In Vivo Gene Transfer into the Ocular Ciliary Muscle Mediated by Ultrasound and Microbubbles

Ultrasound in Med. & Biol., Vol. 37, No. 11, pp. 1814–1827, 2011 Copyright Ó 2011 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter

doi:10.1016/j.ultrasmedbio.2011.07.010

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Original Contribution IN VIVO GENE TRANSFER INTO THE OCULAR CILIARY MUSCLE MEDIATED BY ULTRASOUND AND MICROBUBBLES LAURA KOWALCZUK,*yz MICH
ELE BOUDINET,x MOHAMED EL SANHARAWI,*yz ELODIE TOUCHARD,*yz MARIE-CHRISTINE NAUD,*yz AMENA SA€IED,x JEAN-CLAUDE JEANNY,*yz FRANCINE BEHAR-COHEN,*yzk and PASCAL LAUGIERx * Inserm U872, Physiopathology of Ocular Diseases: Therapeutic Innovations, Paris, France; y Centre de Recherche des Cordeliers, Universite Pierre et Marie Curie, Paris, France; z Universite Paris Descartes, UMR S 872, Paris, France; x CNRS otel-Dieu, Department UMR 7623, Laboratoire d’Imagerie Parametrique, Universite Pierre et Marie Curie, Paris, France; and k H^ of Ophthalmology, Paris, France (Received 14 January 2011; revised 17 June 2011; in final form 23 July 2011)

Abstract—This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, b-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm2, 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US1MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (32.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases. (E-mail: francine. [email protected]) Ó 2011 World Federation for Ultrasound in Medicine & Biology. Key Words: Non-viral gene therapy, Eye, Ciliary muscle, Sonoporation, Ultrasound, Microbubble, Targeted protein delivery.

explored to reduce the frequency of intravitreous injections. Gene therapy is an optimal option for eye diseases and non-viral gene vectors are promising to overcome known limitations of viral vectors which are associated with immunogenicity, toxicity and potential risk of insertion in oncogenic sequences (Mehier-Humbert and Guy 2005) and limitation in gene size. Ultrasound (US)-assisted gene delivery has been recently developed as an alternative to non-viral approach (Newman et al. 2001). It has been known since the 1980s that ultrasound can promote the introduction of exogenous DNA to cells in vitro (Fechheimer et al. 1987). Cells exposed to US showed multiple surface pores (Tachibana et al. 1999). This process referred to as sonoporation is a physical method using an external US energy source to induce small and transient cell permeability, allowing foreign DNA to enter the cell (Miller et al. 2002). Sonoporation is potentiated by microbubbles (MB), which are stabilized gas bubbles of around 2.5 mm in diameter, originally developed as ultrasound contrast agents (UCA) for

INTRODUCTION In recent years, there have been exciting new advances for the treatment of blinding ocular diseases such as agerelated macular degeneration (de Jong 2006) and diabetic retinopathy (Frank 2004). Repeated intravitreal injections of therapeutic proteins are rapidly increasing in frequency as a route of intraocular delivery of therapeutic proteins. Intravitreous injections are commonly performed and well tolerated but they are associated with rare but severe complication risks such as retinal lesions, cataract formation and eye infection (Jonas et al. 2008). More problematic is the poor compliance of frequently repeated injections. A wide field of innovative methods is being

Address correspondence to: Francine Behar-Cohen, INSERM UMRS 872, Centre de Recherche des Cordeliers, Team 17, Physiopathology of Ocular Diseases: Therapeutic Innovations, Universite Pierre et Marie Curie, Universite Paris Descartes, 15 rue de l’ecole de medicine, F-75006 Paris, France. E-mail: [email protected] 1814

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medical imaging (Bao et al. 1997; Greenleaf et al. 1998; Lawrie et al. 2000). Promising results have been reported using microbubble-mediated sonoporation for gene transfer in various animal tissues in vivo, including heart (Shohet et al. 2000; Bekeredjian et al. 2003; Chen et al. 2003), carotid artery (Taniyama et al. 2002a; Huber et al. 2003), skeletal muscle (Taniyama et al. 2002b; Li et al. 2003; Lu and Blomley 2003; Pislaru et al. 2003; Wang et al. 2005), kidneys (Koike et al. 2005), brain (Manome et al. 2005), liver (Hauff et al. 2005), pancreas (Chen et al. 2006), knee joint (Saito et al. 2007) and tumor model (Duvshani-Eshet and Machluf 2007). Several recent studies have reviewed in detail the applications of low-intensity US in gene delivery (Newman and Bettinger 2007; O’Brien 2007; Pichon et al. 2008; Wu and Nyborg 2008). The intensity of ‘‘low-intensity’’ ultrasound is within the range of 0.5 to 3 W/cm2 and approved for medical applications (Ng and Liu 2002). The mechanisms by which low-intensity ultrasound (in a ‘‘non-thermal’’ context) mediate DNA transfer into target cells are not clearly understood. It is widely believed that a combination of cavitation and acoustic streaming are involved. It has been suggested that the main mechanical effect induced by ultrasound on cell permeability is related to acoustic cavitation (Paliwal and Mitragotri 2006). Cavitation is the interaction between an ultrasonic field in a liquid and gaseous inclusion (i.e., microbubble) within the insonated medium (Miller and Thomas 1995; Miller et al. 1996). At relatively low-intensity ultrasound, oscillating microbubbles on nearby cells undergo volumetric oscillations that can produce mechanical stress on nearby cell membranes. This has been recently shown through electrophysiologic studies to be associated with increased cell membrane permeability (Juffermans et al. 2006, 2008; Tran et al. 2007, 2008). At higher acoustic pressures, violent cavitation activity with collapse of US contrast agent microbubbles (inertial cavitation) can be generated (Miller 2007). Such bubble collapse can generate shock waves and fluid microjets, which are projected on nearby cells, producing openings in the cell membrane (Postema et al. 2005; Ohl et al. 2006; Postema and Gilja 2007). Most of the studies performed with insonation within the low megahertz range (thermal effects are minimized at these frequencies) showed the presence of a threshold for the occurrence of mechanical bioeffects that approximately coincides with the threshold for cavitation (Miller et al. 2002). In their study, Forbes et al. (2008) have reported that inertial cavitation is not the responsible mechanism of sonoporation and they hypothesize that microstreaming as a result of bubble oscillations is principally responsible (Forbes et al. 2008). Acoustic microstreaming produces fluid motion around acoustically driven microbubbles (Zauhar et al. 1998; Baker et al. 2001;

