Successful and optimized in vivo gene transfer to rabbit ... - Nature

Gene Therapy (2001) 8, 1174–1179  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt

RESEARCH ARTICLE

Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse T Matsumoto1, K Komori1, T Shoji1, S Kuma1, M Kume1, T Yamaoka1, E Mori1, T Furuyama1, Y Yonemitsu2 and K Sugimachi1 1

Department of Surgery and Science, and 2Division of Pathophysiological and Experimental Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Several gene transfer methods, including viral or nonviral vehicles have been developed, however, efficacy, safety or handling continue to present problems. We developed a nonviral and plasmid-based method for arterial gene transfer by in vivo electronic pulse, using a newly designed T-shaped electrode. Using rabbit carotid arteries, we first optimized gene transfer efficiency, and firefly luciferase gene transfer via electronic pulse under 20 voltage (the pulse length: Pon time 20 ms, the pulse interval: Poff time 80 ms, number of pulse: 10 times) showed the highest gene expression.

Exogenous gene expression was detectable for at least up to 14 days. Electroporation-mediated gene transfer of E. coli lacZ with nuclear localizing signal revealed successful gene transfer to luminal endothelial cells and to medial cells. Histological damage was recognized as the voltage was increased but neointima formation 4 weeks after gene transfer was not induced. In vivo electroporation-mediated arterial gene transfer is readily facilitated, is safe and may prove to be an alternative form of gene transfer to the vasculature. Gene Therapy (2001) 8, 1174–1179.

Keywords: gene transfer; electroporation; arterial wall

Introduction Human gene therapy for cardiovascular disease is now a reality, however, gene transfer technologies related to efficacy and safety continue to present problems. Viral vectors,1,2 including adenoviral vectors,3 can achieve efficient arterial gene transfer in vivo, but there is the potential for immunologic and cyototoxic complications.4 Nonviral methods for in vivo gene transfer, such as hydrogel balloon-mediated gene delivery5 or cationic lipid,6 have low efficiencies of plasmid DNA transfer into the arterial wall. Electroporation-mediated gene transfer has been widely used to introduce DNA into various types of cells in vitro,7–11 and in vivo application of electronic pulsemediated gene transfer, using specially designed electrodes, has been shown to be effective for mouse muscle,12,13 chick embryos,14 rat liver,15 cardiac tissue,16 and solid tumors.17–19 Information on the usefulness of electroporation in arterial gene transfer has not been available. Electroporation has a variety of advantages over viral vectors, as follows: (1) any type of cell and tissue could theoretically be targets; (2) easy and rapidly facilitated techniques; and (3) potentially repeatable. Therefore, electroporation-mediated gene transfer technique may have the potential for use as a current vector for human studies. Correspondence: K Komori, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan Received 27 July 2000; accepted 10 May 2001

The present study was designed to examine: (1) whether electroporation is applicable for arterial gene transfer in vivo; (2) the optimal conditions of in vivo gene transfer, including voltage, pulse-on time (Pon time), pulse-off interval (Poff time), and concentration of plasmid DNA; (3) target cell species of electronic pulsemediated gene transfer; (4) histological damage, using a specially designed T-shaped electrode; and (5) whether or not neointima formation is induced by in vivo electroporation. We describe here the optimal condition of successful electronic pulse-mediated arterial gene transfer, using rabbit carotid arteries, and potential advantages of this novel technology for vascular gene transfer are discussed.

Results Optimal conditions for electroporation Effects of voltage: To optimize the voltage of electric pulses used for electroporation in vivo, we assessed luciferase activity 2 days after electroporation at various electrode voltages. In this experiment, the pulse length (Pon 5 ms), number of pulses (10 times) and DNA concentration (200 ␮g/ml), which can all affect efficiency of gene transfer, were fixed. The luciferase activity increased nearly in proportion to the voltage up to 20 V, however, administration of over 30 V resulted in a marked decrease of gene expression (Figure 1). Optimal gene expression was achieved at 20 V, the luciferase activity being 5.97 × 106 (RLU/mg) (n = 6). Luciferase activity at 0 V which rep-

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Figure 1 Luciferase activity in rabbit carotid artery 2 days after in vivo electroporation. The ordinate indicates light units measured using a luminometer, and the abscissa represents the voltage applied in gene transfer (Pon 5 ms, Poff 95 ms, No. 10, 200 ␮g/ml).

