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Use of Ultrasound to Enhance Nonviral Lung Gene Transfer in Vivo

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Use of Ultrasound to Enhance Nonviral Lung Gene Transfer in Vivo 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 1Department 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 being attenuated by air in the lung, sufficient energy enhance nonviral gene transfer to the mouse lung. Cationic penetrates the tissue to increase gene transfer. US-induced lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked lung haemorrhage, assessed histologically, increased with plasmid DNA (pDNA) were delivered via intranasal instilla- prolonged US exposure. The left lung was more affected tion, mixed with Optison microbubbles, and the animals were than the right and this was mirrored by a lesser increase then exposed to 1 MHz US. Addition of Optison alone in naked pDNA gene transfer, in the left lung. The positive significantly reduced the transfection efficiency of all three effect of US was dependent on Optison, as in its absence US gene transfer agents. US exposure did not increase GL67/ did not increase naked pDNA transfection efficiency. We pDNA or PEI/pDNA gene transfer compared to Optison- have thus established proof of principle that US can increase treated animals. However, it increased naked pDNA trans- nonviral gene transfer, in the air-filled murine lung. fection efficiency by approximately 15-fold compared to Gene Therapy (2007) 14, 768–774. doi:10.1038/sj.gt.3302922; Optison-treated animals, suggesting that despite ultrasound published online 15 February 2007 Keywords: sonoporation; ultrasound; gene transfer; nonviral vectors; lung; cystic fibrosis Introduction Both viral and nonviral vectors have been used in CF clinical trials, delivered either to the nasal epithelium Our aim is to establish a clinically relevant treatment for or the lung of CF patients.5,6 Our particular focus is on cystic fibrosis (CF), a monogenic, lethal disease, in which nonviral vectors because of their capacity to be repeat- pulmonary failure is the major cause of mortality. We are, edly administered,7 a critical need in the treatment of a thus, interested in optimizing lung gene transfer, and lifelong condition. Proof of principle for the delivery and more specifically, gene transfer to the respiratory airway expression of cystic fibrosis transmembrane conductance epithelium. The lung offers the advantage of allowing for regulator (CFTR), the gene mutated in CF, with nonviral topical administration of therapeutic agents, usually vectors has been established, in preclinical models8 as via nebulization. In spite of this, it has proved to be well as in clinical trials.5,9 However, in general, nonviral a formidable target for gene delivery, with a series of gene transfer agents are not as efficient as viruses, since extracellular barriers such as mucociliary clearance and they lack the natural mechanisms to invade cells, and mucus, and in the case of CF, a layer of infected sputum. CFTR expression in clinical trials resulted in partial In addition, there are several intracellular obstacles, correction of the bioelectrical defect.10 Attempts to including clearance by uptake into endosomes, and the improve the efficiency of nonviral vectors are being nuclear membrane that must be breached.1 Efforts made, including the addition of peptide ligands to target to overcome these barriers with the use of mucolytic the vector to receptors localized on the apical membrane agents,2 viscoelastic gels,3 or agents that disrupt tight of airway epithelial cells,11 and the development of junctions, thus allowing access to receptors present on nanoparticles to improve nuclear entry.12 Here we have the basolateral membrane,4 have enhanced transfection assessed, if sonoporation, the use of high-frequency efficiency to airway epithelium, but further improve- ultrasound (US) increases nonviral gene transfer to the ments would always be welcome. lung. US is a versatile technique, and has several medical applications. High-frequency (41 MHz), low-intensity Correspondence: Dr U Griesenbach, Department of Gene Therapy, US is commonly used for imaging, for example to detect National Heart and Lung Institute, Imperial College, 1B Manresa foetal abnormalities. In contrast, US shock waves, at Road, London SW3 6LR, UK. E-mail: [email protected] lower frequencies but higher