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Van Bavel 2007; Duck 2008), which may create transient pores in proximate cell membrane. This has been shown to facilitate molecular transport (Marmottant and Hilgenfeldt 2003; Van Wamel et al. 2006; ter Haar 2007). Thus, while the role of microbubbles in USassisted gene transfer seems to be real, the actual mechanisms of sonoporation still need to be elucidated. Eye tissues are a recently developed target for in vivo gene transfer. In this context, Bloquel et al. (2006) have developed a novel electrotransfer (ET) technique in the rat eye using for the first time the ciliary muscle as a target tissue for gene delivery and as a reservoir for production of therapeutic proteins into the ocular media: aqueous and vitreous humours (Bloquel et al. 2006). This tissue is an attractive target for non-viral gene therapy because it offers two main advantages. First, it is an easily accessible non-neuronal tissue without penetration in the vitreous cavity. Second, this muscle is located at the junction between the anterior and posterior segments of the eye, allowing protein secretion toward the aqueous humor as well as the vitreous and retina. The skeletal muscle fibers are good candidates for gene transfer by electroporation and for long-term, high level production of proteins (Mir et al. 1998, 1999). In the eye, the ciliary muscle is a particular smooth muscle with some characteristics of striated skeletal muscle (Bejjani et al. 2007). Electrotransfer for specific transfection of ocular ciliary muscle consisted in application of an electrical field through the ciliary muscle, performed between specifically designed electrodes after intramuscular DNA injection. Its efficacy was demonstrated by evaluating the effects of intraocular production of TNF receptors in two rat models of ocular inflammation (Bloquel et al. 2006; Kowalczuk et al. 2009; Touchard et al. 2009). Although electrotransfer to the ciliary muscle is an effective physical method of gene transfer, it requires invasive needle electrode placement into the target tissue to deliver electric pulses. In contrast, sonoporation should be less invasive than electrotransfer since the US energy is applied at the site of interest from an external ultrasound probe. Effective reporter gene transfer using US exposure in conjunction with microbubbles has been reported in cornea (Sonoda et al. 2006), conjunctiva (Yamashita et al. 2007) and retina (Sonoda et al. 2005). Recently, Hirokawa et al. (2007) have shown transit changes in vascular permeability induced by the combination of ultrasound and an intravascular microbubble contrast agent in the rabbit eye. However, sonoporation has not yet been applied to target the ciliary muscle. In the present study, we investigate the feasibility of sonoporation to the ciliary muscle of rat eyes. To our knowledge, there has been no previous study of whether lowintensity ultrasound exposure combined with commercial microbubble is able to increase gene transfer into the

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smooth ciliary muscle after intramuscular delivery of reporter genes. Plasmids encoding for reporter genes were used to evaluate transfection rate and localize transfected cells. Then, temperature measurements and detection of apoptotic cells were performed to evaluate the treatment tolerance. Lastly, results were discussed and compared with the state-of-knowledge of in vivo ocular gene transfer. Several limitations of the present study are identified and research directions are suggested for future work. MATERIALS AND METHODS Materials Animals. Female Lewis rats, weighing 150 to 200 g, were purchased from Janvier (Le Genest-Saint-Isle, France) at 7 weeks of age and roomed in our facility for 1 week before inclusion in the study. Experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Ethics Committee in Animal Experiment Charles Darwin (approval Ce5/2011/003). For experiments, rats were anesthetized by IM injection of a mixture of Ketamine 1000 (80 mg/kg; Virbac, Carros, France) and Largactil (0.5 mg/kg; Sanofi-Aventis, Paris, France). At the end of the experiments, rats were sacrificed by carbon dioxide inhalation. Plasmids. Three commercially available plasmids were used to study reporter gene expression in the ciliary muscle: the pCMV-Gluc-1 (Nanolight Technology, Pinetop, AZ, USA) encoding a secreted Gaussia luciferase under control of a cytomegalovirus (CMV) promoter (Clontech, Palo Alto, CA, USA), the pVAX1-LacZ (Invitrogen, Carlsbad, CA, USA) containing the LacZ gene under control of a CMV promoter (Clontech) and the plasmid pEGFP-C1 that is a 4.7 kb plasmid carrying the green fluorescent protein (GFP) gene under control of a CMV promoter (Clontech). After amplification in Escherichia coli DH5-alpha, plasmids were purified with Nucleobond AX1000 kits (Macherey-Nagel GmbH & Co. KG, Hoerdt, France) according to the manufacturer’s instructions and then prepared at appropriate concentrations, to inject 15 mg of plasmids in 10 mL of saline, i.e., 1.5 mg/mL without MB or 3 mg/mL with 50% MB. Microbubbles (MB). We used for all our experiments commercially available, ready-to-use, a second-generation microbubbles provided by Artison Corp. (Inola, OK, USA). The Artison MB is a lipid-shell US contrast agent filled with perfluorocarbon gas and measuring around 2.4 mm in diameter. The MB solution is approximately composed of 13.108 microspheres/mL. MB were prepared according to the manufacturer’s instructions. Artison MB were first re-dispersed by gently