resented naked DNA infiltration showed a low level of gene expression. Effects of DNA concentration: Electronic pulse-mediated in vivo arterial gene transfer strongly depended on the amount of DNA infusion into the artery (Figure 2). Maximum luciferase activity was obtained at a concentration of 200 ␮g/ml of plasmid (n = 6). In this experiment, the pulse length (Pon 5 ms), number of pulses (10 times) and voltage (20 V) were fixed. Effects of pulse length: Next, we compared the effect of duration of pulse on time and pulse off time (Figure 3). The luciferase activity increased nearly in proportion to Pon time up to 20 ms; however, administration of Pon time 50 resulted in a decrease in luciferase activity. Thus, optimal gene expression was achieved at 20 ms, resulting in the luciferase activity of 9.04 × 106 (RLU/mg) (n = 5). Time-course of luciferase activity by in vivo electroporation Luciferase activity in the carotid artery was assayed at various time-points after gene transfer, under determined optimal conditions (voltage = 20 V, Pon = 20 ms, Poff 80

Figure 2 Dose dependence of luciferase activity in the rabbit carotid artery 2 days after in vivo electroporation of different amounts of luciferase (Pon 5 ms, Poff 95 ms, No. 10, voltage 20 V).

Figure 3 Effect of duration of pulse on time and pulse off time for luciferase activity in rabbit carotid artery 2 days after in vivo electroporation (voltage 20 V, No. 10, 200 ␮g/ml).

ms, and pulse frequency = 10) (Figure 4). The level of luciferase activity in the carotid arteries reached a maximal value after 48 h, followed by a decrease to less than 5%of the peak activity during the first week (n = 4). This level of expression remained stable during the next week (Figure 4) (n = 4). Nuclear targeted LacZ gene transfer to rabbit carotid arteries To confirm the localization of transgene expression in the rabbit carotid artery wall, pAct-NLS-LacZ was transferred into the carotid arterial wall of rabbit by electroporation, under determined optimal conditions (voltage = 20 V, Pon = 20 ms, Poff 80 ms, and pulse frequency = 10). Gross observation of the vessels transferred by electroporation and stained with X-gal solution revealed a diffuse and intense blue stain in the vascular wall, and under a dissecting microscope this blue stain was observed along the luminal surface of the artery (Figure 5a and b). In contrast, arterial segments infused with plasmid DNA (LacZ) only under distending pressure (Figure 5c) and infused with balanced salt saline (BSS) containing no plasmid DNA (Figure 5d) showed no blue area.

Figure 4 Luciferase activity in rabbit carotid artery on days 2, 7, 14, 21. The ordinate indicates light units measured by a luminometer, and the abscissa represents period (day) after transfer (voltage 20 V, Pon 20 ms, Poff 80 ms, No. 10, 200 ␮g/ml). Gene Therapy


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Figure 5 Dissecting microscopic (a, c and d) and light microscopic (b) findings of pAct-NLS-LacZ gene transferred rabbit arterial wall under exposure to voltage 20, Pon 20 ms, Poff 80 ms, No. 10. The arterial segments harvested 3 days after gene transfer, reacted with X-Gal solution. (a, b) LacZ, electroporation (+); (c) LacZ, electroporation (−); (d) BSS, electroporation (+). (a) Intense blue stain is diffuse on the arterial wall. Original magnification × 6. (b) High-powered view of X-Gal-stained arterial wall. Intense blue signal is detected in all layers of the arterial wall. Original magnification × 18. (c, d) No blue staining is visible in the arterial wall. Original magnification × 6.

Figure 7 Effect of in vivo electroporation on neointima formation 4 weeks after gene transfer. All arterial segments were fixed under continuous pressure and embedded in paraffin. Sections of (a, b) were treated with electroporation under determined optimal conditions (voltage = 20 V, Pon = 20 ms, Poff 80 ms, and pulse frequency = 10, 200 ␮g/ml). Section (c) was treated with naked DNA (luciferase) only under conditions of distending pressure. (a) Luc, electroporation (+); (b) BSS, electroporation (+); (c) Luc, electroporation (−). (a) Cross-section of luciferase (Luc) transferred artery by in vivo electroporation. (b) Cross-section of control artery (balanced salt saline: BSS) treated with electroporation. (c) Cross-section of a control artery treated with naked DNA (luciferase) only under conditions of distending pressure. Original magnification × 40. Counter-stained with elastica van Gieson.

