13 In terms of gene transfer, US has been applied in several in vitro and in vivo systems. Both shock waves and HIFU have been used.13,14 In most gene delivery studies, however, the US applied has been in the range of 1–2 MHz. Sonoporation has increased gene transfer of naked plasmid DNA (pDNA) and cationic liposomes in several cell lines,15 including vascular,16 and skeletal muscle cells.17 It has also been used to enhance nonviral gene transfer to tumours,18 the arterial wall,19 skeletal muscle,20 and liver,21 in vivo. US has also been successfully used to increase adenoviral transfection efficiency to the myocardium.22 US is thought to act by transiently permeabilizing the cell membrane, thus increasing vector uptake. The mechanism behind this effect is thought to be cavitation, the formation and oscillation of gas bubbles, in a liquid medium. The size of the bubbles fluctuates in response to the pressure fluctuations of the US wave, and ultimately the bubbles collapse, creating pores in cell membranes.23 The theory that cavitation is responsible for sonoporation effects is also supported by the fact that addition of gasfilled microbubbles, further augments US-mediated gene delivery. Thus, microbubbles, and in particular, Optison, have been used in several sonoporation studies.13,24 Optison is an US contrast agent, used for medical imaging, consisting of albumin microspheres, filled with octafluoropropane.25 When compared to similar available agents, Optison was superior for US-mediated gene transfer to skeletal muscle in vivo.26 A recent study also demonstrated that the effects of US on the cell membrane are much more profound in the presence of Optison, providing further support for the role of cavitation.27 Thus, we have tested, for the first time, the effect of sonoporation on nonviral gene transfer to the lung. It is well established, that ultrasound is greatly attenuated when travelling through air.28 Reflection of the wave, and therefore, energy loss, will also occur as it passes through air/ tissue interfaces.28 Nonetheless, we assessed whether the energy that is able to penetrate the lung tissue was sufficient to enhance the gene delivery of cationic lipid 67 (GL67)/ pDNA or polyethylenimine (PEI)/pDNA complexes, and naked pDNA, all previously used for lung gene transfer.29–31

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0.1 Figure 1 Optison reduces the transfection efficiency of naked pDNA, PEI and GL67/pDNA complexes. Animals were instilled with naked pDNA (100 mg/100 ml), PEI/pDNA (10 mg/100 ml) or GL67/pDNA (40 mg/100 ml) complexes, in the absence (closed bars) or presence (open bars) of Optison (1:1 (v/v) ratio). ***Po0.001, compared to the respective control group of vector alone. n ¼ 6–8 mice/condition.

appeared healthy, we decided to apply US for 20 min in further experiments. We, thus, went on to instill a mixture of GL67/pDNA and Optison (40 mg pDNA/ 100 ml, 1:1 (v/v)), or PEI/pDNA and Optison (10 mg pDNA/100 ml, 1:1 (v/v)) complexes with subsequent exposure to US for a total of 20 min. Sonoporation, however, did not increase GL67 (GL67+Optison: 5.4571.00 RLU (relative light units)/mg protein, GL67/ pDNA+Optison+US: 4.8571.69 RLU/mg protein) or PEI-mediated gene transfer (PEI/pDNA+Optison: 3.1470.90 RLU/mg protein, PEI/pDNA+Optison+US: 2.9870.47).

Sonoporation increases naked pDNA gene transfer in the air-filled lung when mixed with Optison We also tested the effect of sonoporation on naked pDNA (100 mg pDNA/100 ml, 1:1 (v/v)). Importantly, a 20-min US exposure led to a significant (Po0.001, n ¼ 7–8 mice/ condition) increase in gene expression, of approximately 1.5 logs (Figure 2). To assess if we could further enhance this effect, animals were exposed to US for 1 h. However, 2/2 animals died after 35–40 min of exposure, and thus, we did not continue with this. In addition, we included a group of animals exposed to US for 2 min, to determine if the positive effect previously seen with 20 min, could also be achieved at shorter exposures. As shown in Figure 2, the 2-min exposure also resulted in a significant (Po0.001, n ¼ 7–8 mice/group) increase in luciferase expression, compared to the control group. However, the levels of expression obtained with 2 min exposure were lower than with 20 min, although that difference was not statistically significant. Prolonged US exposure causes lung haemorrhage A common side effect of US exposure is microvessel damage and tissue haemorrhage. Therefore, we assessed if haemorrhage had occurred in the mouse lung. US exposure for 2 min did not increase haemorrhage compared to control groups (Figure 3), but this was visible after 20 min of US exposure. Interestingly, the degree of haemorrhage was greater in the left lung than Gene Therapy

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in the right (Figure 4a). The area affected also increased with exposure time and, once again, the left lobe was more affected than the right (Figure 4b). This is likely owing to the fact that it lies directly beneath the rib cage and may be more exposed than the right lobes, which are smaller and fold onto each other. Low levels of haemorrhage were seen in animals treated with naked pDNA alone, but the same levels were also observed in untreated controls (data not shown).