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shaking the vial for 10 s, until obtaining a homogenous milky white suspension after which, the desired volume of the dispersion was withdrawn from the vial. Then, the bubbles were added to the plasmid solution and mixed gently for 10 s to allow their combination immediately prior to their injection. Ultrasound apparatus. Ultrasound (US) treatment was performed using a sonoporation system designed for research: the Sonitron 2000, from Artison with a plane unfocused transducer operating at either 1 MHz or 3 MHz (switchable). A 3-mm diameter probe was selected to deliver the acoustic energy to a small area for rat experiments. The transducer had output intensity Isata (spatial-average temporal-average intensity) ranging from 0 to 5 W/cm2, adjustable in 0.1 W/cm2 increments with duty cycles (percentage of on-time over one pulse period) varying from 5% to 100% and variable pulse-repetition-frequency. The advantage of a pulsed mode of US application is that adverse thermal effects can be reduced. The treatment time was adjustable from 0 to 20 min. Methods In vivo sonoporation to eye ciliary muscle. The experimental procedure, applied in both eyes by the same operator, is illustrated in Figure 1. During treatment, the eye was held in position using a surgical sheet. Fifteen mg of plasmid DNA alone or mixed with MB was first injected under a surgical microscope in the ciliary muscle of both rat eyes, using a 30-gauge disposable needle on a 100-mL microsyringe (BD-microfine syringe, NM Medical, Asni
eres, France). To reach the ciliary

Fig. 1. Schematic representation of the in vivo sonoporation procedure. (1) Fifteen mg of plasmid DNA, alone or mixed with MB, are injected in the rat ciliary muscle under a surgical microscope, using a 30-gauge disposable needle on a 100-mL micro-syringe. (2) The ultrasound tip is applied directly over the ocular surface after interposition of a transparent coupling gel. The US settings applied are: 1 MHz, 2 W/cm2 Isata for 2 min, duty cycle of 50% with a pulse width of 5 ms (on-time) and a pulse interval of 5 ms (off-time) and fixed PRF of 100 Hz. MB 5 microbubbles; PRF 5 pulse-repetition-frequency.

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muscle just below the sclera posterior to the limbus (junction where the transparent cornea joins the white opaque sclera), the injection was performed in the temporal superior side of the limbus. Then, the ultrasound tip was applied directly over the ocular surface after interposition of a transparent coupling gel (Gelaser, Alcon, RueilMalmaison, France) and the region injected was exposed to US. The US probe was not moved during treatment. We conducted all our experiments with best conditions of US plus MB-mediated gene transfer close to those reported in the literature. In all experiments, the US settings applied were as follows: 1 MHz, 2 W/cm2 Isata for 2 min, duty cycle of 50% with a pulse width of 5 ms (on-time) and a pulse interval of 5 ms (off-time) and fixed pulse-repetition-frequency (PRF) of 100 Hz. The peak-negative acoustic pressure was 0.7 MPa when measured with a membrane hydrophone (Precision Acoustics LTD, Dorchester, UK) in a tank containing distilled degassed water at room temperature (Table 1). Experimental design. Three sets of experiments were carried out on a total of 46 Lewis rats. Eyes were examined by naked eye observation immediately after each experiment and then 7 and 30 days after gene transfection. The first set of experiments aimed at assessing the kinetics of luciferase secretion by the ciliary muscle cells at day 7 (n 5 20 rats) and 30 (n 5 12 rats) after sonoporation. For this purpose, the pCMV-Gluc-1 plasmid was used and four experimental groups were formed for the day 7 assay. In the first group, after plasmid injection in the ciliary muscle, no additional treatment was performed to serve as controls (control group, n 5 10 eyes). In the second group, the injection was immediately followed by US exposure (US group, n 5 10 eyes). In the two last groups, plasmid DNA mixed with microbubbles were co-injected into the ciliary muscle followed by US treatment (US1MB group, n 5 12 eyes) or without US exposure (MB group, n 5 8 eyes). Similar groups: control group (n 5 8 eyes), US group (n 5 8 eyes) and US1MB group (n 5 8 eyes) were set up for the day 30 assay. The effect of microbubbles alone was not evaluated at this time point. One additional untreated rat was used as negative control for luciferase expression at each time point. All animals were sacrificed on day 7 or day 30 after treatment. Eyes were enucleated, then, ocular fluids (aqueous and vitreous humours) were removed for evaluation of luciferase activity by luminometry. In the second sets of Table 1. Summary of experimental ultrasound conditions used in the present study Frequency (MHz)

Intensity (W/cm2)

Peak negative pressure (MPa)

Exposure time (min)

Cycle (%)

PRF (Hz)

1

2

0.7

2

50

100

PRF 5 pulse-repetition-frequency.

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experiment, rat eyes were used to analyze the localization of reporter genes 1 week after sonoporation. After injection of the plasmid containing the LacZ gene into the ciliary muscle, eyes were exposed either to US plus MB (n 5 2 eyes), either to US (n 5 2 eyes) or to MB alone (n 5 2 eyes). The remaining rat, receiving no injection or exposure to US, served as a control. At day 7 after transfection, eyes were enucleated and prepared for histochemical analysis. Two more rats were treated in both eyes to localize GFP expression 7 days after sonoporation in presence of MB. In the last set of experiments, temperature rise was measured in the rat lens (n 5 3 eyes) and in the ciliary muscle region (n 5 4 eyes) during US exposure at the applied parameters. Additionally, at 1 and 8 days after saline injection containing 50% of MB in the ciliary muscle (n 5 8 eyes), with or without sonoporation, apoptotic cells were detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in the treated area and histological analysis were performed. Measurement of luciferase activity. At day 7 or 30 after treatment, the eyes treated with intramuscular injection of 15 mg pCMV-Gluc-1, with or without US and/or MB, were enucleated. Ocular fluids (aqueous and vitreous humors) were removed and, after centrifugation at 5000 g for 5 min at 4 C, supernatants were kept at –20 C until used. The luciferase activity was assessed on 10 mL from each sample placed in a white-96-well plate (CostarÒ, tissue-culture treated, white plate) with Renilla luciferase assay system according to manufacturer’s protocol (Promega, Charbonni
eres, France). The detector was a luminometer Wallac Victor2, 1420 Multi-label Counter (EG&G Wallac, Evry, France) which adds 50 mL of luciferase assay substrate (Promega) to the sample and integrates the light produced by the sample over 10 s with 2-s delay. Data analysis was performed using the Wallac 1420 workstation software. Results are given for each sample in counts per second (cps). Background luminescence was less than 120 cps. In situ histochemical analysis of reporter gene expression in the ciliary region Localization of beta-galactosidase activity. Seven days after treatment, the eyes were enucleated, fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in phosphate-buffered saline (PBS) for 1 h at 4 C and then rinsed three times in PBS. The whole eyes were subsequently incubated overnight in the dark at room temperature (37 C) in the presence of a chromogenic X-gal reagent (1 mg/mL of 5-bromo-4-chloro-3-indolyl-dgalactopyranoside, Sigma, Saint-Quentin-Fallavier, France) in PBS containing 5 mM of K3Fe(CN)6, 5 mM of K4Fe(CN)6, 2 mM of MgCl2 and 0.02% NP-40 for