Figure 6 A microscopic view of rabbit carotid arteries, and which corresponds to the respective vessels shown in Figure 5. All sections were counterstained with Kernechtorot for nuclei after X-Gal staining. (a) A crosssection of the pAct-NLS-LacZ gene transferred artery at 20V shows blue staining (arrows) in endothelial and medial layers. Intense blue staining is evident in the cytoplasm of endothelial and medial smooth muscle cells. Original magnification × 200. (b) No blue stained cells are visible throughout any layer of the vessel infused with balanced salt saline (BSS) only. Original magnification × 200. lu, lumen; m, media; ad, adventitia.

Microscopic examination of the vessels demonstrated frequent blue signals in endothelial and medial layers (Figure 6a). Control vessels, which were infused with BSS (Figure 6b) and with plasmid DNA (LacZ) only (data not shown), showed no blue staining throughout all layers of the vessel wall. Effect of in vivo electroporation on neointima formation Histological examination was made on arterial sections harvested 4 weeks after gene transfer under the conditions voltage = 20 V, Pon = 5 ms, Poff 95 ms, and pulse frequency = 10. Neointima formation was not induced in rabbit carotid arteries subjected to electroporation (Figure 7a and b) and with naked DNA (luciferase) only under distending pressure (Figure 7c). These arterial sections were evaluated quantitatively using Mac Scope (Figure 8). Five sections spaced 5 mm per artery (each group contains total 15 sections) were examined. The ratio of neointima to medial area did not differ in either group (Luciferase, 0.14 ± 0.03; BSS, 0.16 ± Gene Therapy

Figure 8 Quantification of the intima-to-media ratio in luciferase, buffer(BSS)-treated arteries by in vivo electroporation, and the naked DNA-treated artery.

0.04; naked DNA 0.12 ± 0.03). There were no significant differences between them. Histological damage induced by in vivo electroporation Decrease in luciferase activity at 30 V (Figure 1) may have damaged smooth muscle cells (SMCs). There was slight


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Discussion

Figure 9 Histological damage 2 days after gene transfer by in vivo electroporation. (a) Histological damage is not evident throughout any layer of the vessel at 0 V. (b) Degeneration and disappearance of smooth muscle cells are recognized in a part of layers of the vessel at 20 V (arrows). (c) Degeneration and disappearance of the smooth muscle cells are frequently evident throughout all layers of the vessel at 40 V (arrows). (Pon 5 ms, Poff 95 ms, No. 10,200 ␮g/ml). Original magnification × 400. Counterstained with hematoxylin–eosin.

damage below 20 V (Figure 9a and b), but 40 V pulses caused extensive damage to SMCs directly sandwiched between the electrode plate (Figure 9c). Damage to SMCs was more extensive as the voltage was increased, and even increased when the duration of each pulse time was increased (data not shown). In this experiment, the pulse length (Pon 5 ms), number of pulses (10 times) and DNA concentration (200 ␮g/ml) were fixed. Histological damage was evaluated quantitatively using the ratio of the intima and media hematoxylin-positive cell numbers to intima and media areas. The intima and medial areas of both groups did not differ (control, 0.44 ± 0.03 mm2; 0 V, 0.49 ± 0.01 mm2; 20 V, 0.43 ± 0.02 mm2; 40 V, 0.47 ± 0.04 mm2; n = 3 for each group), whereas the intima and media hematoxylin-positive cells were reduced as the voltage was increased (control, 1018.3 ± 9.7 cells; 0 V, 927.2 ± 73.1 cells; 20 V, 805.1 ± 47.2 cells; 40 V, 462.1 ± 43.4; n = 3 for each group). The ratio of the intima and media hematoxylin-positive cell numbers to intima and media areas was gradually decreased (control, 2332.5 ± 157.0 cells/mm2; 0 V, 1920.1 ± 185.2 cells/mm2; 20 V, 1871.6 ± 170.1 cells/mm2; 40 V, 993.4 ± 121.5 cells/mm2; n = 3 for each group) (Figure 10) (*P ⬍ 0.05).

Figure 10 Quantification of the ratio of intima and media hematoxylinpositive cell numbers to intima and media area in the control artery (Cont), 0 V, 20 V and 40 V. (Pon 5 ms, Poff 95 ms, No. 10, 200 ␮g/ml) (*P ⬍ 0.05). The data are mean ± s.e.m., and a value of P ⬍ 0.05 indicated a significant difference.