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US-mediated naked pDNA gene transfer produces a reduced effect Because of the ‘uneven’ distribution of US-mediated toxicity in the lung, we determined whether there were 12 11 10 9 8 7 6 5 4 3 2 1 0

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Figure 2 Sonoporation of naked pDNA in the presence of Optison. Animals instilled with a mixture of naked pDNA and Optison (1:1 (v/v) ratio) were exposed to US for 2 or 20 min. The bars represent the levels of luciferase achieved in the trachea and right lobes. The open bar represents the luciferase levels with pDNA alone. ***Po0.001; n ¼ 7–8 mice/condition.

differences in the levels of gene expression achieved by sonoporation of naked pDNA, between the right and left lobes (Figure 5). Although there were no significant differences between right and left lungs in luciferase expression in any group, there was a trend towards reduced gene expression in the left lung. In keeping with this, exposure to US for 2 min did not enhance gene transfer in the left lung, only the 20-min exposure produced a significant (Po0.05, n ¼ 7–8 animals/condition) increase of luciferase expression.

Sonoporation has no effect on gene transfer in the absence of Optison As seen above (Figure 1), Optison reduced the transfection efficiency of all three, gene transfer agents. In addition, Optison microbubbles are very ‘fragile’ and would most likely not withstand nebulization, the likely relevant method of vector delivery for CF clinical trials. We, therefore, assessed whether US could improve gene transfer, in the absence of Optison. Immediately after nasal instillation, animals were exposed to US for a total of 20 min. Sonoporation did not increase reporter gene expression of any of the gene transfer agents (pDNA: 4.1570.78 RLU/mg protein, pDNA+US: 6.1470.79; PEI/pDNA: 3.4970.86 RLU/mg protein, PEI/pDNA+US: 1.6670.23; GL67/pDNA: 31.0174.47 RLU/mg protein, GL67/pDNA+US: 41.017 8.28 RLU/mg protein; n ¼ 6–10 mice/condition).

Discussion Sonoporation has been successfully applied to increase gene transfer in several organs, other than lung, using both viral and nonviral vectors. We assessed, for the first time, whether the technique could also be used to enhance nonviral gene transfer to the lung. Traditionally,

Figure 3 Prolonged US exposure exacerbates lung haemorrhage. Lung sections from animals treated with (a) naked pDNA, (b) pDNA+Optison (1:1 (v/v) ratio), (c) pDNA+Optison (1:1 (v/v))+2 min US and (d) pDNA+Optison (1:1 (v/v))+20 min US, were stained with H&E to assess haemorrhage, 24 h after treatment. AW, airways, BV, blood vessel. The pictures are representative of n ¼ 6–8 animals/group. Gene Therapy

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Luciferase (RLU/mg protein)

Score for Degree of Haemorrhage

a 3.0 2.5 2.0 1.5 1.0 0.5 0.0 pDNA

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Figure 5 Comparison of naked pDNA sonoporation between the left and right lung. Animals were exposed to US for 2 or 20 min, immediately after the administration of pDNA+Optison (100 mg/ 100 ml, 1:1 (v/v)) and luciferase expression was measured 24 h later. The closed bars represent the right lung and the open bars the lower half of the left lobe (*Po0.05, compared to the pDNA+Optison group; n ¼ 7–8 animals/condition).

2.5 2.0 1.5 1.0 0.5 0.0 pDNA

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Figure 4 Histological score of US-induced bioeffects following naked pDNA gene transfer. The degree of haemorrhage (a) and the affected lung area (b), were assessed 24 h after sonoporation with naked pDNA. The distribution of the effects between the right and left lobes are shown. Each symbol represents one animal, with the mean score of the group shown as a horizontal line. Closed diamonds represent the smallest right lobe and open diamonds the upper half of the left lobe.