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colorimetric detection of b-galactosidase activity. Next, the eyes were embedded in paraffin and sliced using a Microm HM-340E microtome (Leica Microsystems, Nussloch GmbH, Germany). Longitudinal 10-mm thick sections were cut parallel to the optic axis. One half of paraffin sections from each sample was mounted in glycerol/PBS (1:1) and the other half was stained with haemalum-eosin to respectively stain the nuclei and cytoplasm. During examination under a Leitz Aristoplan photomicroscope, images were captured using a Leica DFC 480 digital camera with either a 310 or 325 objective lens and an exposure time kept respectively at 2.55 ms and 14.9 ms. Localization of GFP expression. The eyes treated by pEGFP-C1sonoporation were enucleated and immediately embedded in Tissue-TekÒ Optimum Cutting Temperature (OCT) compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands). Longitudinal 8 mm sections were performed, parallel to the optic axis. To visualize cell nuclei, sections were stained for 5 min with 40 ,6-diamino-2-phenylindole (DAPI) solution diluted 1/5000 (Sigma-Aldrich, St-Quentin Fallavier, France), washed, mounted in glycerol/PBS (1/1) and examined under the fluorescence microscope Olympus BX51 coupled with a CCD camera (Olympus DP70). Temperature measurements. To measure the temperature increase during ultrasound exposure in the tissues of interest (i.e., lens and ciliary muscle), a digital thermometer with a 23-gauge T-type thermocouple needle (Fisher Scientific, Illkrich, France) was used in our experiments. After anesthesia, the miniature thermocouple was implanted laterally into the lens or ciliary region. The temperature was recorded prior to, during and following US exposure. Detection of apoptotic cells after sonoporation. The eyes were enucleated and immediately embedded in Tissue-TekÒ OCT. Frozen unfixed 8 mm sections were fixed 5 min at 220 C in methanol/acetic acid (2/1), incubated 2 min with 0.1% Triton X-100 in 0.1% sodium citrate, washed and then incubated for 60 min at 37 C with the reaction mixture allowing the terminal deoxynucleotide transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL enzyme plus TUNEL label; Roche Diagnostics, Meylan, France). After washing, sections were stained for 5 min with DAPI solution, washed again and mounted in glycerol/PBS (1:1) and examined under the fluorescence microscope. Histologic analysis. Eyes were fixed in PAF 4% and glutaraldehyde 0.5% solution in saline for 2 h, dehydrated and embedded in historesin (Leica Microsystems, Nussloch GmbH, Germany). Sections of 5 mm were stained with 0.5% toluidine blue (Serva, Paris, France)

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and observed with an Aristoplan microscope (Leitz) coupled with the Leica DFC480 camera. Statistical analysis. The program used for the analysis of the data was GraphPad Prism (GraphPad Software, San Diego, CA, USA). Results are expressed as mean 6 standard error of the mean (SEM). Data analysis was performed using a nonparametric one-way analysis of variance (ANOVA, Kruskal-Wallis test) followed by a Dunn’s multiple comparison test. Prior to the ANOVA, extreme outlier values were identified by Grubb’s test and excluded from the dataset (one point per group). Statistical significance was set at p , 0.05. RESULTS Clinical examination of treated eyes immediately and on days 1, 7 and 30 after treatment showed the absence of gross ocular damage or cataract. Assessment of the feasibility to transfer genes in the ciliary muscle by sonoporation Kinetics of luciferase activity in ocular fluids. Figure 2 shows Gaussia-luciferase luminescence in the rat ocular fluids, 7 days after injection of the pCMV-Gluc-1 plasmid (15 mg) into the ciliary muscle in four different treatment groups. The mean level of luciferase activity in ocular fluids was increased in both groups treated with US exposure, with or without MB, compared with the control group (DNA injection alone). In eyes that received US application after co-administration of

Fig. 2. Luciferase expression levels in ocular fluids, 7 days after treatments. Four different groups were analyzed: pCMV-Gluc-1 plasmid (DNA) injection alone (control group), DNA1MB (MB group), DNA1US (US group) and DNA1MB1US (US1MB group). Ultrasonic conditions were set at 1 MHz, 50% duty cycle, 2 W/cm2 Isata for 2 min with 50% MB. Black bars represent mean values in each experimental group. Hooks indicate significant increase in luciferase level for the different groups and associated p values (Kruskal-Wallis, Dunn’s test). n 5 number of eyes in each group (n 5 7 to 11). US 5 ultrasound; MB 5 microbubble (Artison).

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plasmid and MB (n 5 11), luciferase activity in ciliary muscle was significantly higher by approximately 2.6-fold compared with the control group (n 5 9, p , 0.05) or to the MB group (n 5 7, p , 0.005). In contrast, US alone (n 5 9) induced a lower increase in luminescence level (of 1.5-fold compared with the control group with no statistically significant difference). Only the difference with the MB group was statistically significant (p , 0.005). Little luciferase activity was detected without US exposure after injection of plasmid alone or mixed with MB. Our results show a high dispersion in luciferase activity, in particular for the US-treated groups. On day 30, luciferase gene expression has dropped in each experimental group by around 50% between days 7 and 30 after treatment (Fig. 3). ANOVA statistical analysis showed that the factor ‘‘time’’ has a very significant effect and accounts for 14.16% of the total variance (p 5 0.0029) and that the factor ‘‘group’’ has a significant effect and accounts for 10.84% of the total variance (p 5 0.0304). The interaction between both factors is considered not significant. Bonferroni post tests determined that luminescence was significantly decreased between day 7 and day 30 only in the ‘‘US1MB group’’ (p , 0.01). Localization of reporter proteins in the ciliary region. Seven days after ciliary muscle targeted sonoporation of a plasmid containing the LacZ reporter gene, b-galactosidase activity revealed by blue staining was detected within the field of US application in the ciliary region cells (Fig. 4A–F). This method led to effective expression of LacZ in the ciliary region in the case of US1MB application (Fig.4A, B, D and E) and US application (not shown), whereas no detectable b-galactosidase activity was observed in the ciliary region without any US application (Fig. 4C and F).