intensities, are used to Received 5 September 2006; revised 21 December 2006; accepted 21 destroy gallbladder or kidney stones (lithotripsy), December 2006; published online 15 February 2007 whereas a similar type known as high-intensity focused Use of ultrasound to enhance nonviral lung gene transfer in vivo S Xenariou et al 769 ultrasound (HIFU) is currently being evaluated in 100 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 13,14 have been used. In most gene delivery studies, 10 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 1 muscle cells.17 It has also been used to enhance nonviral gene transfer to tumours,18 the arterial wall,19 skeletal 20 21 muscle, and liver, in vivo. US has also been success- Luciferase (RLU/mg protein) fully used to increase adenoviral transfection efficiency to the myocardium.22 0.1 US is thought to act by transiently permeabilizing Naked pDNA PEI/pDNA GL67/pDNA the cell membrane, thus increasing vector uptake. The Figure 1 Optison reduces the transfection efficiency of naked mechanism behind this effect is thought to be cavitation, pDNA, PEI and GL67/pDNA complexes. Animals were instilled the formation and oscillation of gas bubbles, in a liquid with naked pDNA (100 mg/100 ml), PEI/pDNA (10 mg/100 ml) or medium. The size of the bubbles fluctuates in response to 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, the pressure fluctuations of the US wave, and ultimately compared to the respective control group of vector alone. n ¼ 6–8 23 the bubbles collapse, creating pores in cell membranes. mice/condition. The theory that cavitation is responsible for sonoporation effects is also supported by the fact that addition of gas- filled microbubbles, further augments US-mediated gene appeared healthy, we decided to apply US for 20 min delivery. Thus, microbubbles, and in particular, Optison, in further experiments. We, thus, went on to instill a have been used in several sonoporation studies.13,24 mixture of GL67/pDNA and Optison (40 mg pDNA/ Optison is an US contrast agent, used for medical 100 ml, 1:1 (v/v)), or PEI/pDNA and Optison (10 mg imaging, consisting of albumin microspheres, filled with pDNA/100 ml, 1:1 (v/v)) complexes with subsequent octafluoropropane.25 When compared to similar avail- exposure to US for a total of 20 min. Sonoporation, able agents, Optison was superior for US-mediated gene however, did not increase GL67 (GL67+Optison: transfer to skeletal muscle in vivo.26 A recent study also 5.4571.00 RLU (relative light units)/mg protein, GL67/ demonstrated that the effects of US on the cell membrane pDNA+Optison+US: 4.8571.69 RLU/mg protein) or are much more profound in the presence of Optison, PEI-mediated gene transfer (PEI/pDNA+Optison: providing further support for the role of cavitation.27 3.1470.90 RLU/mg protein, PEI/pDNA+Optison+US: Thus,wehavetested,forthefirsttime,theeffectof 2.9870.47). sonoporation on nonviral gene transfer to the lung. It is well established, that ultrasound is greatly attenuated when Sonoporation increases naked pDNA gene transfer in travelling through air.28 Reflection of the wave, and there- the air-filled lung when mixed with Optison fore, energy loss, will also occur as it passes through air/ tissue interfaces.28 Nonetheless, we assessed whether the We also tested the effect of sonoporation on naked pDNA m m energy that is able to penetrate the lung tissue was sufficient (100 g pDNA/100 l, 1:1 (v/v)). Importantly, a 20-min ¼ to enhance the gene delivery of cationic lipid 67 (GL67)/ US exposure led to a significant (Po0.001, n 7–8 mice/ pDNA or polyethylenimine (PEI)/pDNA complexes, and condition) increase in gene expression, of approximately naked pDNA, all previously used for lung gene transfer.29–31 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, Results we did not continue with this. In addition, we included a group of animals exposed to US for 2 min, to determine Optison reduces nonviral gene transfer in the mouse if the positive effect previously seen with 20 min, could lung also be achieved at shorter exposures. As shown in Initial studies were carried out to investigate the effect of Figure 2, the 2-min exposure also resulted in a significant Optison on nonviral vectors. Naked pDNA, PEI/pDNA (Po0.001, n ¼ 7–8 mice/group) increase in luciferase and GL67/pDNA, mixed with Optison at 1:1 (v/v) ratio, expression, compared to the control group.
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