the lung poses challenges for US applications, including imaging, because US waves are greatly attenuated when travelling through air. We show here, that despite these limitations, sonoporation increased naked pDNA gene transfer, when mixed with microbubbles (Optison), but did not enhance GL67/pDNA or PEI/pDNA transfection efficiency. Microbubbles, and Optison in particular, are often used in sonoporation studies. They are thought to lower the cavitation threshold and thus to enhance the effects of US on gene transfer. However, in contrast to previously published data,32 addition of Optison significantly reduced the transfection efficiency of PEI and GL67/pDNA complexes, as well as naked pDNA. Optison alone has been shown to increase naked pDNA gene transfer to the skeletal muscle of young (4 weeks), but not older (6 months) mice.32 Interestingly, Optison did not affect PEI/pDNA transfection efficiency in these studies. Although the mice in our experiments were only 6–8 weeks old, their lungs were fully developed and differentiated. The discrepant results are most likely a reflection of organ-specific differences. A vector formulation that is efficient in transfecting skeletal muscle is not necessarily as effective in the lung, given that the cell morphology and function, as well as the extracellular

barriers to gene transfer, are different. In addition, it is not yet known how Optison interacts with gene transfer agents. PEI/pDNA and GL67/pDNA complexes have been careful optimized with regard to biophysical properties and hence lung gene transfer efficacy. Addition of Optison likely disrupts these properties, thus reducing their transfection efficiency. We next assessed the effect of sonoporation on nonviral gene transfer in the presence of Optison. US exposure failed to enhance GL67/pDNA and PEI/ pDNA gene transfer over and above the reduced levels produced by Optison. However, naked pDNA transfection efficiency was significantly enhanced. The experiments presented here were carried out by measuring luciferase reporter gene activity in lung homogenate. This did not allow us to determine if sonoporation increased the number of cells transfected, or increased the amount of reporter gene expression per cell. Although green fluorescent protein (GFP) is the most appropriate reporter gene to address these important questions, in our experiments, the levels of luciferase expression detected would not have translated into detectable GFP expression (Dr Lee Davies, personal communication). The positive effect of sonoporation on naked pDNA suggests that in spite of the attenuation caused by air in the lungs, the remaining energy is sufficient to increase gene transfer in our model. Despite this, however, the overall levels of sonoporation-mediated gene transfer did not exceed those of naked pDNA alone, owing to the inhibitory effect of Optison. Nonetheless, the positive effect of US on gene transfer was entirely dependent on the presence of Optison, suggesting that the levels of cavitation required to increase gene transfer can only be achieved with the use of microbubbles. Although it is unlikely that the fragile Optison microbubbles will withstand nebulization through standard jet nebulizers, such as the Pari LC, the latest generation of single-pass mesh nebulizers (e.g. Pari eFlow) might be suitable for nebulization of Optison. Gene Therapy

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The vector-specific effects of US cannot be simply attributed to restrictions in the size of the pores caused by sonoporation. Several studies have assessed the effects of US on the cell membrane, as well as the cellular uptake of different size particles and vectors after sonoporation in vitro. These studies have confirmed that naked pDNA is directly transferred into the cytoplasm after sonoporation, probably through USinduced pores.33 This process is much faster than lipofection, which involves endocytosis. In similar studies, fluorescent latex nanospheres ranging from 25 to 75 nm, as well as fluorescently labelled dextran molecules with a molecular mass of 77–464 kDa, were also internalized via US-induced pores in the cell membrane.34 The size of PEI/pDNA complexes, at a N/P ratio of 10:1, has been shown to be around 60 nm,35 which, given the above studies, should theoretically allow them to enter cells via sonoporation-induced pores. It is not known, however, how ultrasound or the addition of Optison may affect the size or charge of our gene transfer agents. We have shown that US-induced haemorrhage increased with exposure time, which in turn may reduce gene transfer efficiency. There was also some haemorrhage, albeit at low levels, in control animals that were not exposed to US. This was most likely owing to the culling method, since the same observations were made in untreated controls, although the actual instillation might also contribute. However, we had already established that in our mouse strain culling by cervical dislocation causes similar lung haemorrhage as exsanguinations, and less than lethal injection (data not shown). US exposure affected the left lobe more severely than the right lung, which may explain why sonoporation had a more modest effect on naked pDNA gene transfer in the former. The appearance of haemorrhage in all areas of the lungs provides indirect evidence that US penetrates deep into the lung tissue of mice. However, these findings also suggest that there may be a narrow toxicity/efficacy window for US-mediated gene transfer to the mouse lung. US-induced bioeffects in the lung have been previously studied but whether cavitation is responsible for these effects remains unclear. Although air within the lungs could theoretically give rise to ‘microbubbles’, the gas is so closely packed in the alveoli that this is unlikely.36 More likely, the mechanical stress on the tissue is caused by reflection of the US energy at air/tissue interfaces.37 However, cavitation might still play a role in our system as we are delivering microbubbles topically into the lung. Interestingly, the threshold for haemorrhage in the mouse lung has been determined to be approximately 1 MPa.38 This is higher than the pressure applied in our studies (0.74 MPa), but the presence of Optison might allow for bioeffects to be induced at lower pressures. US-induced toxicity may be overcome by further optimization of the US conditions, such as the duty cycle, the total exposure time and the acoustic intensity. However, damage may be necessary for US-mediated gene delivery to take place. In retrospect, we do not believe that mice are the most suitable model to assess these issues. Firstly, the size of the mouse lung and therefore, the distance that US has to travel, is much smaller than in comparison to man. Secondly, there is