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As shown in (Fig. 4G–I), the localization of transfected cells was confirmed by microscopic observation of GFP expression, 7 days after sonoporation of the pEGFP-C1 plasmid mixed with MB. Characteristics and tolerance of ciliary muscle sonoporation Temperature rise measurements. The absorption of the ultrasonic sound energy may lead to tissue heating (Ter Haar et al. 2011) that may be damaging for ocular structures. To evaluate this potential risk, the per procedure mean temperature rise was recorded in vivo in rat lens (n 5 3) and in the ciliary muscle (n 5 4) region. Using chosen US parameters for insonation the recorded increases were 3.7 6 2.0 C and 7.3 6 1.5 C, respectively, during the 2 min of treatment. Tolerance of sonoporation into the ciliary muscle. Just after sonoporation of saline with MB, no structural damage was observed near the site of injection (Fig. 5A). At day 1, histologic sections showed that MB were not eliminated from the injection site and on the contrary had diffused in the ciliary body and up to the anterior vitreous (Fig. 5B and C, stars). However, only sparse and occasional TUNEL positive apoptotic cells were detected in both the control injected (Fig. 5E) and the treated eyes at (Fig. 5F) at day 1 but not at day 8 (Fig. 5G) demonstrating that either MB or US did not induce significant damages to ocular tissues. Importantly, no apoptotic cells were detected in the retina. By contrast, the TUNEL labeling highlighted apoptotic nuclei at the sclera surface one day after injection, alone (Fig. 6A) or combined with insonation (Fig. 6B). Very few apoptic cells were detected at the same level 1 week after these treatments (Fig. 6C and D). DISCUSSION

Fig. 3. Kinetics of luciferase expression in ocular media. Fifteen mg of pCMV-Gluc-1 were injected in the ciliary muscle of both eyes. The injection was followed by US alone or US in combination with MB compared with control rats receiving the plasmid vector alone. Values are mean 6SEM. Control group (n 5 8) is shown in white bars, US group (n 5 8) in grey bars and US1MB (n 5 7) in black bars. US 5 ultrasound; MB 5 microbubbles.

The main objective of the present study was to investigate the feasibility of gene transfer into ciliary muscle by sonoporation. To our knowledge, this is the first study on in vivo plasmid DNA transfer targeted to this ocular tissue mediated by US and MB. Our experiments involving the co-administration of microbubbles and plasmid DNA were carried out with reporter genes widely used as markers to assess the efficiency of gene-therapy technology. We have shown that external ultrasound exposure on the ciliary region following a single intramuscular injection of a plasmid mixture containing a reporter gene and MB is a simple and effective method to facilitate in vivo gene transfer into the rat ciliary muscle. The observation of protein secretion in ocular fluids (aqueous vitreous and humours) at day 7 after sonoporation of 15 mg plasmid

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Fig. 4. Microscopic localization of reporter gene expression in the ciliary region, 7 days after sonoporation with MB: 1 MHz, 50% duty cycle, 2 W/cm2 Isata for 2 min. (A–E) Localization of b-galactosidase activity on longitudinal paraffin sections, after injection of pVAX1-LacZ plasmid mixed with MB (50%), with (A, B, D and E) or without (C and F) sonoporation (A, B, D and E). Staining with haemalum-eosin (A and D) allowed localization of the ciliary muscle on slides directly mounted in glycerol/PBS (B and E). b galactosidase activity was revealed by blue staining and detected in muscle cells (arrows) and in few sparse cells around the ciliary body (arrow head). (C and F) No staining was seen in the control eye, excepted in corneal epithelium which was considered as no specific staining in all samples (*). (G–I) GFP localization on longitudinal cryosections. After nuclei staining with DAPI (G, injected control eye), GFP was detected in the fibers of the ciliary muscle (H and I, white arrows) and in few cells around the ciliary body (I, white arrowheads). Scale bars 5 100 mm. cb 5 ciliary body; cm 5 ciliary muscle; MB 5 microbubbles; GFP 5 green fluorescent protein.

reporter delivered into the ciliary muscle confirms the feasibility of this method. Luciferase uptake into the ocular media was about 2.6-fold higher in US1MB group compared with controls treated with luciferase injection alone. When no US was applied after DNA injection, gene expression was low. US and MB-mediated gene transfer to the ciliary muscles showed a relatively limited transfection efficacy and lack of sustained gene expression in comparison with ocular gene electrotransfer. Indeed, 1 week after electrotransfer (200 V/cm) of 15 mg pCMV-GLuc-1 in the ciliary muscle, luminescence measured in ocular media was 20-fold higher than after sonoporation (Touchard et al. 2010). Moreover, whereas electrotransfer allows a long-lasting gene expression, at least 8 months,

our results demonstrated a relatively short duration of gene expression since at day 30 post-transfer, the levels of gene expression dropped significantly. Nevertheless, a similar course of expression was observed by Sonoda et al. (2006) in an in vivo evaluation of GFP expression in the rabbit cornea. In contrast, Yamashita et al. (2007) observed that GFP expression in rat conjunctiva appeared on day 2 and returned to the control level on day 8 after sonoporation (Yamashita et al. 2007). These differences may be related to the use of different conditions including the administration procedure, the target tissue, US conditions or MB formulation. Data reported in the literature using sonoporation to increase gene transfer in several animal organs in vivo show various transfection rates. However, most experimental studies achieved

Sonoporation of eye ciliary muscle d L. KOWALCZUK et al.