Gene therapy can be effective for the treatment of cardiovascular disease, such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Although reports have suggested successful gene therapy strategies for vascular disease in preclinical animal models, there are limitations of use of gene transfer techniques for vasculature, with regard to efficacy and safety for humans.20 In vivo transfection of plasmid DNA into the arterial wall appears to be safe and has been shown to have significant biological effects in several animal models,21–24 however, currently available nonviral gene transfer methods, such as hydrogel balloon delivery or cationic lipid showed low gene transfer efficiency in vivo.5,6 Thus we asked if electronic pulses would enhance nonviral gene transfer into the arterial wall. In the present study, we characterized this nonviral in vivo gene transfer technique into the rabbit arterial wall. Although voltage-dependent damage in the arterial wall was apparent, this technique was reported to be applicable to arteries of living animals, as well as to skeletal muscles or livers.12–15 As we described here, voltage, Pon time, and plasmid DNA concentration were critical parameters for the efficiency of electronic pulse-mediated arterial gene transfer, and evaluation concerning each target vessel, especially human vessels will be needed before clinical application can be considered. We find that transgene expression was increased in nearly a voltage-dependent manner up to 20 V; however, administration of over 30 V resulted in a marked decrease in luciferase activity, possibly because of cell death in the arterial wall due to apoptosis induced by electroporation.25,26 Histological examination and histochemical X-gal staining with 20 V resulted in mild tissue damage, and cells seem to be diffusely distributed in endothelial and medial layers. Gene expression in the adventitial layer was not apparent, probably due to lack of plasmid DNA between the electrode and outside of the arterial wall. However, we did achieve gene transfer to the adventitial layer with infusion of plasmid DNA between the electrode and outside of the arterial wall (data not shown). The total number of transfected cells in the positive electrical field exceeded that in the negative electrical field. With opposite polarity, we achieved equal transfer of the gene into the arterial wall (data not shown). The level of luciferase activity in the carotid arteries was maximal at 48 h, followed by a decrease to less than 5%of the peak activity during the first week. Gene transfer by electroporation, which makes use of plasmid DNA as the vector, has several advantages, compared with use of viral vectors. A large quantity of highly purified plasmid DNA has been used in ongoing clinical studies. Also, gene transfer can be repeated without apparent immunologic responses to the DNA vector. There is less likelihood of recombination events with the cellular genome, thus eliminating the risk of the insertional mutagenesis that is associated with use of viral vectors. The one disadvantage of the vector is, as shown in this study, transient transgene expression. However, recent studies showed that the combination of Epstein– Barr virus (EBV) replicon vector based on the plasmid resulted in sustained gene expresion in nonlymphocytic cultured cells and the mouse liver;27 thus we are now Gene Therapy


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assessing the sustained gene expression using an EBV replicon vector in a similar animal model. In summary, we developed a method for gene transfer using electronic pulse, an approach which proved to be safe and more efficient than that using naked DNA. To our knowledge, this is the first demonstration that electroporation can achieve gene transfer into the arterial wall.

Material and methods Plasmid construction The full coding region of the firefly luciferase gene was isolated from pGL2 promotor vector (Promega, Madison, WI, USA) by HindIII and BamHI digestion. pcLuc was constructed by subcloning this luciferase gene into a multicloning site of pcDNA3 (InVitrogen, Carlsbad, CA, USA). A nuclear targeted LacZ gene driven with the chicken ␤-actin promoter, named pAct-NLS-LacZ, was as described.28 All these plasmids were purified using Qiagen Mega Kits (Qiagen, Hilden, Germany), and stored at −80°C until use. Electric pulse delivery and newly designed electrode Electric pulses were delivered through an electric pulse generator (CUY 201 BTX, San Diego, CA, USA). Pulses were delivered to rabbit carotid arteries at a rate of 10 pulses per second with newly designed T-type electrodes consisting of a pair of stainless steel flat plates of 2.5 cm long and 0.5 cm wide fixed with a 1 mm distance between them. Animals and procedures used for in vivo gene transfer The following experiments were reviewed by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University and were carried out under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University and The Law (No. 105) and Notification (No. 6) of the Government. The ‘Principles of Laboratory Animal Care’ and the ‘Guide for the Care and Use of Laboratory Animals’ (publication No. NIH 80–23, revised 1985) were also followed. Male Japanese white rabbits (2.5 to 3.5 kg body weight) purchased from Kyudo Co. (Tosu, Saga, Japan) were used. A midline incision to the neck was made, and the right common carotid artery was exposed. These rabbits had been thoroughly anesthetized by giving an intramuscular injection of xylazine (15 mg/kg) and ketamine (0.5 ml/kg). Some small branches of the common carotid artery were ligated and cut. After clamping the proximal site of the common carotid and external and internal carotid artery with clips, an 18-gauge double lumen catheter (Arrow International, Reading, PA, USA) was inserted through the first branch above the external and internal carotid artery bifurcation. The luminal space of the isolated arterial segment was rinsed with 5 ml of physiological saline containing 5 U/ml of heparin to wash out blood from the vessels. Plasmid DNA solution was instilled into the arterial space, the carotid artery was sandwiched between the electrode and electric pulses were deliverd to each carotid artery. Then the clips were removed, the branch was ligated, and arterial circulation was restored. An additional 2000 U of heparin were Gene Therapy