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evidence to suggest that mice are more susceptible to US-induced lung damage than other species, owing to differences in lung structure. For instance, mice, rats monkeys are more vulnerable than sheep, pigs and humans, because they have thinner visceral pleura. In addition, their airways are shorter, thinner and less flexible, which also seems to contribute to their increased susceptibility.39 To address the issues of toxicity and clinical feasibility, studies in a larger animal model may be necessary. Sheep, in particular, are an attractive option, as proof of principle for nonviral gene transfer to the ovine lung has already been established.29 Also, their lung is similar to humans both in structure and size. Sonoporation studies in this model could assess how US is applied, for instance endobronchially or via the chest wall, and optimize US conditions for lung gene transfer while assessing any US-induced side effects. They would thus provide valuable information on the potential clinical use of sonoporation.

Materials and methods Vectors The gene transfer agents used in these studies were: PEI, 25 kDa, branched; Sigma, Poole, UK), cationic lipid 67 (GL67; consisting of GL67/DOPE/DMPE-PEG5000 at molar ratios of 1:2:0.05; Genzyme Corporation, Framingham, MA, USA) and naked plasmid DNA (pDNA). The plasmid DNA carried a luciferase reporter gene under the control of a cytomegalovirus immediate early promoter. Equal volumes of PEI and pDNA, in sterilized water for injection (Arnolds Veterinary Products, Shrewsbury, UK), were mixed at nitrogen (N):phosphate (P) ratio of 10:1.40 The solution was vortexed and incubated for 20 min at room temperature. Similarly, equal volumes of GL67 (1.2 mM) and pDNA (4.8 mM, 1.6 mg/ml) were mixed at a molar ratio of 1:4 (GL67:pDNA) in sterilized water for injection and incubated at 301C, for 15 min.41 In some cases, Optison (Amersham Health, Oslo, Norway), an ultrasound contrast agent, was mixed at a 1:1 volume: volume (v/v, DNA/Optison) ratio with the gene transfer agents described above, in a total volume of 100 ml. US equipment Pulsed US at 1 MHz frequency, with a pulse repetition frequency of 100 Hz and 20% duty cycle, was used (Electro-Medical Supplies, Abingdon, UK). The acoustic intensity was 3 W/cm2 and the peak pressure was 0.74 MPa. The diameter of the probe was 1.3 cm. In vivo transfection and sonoporation Male Balb/c mice (6–10-weeks old) were anaesthetized by intraperitoneal injection of ketamine/metetomidine (76 and 1 mg/kg, respectively; National Veterinary Services Ltd, Stoke, UK) and the vector solution was delivered to the lungs by nasal instillation. Briefly, the animals were positioned upright, and their mouth was held closed between the thumb and forefinger. A total of 100 ml of solution were delivered drop-wise onto the tip of the nose and were inhaled. The animals that were exposed to US had their back and chest shaven, before the nasal instillation. Immediately after the vector was

Use of ultrasound to enhance nonviral lung gene transfer in vivo S Xenariou et al