Fig. 5. Histologic evaluation of the ciliary muscle sonoporation tolerance. (A–D) Histologic sections stained with toluidine blue of the treated area, 30-min after sonoporation (A), 1 day after injection of saline plus MB (1:1,) without (B) and with sonoporation (C), and 8 days (D) after sonoporation. White arrows indicate the injection site. Histologic sections revealed no structural damage just after the treatment (A) and allowed the localization of the site of injection at day 1 after the injection (B and C), which is characterized by the presence of MB (stars), but not at day 8 (D). (E–G) Fluorescence microphotographs of TUNEL assay on longitudinal sections of the ciliary region. One day after injection of saline plus MB (1:1,) without (E) and with sonoporation (F), One day after injection of saline plus MB (1:1,) without (E) and with sonoporation (F), sparse TUNEL-positive cells are observed in the ciliary muscle region (arrow heads) but not 8 days after sonoporation (G). Nuclei are stained with DAPI. Scale bars 5 100 mm. cb 5 ciliary body; cm 5 ciliary muscle; MB 5 microbubbles.

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Fig. 6. Fluorescence microphotographs of TUNEL assay on longitudinal sections of the sclera posterior to the limbus. One day after injection of saline plus MB (1:1,) without (A) and with sonoporation (D), numerous TUNEL-positive cells are observed at the surface of the injected site. Eight days after treatment, sparse TUNEL-positive cells were detected at this level in control (C) as in treated eyes (D). Nuclei are stained with DAPI. Scale bars 5 100 mm. MB 5 microbubbles.

approximately 10-fold enhancement in transfection efficiency in vivo (Shohet et al. 2000). Optimization of ultrasound parameters was beyond the scope of this feasibility study. We, therefore, conducted all our experiments with best conditions of US plus MB-mediated gene transfer close to those reported in the literature. It has been shown that an efficient and safe in vivo gene delivery depends on several parameters, including (1) the ultrasound exposure parameters (transmit frequency, mode of US, acoustic intensity, total exposure time); (2) the bubble properties; and (3) the therapeutic agent (plasmid DNA concentration). Efficient sonoporation has been reported with ultrasound at frequencies ranging from 0.5 to 4 MHz. Sonoporation efficiency near or at 1 MHz has been recently demonstrated in vivo in several organs including the cardiovascular system (Taniyama et al. 2002a; Bekeredjian et al. 2003; Tsunoda et al. 2005; Vancraeynest et al. 2006) and striated muscle (Taniyama et al. 2002b; Lu and Blomley 2003). Optimal gene delivery to the rat heart has been achieved with a relatively low-transmission frequency (1.3 MHz) compared with that obtained at higher frequencies (5 and 12 MHz) (Chen et al. 2003). The frequency of 1 MHz selected for our experiments is thus within the frequency range demonstrated to provide efficient sonoporation and optimal expansion of contrast microbubbles prior to destruction (Chomas et al. 2001).

The mode of ultrasound application is known to influence gene transfection. In an in vivo study, Chen et al. (2003) have observed higher efficiency gene delivery to the rat cardiac muscle in pulse-echo mode compared with continuous mode. By sonicating human glioblastoma cell line, cell transfection was enhanced by five times from 10% to 40% of duty cycle but at 75% duty cycle, high transfection was associated with high cell mortality (Kaddur et al. 2007). In another study using the voltage clamp technique and Xenopus oocyte model, the transmembrane current was increased with increasing duty cycle, while the recovering process of membrane pores and cell survival rate decreased at higher duty cycles (Pan et al. 2005). Similar observations were reported by Karshafian et al. (2009) using an in vitro cell model in suspension (Karshafian et al. 2009). In another report, Sonoda et al. (2006) have shown that a duty cycle of 100% was most effective for gene transfer to the corneal cells but cell damage was high. Therefore, it seems that the duty cycle may influence both gene transfection efficiency and transfected cell viability and that a trade-off between efficiency and viability has to be found. The duty cycle of 50% chosen for our in vivo study is within the duty cycle range demonstrated in the literature to provide efficient sonoporation. Further studies are warranted to determine the optimal one. In literature, the level of sonication energy applied to cells or to tissues is expressed in terms of acoustic pressure or intensity using different measurements units:

Sonoporation of eye ciliary muscle d L. KOWALCZUK et al.

such as mechanical index, Pascal or W/cm2 adding difficulties when comparing results. In our study, acoustic intensity is expressed in W/cm2. Many gene delivery studies using US have been conducted in vivo with intensity levels of 0.5–2.0 W/cm2. For example, sonoporation performed at 2 W/cm2 allowed skeletal muscle transfection (Li et al. 2003; Sonoda et al. 2005; Wang et al. 2005) and, in the presence of MB, the most efficient gene transfer to rabbit cornea (Sonoda et al. 2006). Therefore, an acoustic intensity of 2 W/cm2 was selected for the present study. However, cell mortality is correlated to transfection efficiency and increases with acoustic pressure (Kaddur et al. 2007). Additional work is needed to evaluate cell damage induced by US exposure and to optimize the level of intensity delivered to the tissue for an efficient gene transfection. Ultrasound exposure times were found to vary across studies from 1 s (Zarnitzen and Prausnitz 2004) to 30 min (Duvshani-Eshet and Machluf 2005; Duvshani-Eshet et al. 2006). In an in vitro gene delivery process, Duvshani-Eshet et al. (2006) have demonstrated that 30 min US application localizes the plasmid DNA into the cytoplasmic and nuclear compartment of the cell. In an in vivo study (Duvshani-Eshet et al. 2006), Yamashita et al. (2007) have recently reported that US applied for 20 s can transfer reporter gene to the rat ocular conjunctiva with safety and efficacy in the presence of bubble liposome (Yamashita et al. 2007). Kaddur et al. (2007) have found that transfection efficiency increased between 1 and 2 min of sonication while mortality rate does not change significantly in human glioblastoma cells with Br-14 bubbles (Bracco Research SA, Geneva, Switzerland). Considering gene transfer efficiency and cell toxicity, a 2-min exposure time was chosen for in vivo gene transfer to the cornea in presence of Optison (Sonoda et al. 2006). A 2-min ultrasound exposure time was selected for our experiments to provide efficient sonoporation with minimal cell damage. However, it must be noted that short-time application (,5 min) used by most of published studies may not be sufficient to drive the DNA uptake into the cell nucleus (Duvshani-Eshet et al. 2006). Longer treatment times need to be studied for DNA delivery into the nucleus. There seems to be a consensus on the fact that the presence of MB is required to enhance US-assisted gene transfection. The presence of microbubble cavitation nuclei potentiates the effects of US (Greenleaf et al. 1998). Besides US parameters settings, results are also influenced by MB type and concentration. In preliminary experiments conducted on a small number of animals, we have observed higher levels of luciferase expression in a 50% MB group compared with a 20% MB group 7 days after US application (data not shown). Our present results show that ultrasound alone produced