administrated intramuscularly 10 min before arterial clumping and at the time of skin closure. Luciferase assay Luciferase activity of rabbit carotid artery was measured, as described elsewhere.29,30 Briefly, at each point after transfection, the carotid artery was removed, minced and homogenized in 500 ml of 1 × Cell Culture Lysis Regent (Promega) and 20 ␮l of supernatant was examined for luciferase activity, using the Promega Luciferase Assay System and the Lumat LB 9507 (EG&G Berthold, Bad Wildbad, Germany) luminophotometer. All of the luciferase activities represent the number of parallel plates. X-Gal staining and histological examination The common carotid artery was gently and carefully dissected, then was cannulated with an 18 G catheter. Arterial segments were rinsed with normal saline for 10 min and perfused with 2% formaldehyde–0.25% glutaraldehyde (v/v) for 10 min at 150 mmHg positive pressure in vivo, then additional fixation was applied for 10 min. The arterial segments were incubated in X-Gal staining solution (0.2% v/v 5-bromo-4-chloro-3-indolyl-␤-d-galactoside (Nakarai Tasque, Kyoto, Japan), 1 mmol/l MgCl2, 150 mmol/l NaCl, 3.3 mmol/l K4Fe(CN)6, 3.3 mmol/l K3Fe(CN)6, 60 mmol/l Na2HPO4 and 40 mmol/l NaH2PO4) for 12 h at 37°C. The X-Gal-stained arteries were then placed in formaldehyde/ glutaraldehyde fixative and examined and photographed using a Zeiss Stemi 2000-C dissecting microscope (Zeiss, Oberkochen, Germany) then were sectioned into four or five serial segments at 5 mm intervals. These sections were embedded in paraffin and 5 ␮m-thick sections were counter-stained with nuclear fast red. Histological examination Four weeks after gene transfer, the rabbits were killed, the common carotid arteries, which had been gently and carefully dissected, were cannulated with an 18-gauge catheter. The arterial segments were rinsed for 10 min with normal saline and perfused with neutralized 10% formaldehyde for 10 min at a sustained positive pressure of 150 mm Hg in vivo, then additional fixation was applied for 6 h. The arterial segments were cut into four or five serial sections, at 5-mm intervals. These sections (5 ␮m thick) were embedded in paraffin and were stained with hematoxylin–eosin and elastica van Gieson for light microscopic examination. The intimal and medial area of five sections, at 5-mm intervals per artery, were quantified using Mac Scope (Mitanii Corporation, Japan) connected to a Macintosh computer system (Power Machintosh G3; Apple Computer, California, USA). Evaluation of histological damage Two days after the luciferase gene transfer, the rabbits were killed, and portions of the common carotid arteries were perfusion-fixed with neutralized 10% formaldehyde, paraffin-embedded, sectioned to 5 ␮m, and subsequently processed for light microscopy (hematoxylin– eosin staining). Histological damage was evaluated by the ratio of intima and media hematoxylin-positive cell numbers to intima and media areas. The intimal and medial areas were quantified as above.


Statistical analysis The results are expressed as the mean ± s.e.m. Statistical evaluation of the data was made using analysis of variance. If the value was statistically significant, a post hoc test for multiple comparisons (Fischer’s protected least significant difference) was used to identify differences among the groups. The values were considered to be statistically significant when the P value was less than 0.05.

Acknowledgements We thank Dr M Ishida, Department of Surgery and Science,Graduate School of Medical Sciences, Kyushu University for technical assistance and M Ohara for useful comments on the manuscript. This work was supported in part by a Grant-in-aid for General Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan.

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