delivered, a water-based contact gel (Aquasonic, Sonora Medical Systems Inc., Longmont, CO, USA) was applied onto the shaved area and the lungs were exposed to US (1 MHz, 3 W/cm2, 20% duty cycle), for a total of 2 or 20 min, as indicated. The US probe covered most of the chest but during the exposure it was moved around in a circular motion, to ensure that both the right and left lobes were exposed. During the exposure the animals were placed onto heated boards. At the end of the procedure, Antisedan (National Veterinary Services Ltd, UK), an antidote to the anaesthetic, was administered (1 mg/kg), by intraperitoneal injection. All procedures were approved by the Home Office under the Animals (Scientific Procedures) Act 1986. The pDNA doses used in the absence of Optison were: 20 mg/100 ml for PEI/pDNA,42 80 mg/100 ml for GL67/ pDNA,41 and 100 mg/100 ml for naked pDNA.31 When Optison was added, the pDNA doses had to be reduced in some cases, in order to keep the final volume constant. Thus, in the presence of Optison, the pDNA doses were: 10 mg/100 ml for PEI/pDNA, 40 mg/100 ml for GL67/ pDNA and 100 mg/100 ml for naked pDNA. The animals were killed 24 h after transfection, by cervical dislocation, and the trachea and lungs removed. The smallest right lobe and the upper half of the left lobe, were used for histological analysis. The remaining lung was snap-frozen in liquid nitrogen, homogenized (Ultra-Turrax homogeniser, Science Lab, Houston, TX, USA) in 300 ml (trachea and right lobes) or 150 ml (half left lobe) of Reporter Gene Assay Lysis Buffer (Roche Diagnostics GmbH, Mannheim, Germany). The samples were freeze–thawed three times, spun at 16 000 g for 5 min and the supernatant stored at 801C for luciferase measurements.

Reporter gene expression assays Luciferase activity was measured using the Luciferase Assay System (Promega, Southampton, UK) according to the manufacturer’s instructions, and a single-tube luminometer (TD-20e, Turner, Steptech Instruments, Arleysey, UK) and the total protein content of the samples was quantified using the Bio-Rad protein assay kit (Bio-Rad, Hemel Hempstead, UK). Each sample was assayed in triplicate and expressed as RLU/mg of total protein. Histology The smallest right lobe and the upper half of the left lobe, were fixed in 10% formalin, and paraffin embedded. Transverse sections (5 mm) from the middle of the tissue were cut and stained with haematoxylin and eosin (H&E). The tissues were then scored semiquantitatively for degree and area of haemorrhage, using an arbitrary scoring system. The scores assigned for degree of haemorrhage were: 1 ¼ slight increase in alveolar wall thickness; 2 ¼ greater increase in alveolar wall thickness, but alveolar spaces still visible; 3 ¼ no alveolar space, lung solid. The scores for area affected by haemorrhage were: 0.5 ¼ o1/4; 1 ¼ o1/3; 2 ¼ between 1/3 and 2/3; 3 ¼ 42/3. In some cases, samples were given two scores, for instance 1–2 for degree of haemorrhage. In those cases the average score was used for that sample, in this instance 1.5.

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Statistical analysis Gene expression data are expressed as mean7standard error of the mean (s.e.m). Independent sample t-tests or one-way analysis of variance (ANOVA) with a Bonferroni post hoc correction, were carried out for all comparisons. Where necessary a log10 transformation of the raw values was carried out to ensure normal distribution of the data and equal variance between groups. If normal distribution and equal variance were not achieved after the log transformation, a non-parametric Mann–Whitney U-test or a Kruskal–Wallis plus a Dunns post hoc correction, were carried out. The null hypothesis was rejected at Po0.05.

Acknowledgements This work was supported by UK Cystic Fibrosis Trust, through a grant to the UK Cystic Fibrosis Gene Therapy Consortium.

References 1 Ferrari S, Geddes DM, Alton EW. Barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis. Adv Drug Deliv Rev 2002; 54: 1373–1393. 2 Ferrari S, Kitson C, Farley R, Steel R, Marriott C, Parkins DA et al. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium. Gene Therapy 2001; 8: 1380–1386. 3 Sinn PL, Shah AJ, Donovan MD, McCray Jr PB. Viscoelastic gel formulations enhance airway epithelial gene transfer with viral vectors. Am J Respir Cell Mol Biol 2005; 32: 404–410. 4 Wang G, Zabner J, Deering C, Launspach J, Shao J, Bodner M et al. Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo. Am J Respir Cell Mol Biol 2000; 22: 129–138. 5 Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X et al. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995; 1: 39–46. 6 Flotte TR, Zeitlin PL, Reynolds TC, Heald AE, Pedersen P, Beck S et al. Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)CFTR vector in adult cystic fibrosis patients: a two-part clinical study. Hum Gene Ther 2003; 14: 1079–1088. 7 Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE et al. Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Therapy 2000; 7: 1156–1165. 8 Hyde SC, Gill DR, Higgins CF, Trezise AE, MacVinish LJ, Cuthbert AW et al. Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 1993; 362: 250–255. 9 Porteous DJ, Dorin JR, McLachlan G, Vidson-Smith H, Davidson H, Stevenson BJ et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Therapy 1997; 4: 210–218. 10 Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebocontrolled trial. Lancet 1999; 353: 947–954. 11 Ziady AG, Kelley TJ, Milliken E, Ferkol T, Davis PB. Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA Gene Therapy

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