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only a slight enhancement of gene luciferase expression compared to control animals, whereas the combination of 1 MHz ultrasound with 50% Artison MB added to the plasmid DNA solution showed a 2.6-fold increase in luciferase activity (Fig. 2). Adding Artison MB has thus provided more efficient gene transfer. Similar results were obtained for gene transfer to rabbit cornea in vivo by Sonoda et al. (2006). US alone (Sonitron 2000 at 1 MHz, 2 W/cm2, 2 min, 50% duty cycle) enhanced corneal gene transfer slightly while US in the presence of 20% Optison microbubbles (GE Healthcare, Princeton, NJ, USA) increased gene transfer efficiency by twofold to threefold. One study only by Sonoda et al. (2005) has reported that ultrasound alone without MB (Sonitron 2000 at 1 MHz, 2 W/cm2, 480 s, 50% duty cycle) could provide better gene delivery into rat retina compared with US and 50% Optison MB in vivo (Sonoda et al. 2005). Not all commercially available MB exhibit the same efficiencies to achieve gene delivery into cells as shown by Li et al. (2003) and Wang et al. (2005). Microbubble properties (size, gas and shell compositions, and surface rigidity), their lifetime (Li 2003) and concentration could influence their efficiency for gene delivery (MehierHumbert et al. 2007, 2009; Sboros 2008; Alter et al. 2009). In an in vivo study, Yamashita et al. (2007) have recently reported that a newly developed bubble liposome (950 nm in average diameter) exposed to US generated by a Sonitron 2000 (1 MHz, 1.2 W/cm2 for 20 s, 50% duty cycle) can transfer reporter gene to the rat ocular conjunctiva with safety and efficacy. These results show that new developed bubbles, smaller in diameter than conventional MB (size ranges from 1 to 10 mm) could be more effective than conventional microbubbles for ocular gene delivery. Despite the disparity between the US conditions and the results reported in experiments on animals, most studies have been performed using US with commercially available Optison microbubbles (albumin-coated octafluoropropane gas microbubbles) for efficient gene transfer to skeletal muscle (Taniyama et al. 2002b), blood vessel (Taniyama et al. 2002a), kidney (Koike et al. 2005) and spinal cord (Shimamura et al. 2005). Histologic damage was observed in tissue infused with bubble concentration higher than 50% (Koike et al. 2005; Sonoda et al. 2006). A concentration of 50% bubbles in our experiments is thus in good agreement with our preliminary data and within the range demonstrated to obtain acceptable results for transfer efficiency with minimal cytotoxicity. To the best of our knowledge, we have performed the first evaluation of Artison bubbles for this application. Future research directions should include evaluation of other formulations of echo-contrast agents and development of new generation of bubbles as carriers of genes to targeted locations, in which drug or gene can be attached either outside or inside of the bubble shell. MB destruction

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induced by ultrasound application can then release the active material into the target site in a most efficient way (Unger et al. 2001; Bouakaz and de Jong 2007). In the present study, the dose of plasmid DNA (15 mg) was similar to that applied for successful gene electrotransfer in rat muscle studies. Defining a doserelated response for this delivery technique nevertheless remains important. In addition to the benefits of the combined use of ultrasound and microbubbles, our approach offers some advantages specific to ocular gene transfer by sonoporation. First, it requires only intramuscular needle for DNA injection and external US exposure, which is widely accepted in clinical practice. Its safety has been well established. US can be focused with precision within the targeted site, even of small size as are ciliary muscles, to produce local gene delivery and are widely used for clinical examinations and therapies. Several limitations of this study must be addressed.  No attempt was made to measure potential mechanical effects induced by ultrasound, especially to measure acoustic cavitation. Future experiments need to address these important issues through in vitro experiments.  A limited number of US application time (1 or 2 min), bubble concentrations (20% or 50%) were evaluated in preliminary studies on a small number of animals (unpublished data). Further optimization of USmediated gene transfer requires comprehensive studies of influential experimental parameters.  Transgene expression was assessed at only two time points of protein expression (7 and 30 days for the luciferase experiments). Several in vivo publications in different tissues have demonstrated variable levels of luciferase or GFP expression, with a peak expression between day 2 and 10 and then decrease in expression was also widely variable (Bekeredjian et al. 2003; Manome et al. 2005; Tsunoda et al. 2005; Yamashita et al. 2007). Therefore, kinetics of luciferase expression in the transfected muscle should be assessed at several time points during a 2–3-week period but with earlier time points such as at 2 and 4 days, respectively, after treatment. However, our gene transfer approach has two major limitations: the relatively low level of gene expression and the high expression variability.  The level of transfection achieved in this study was relatively low. As gene delivery to ciliary muscle cells has not previously been reported using an ultrasoundmicrobubble-mediated gene transfer method, we cannot make direct comparisons of gene expression levels. Future applications in animal models of ocular diseases should give additional information’s regarding the

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therapeutic potential of the achieved transfection. In the meantime, continued research is needed to increase and prolong transgene expression in ciliary muscle cells, such as (1) optimizing acoustic parameters and microbubbles characteristics (Mehier-Humbert et al. 2005a, 2005b); (2) administration of plasmid at multiple injection sites or repeated deliveries over time (which should be acceptable, given the noninvasiveness of the present procedure) (Bekeredjian et al. 2003) compared with electrotransfer using a needle electrode inserted into the ciliary muscle; (3) injection of plasmid DNA complex with cationic materials to the target site before US exposure (Yamamoto and Tabata 2006); and (4) combination of the sonoporator device with a realtime ultrasound biomicroscopy (UBM) imaging system to allow visualization of the ciliary region and to guide needle injection more precisely for local gene delivery.  Our results suggest a high variability of luciferase activity within each experimental group, especially in the US1MB group (Fig. 2). Potential sources of variability have to be reduced in future experiments. Despite our stringent experimental protocol, the variability in the results remains high. High variability in gene expression may be attributed to two main sources: technical variability and biologic variability. To minimize experimental variability, all experiments were conducted by the same operator with the same equipment and procedure. In particular, special care was taken to ensure reproducible experimental conditions during the various phases of the experimental protocol, including DNA-microbubble mixture preparation, localization of the injected site and ultrasound application. Variations in the injection speed might be an explanation for the variability in DNA expression as shown by Andre et al. (2006). All these sources of variability may be responsible for some variability of gene transfer to the target tissue as well as to the biologic variability, which is a well known large source of uncertainty in experimental studies. Note that a high variability has also been reported with gene electrotransfer (Touchard et al. 2010), suggesting that the variability finds its source not only in the technique itself but also in the diversity of the biologic response. To reduce the biologic variability of gene expression, rats included within each experimental group were as homogeneous as possible regarding age, race, sex and weight. Technical variability of next gene expression studies should be minimized through the rigorous application of study protocols. Close attention must be given to the procedure of preparation of MB-DNA mixture just before intramuscular injection. The injection speed of DNA has to be constant and slow to reduce variability (Andre et al. 2006). The uncertainty may be reduced by

Sonoporation of eye ciliary muscle d L. KOWALCZUK et al.

increasing the sample size with respect to ethical recommendations. Microbubble collapse may induce side-effects such as apoptosis or programmed cell death (Feril et al. 2003, 2007; Yoshida et al. 2007) and secondary cell necrosis (pathologic cell death). Membrane cell damage is the trigger for apoptosis (Tachibana et al. 2008). Cellular injury leading to apoptosis might complicate the evaluation of the efficiency of gene transfer by sonoporation due to brief gene expression before final apoptotic cell death and has to be minimized for ultrasonic gene transfer (Miller and Dou 2009). In our experiments, we did not detect apoptotic cells using the TUNEL assay in the ciliary muscle, suggesting that no apoptosis resulted from either the microbubbles or from their combination with ultrasound at this energy level. On the other hand, we have observed increased apoptosis at the conjunctival surface at one day, resulting from either the injection itself but also from the probe application or from potential damages. The conjunctival surface had totally recovered its normal structure at day 7 demonstrating that basal proliferative cells were not altered by the treatment. Further improvement in the probe geometry and application control should be developed to better analyze potential damages resulting from contact or from thermal effects. Importantly, no damages to the cornea, the ciliary muscle or the retina resulted from the treatment. Ultrasound energy may damage sensitive structures in the eye by being converted into heat. This is especially true for the lens because of its avascular nature (not cooled by blood flow), which through absorption of acoustic energy may become cataractous and for the targeted tissue (i.e., ciliary region) directly exposed to acoustic energy. Mean temperature rise measured in our in vivo experiments was 3.7 6 2.0 C in the lens and 7.3 6 1.5 C in the target tissue. Thus, acoustically induced rise in tissue temperature is not negligible. These temperature increases are consistent with those recorded by Zderic et al. (2004) during ultrasound-mediated drug delivery in vivo in the rabbit eye. Temperature increases of up to 9 C in the cornea were measured during ultrasound exposure (880-KHz, 0.56 W/cm2, continuous mode, 5 min). Interestingly, whilst temperature rose during the procedure into the lens, we did not observe any change in the lens transparency at any of the observation time and up to one month. This might be due to the fact that aqueous humour turn-over may cool down the lens quickly. Indeed, already a few seconds after the end of the procedure the normal lens temperature had recovered suggesting that this temperature elevation was of short duration. However, it is important to maintain safety levels during sonoporation. Therefore, to minimize temperature rises in sensitive tissues, a cooling system for the

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transducer may be a technical option allowing for temperature control during US exposure. The findings of this initial study on sonoporation to the ciliary muscle can guide future research directions for the optimization of DNA transfection in the ciliary muscle. Further studies are required therefore to elucidate what effect different acoustic parameters (frequency, pulse duration, intensity, exposure time, bubble properties) have on enhancing gene transfer with suitable expression levels and minimal cell toxicity in vivo. Optimization of US parameters require the design of a suitable ultrasound transducer, specifically for ocular gene delivery with appropriate characteristics adapted to the geometry of the eyeball, to the small size and superficial location of targeted ciliary muscle. Recent studies have identified optimum ultrasound exposure conditions for cell permeabilization in vitro (Zarnitzen and Prausnitz 2004; Duvshani-Eshet and Machluf 2005; Karshafian et al. 2009). Additional studies are needed to determine comparable conditions under in vivo conditions. In summary, this study demonstrates that the ocular ciliary muscle can be targeted by DNA sonoporation allowing for protein secretion into the ocular sphere. These encouraging results may serve as a guide for future work on the optimization of ultrasound exposure parameters for sonoporation of eye ciliary muscle. Our findings suggest that sonoporation targeted to ciliary muscles has potential as a non-viral, gene delivery procedure for the treatment of various ocular diseases. However, extensive safety studies on MB toxicity and optimization of the procedure through the addition of cooling are required to eliminate any potential harm resulting from heating. Acknowledgments—This work was supported by a grant from Institut National des Sciences de l’Ingenierie et des Syst
emes (INSIS) of the CNRS (PEPS09_301: Projets exploratoires premier soutien). The authors would like to acknowledge the support from Artison that provided the microbubbles.

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