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Eur J Cardiothorac Surg 2005;27:768-773
© 2005 Elsevier Science NL

Application of HVJ-liposome mediated gene transfer in lung transplantation—distribution and transfection efficiency in the lung

Kenichi Ohmori*, Shin-ichi Takeda1, Shinichiro Miyoshi2, Masato Minami, Shigeru Nakane, Mitsunori Ohta, Yoshiki Sawa, Hikaru Matsuda

Department of Surgery (E1), Osaka University Graduate School of Medicine, Yamada-oka 2-2, Suita City, Osaka 565-0871, Japan

Received 20 June 2004; received in revised form 1 December 2004; accepted 23 December 2004.

* Corresponding author. Address: Department of General Thoracic Surgery, Kure Medical Center, Kure National Hospital, Aoyamacho 3-1 Kure, Hiroshima 737-0023, Japan. Tel.: +81 823 22 3111; fax: +81 823 21 0478. (E-mail: omorik{at}kure-nh.go.jp).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: A novel hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer system has been shown to have benefits of a high efficiency of transfection and low immunogenicity. The aims of this study were to determine the effect of re-transfection of the HVJ-liposome system via the airway, and to quantify the distribution of gene expression between transtracheal and transplantation approaches. Methods: ß-Galactosidase (ß-gal) plasmid DNA was introduced into lung tissues using the HVJ-liposome method. Two groups of Sprague–Dawley (SD) rats received intratracheal instillation of 10µg of the ß-gal gene, once on Day 0 in 1 group (Group Tb-1, n=4) and 3 times on Days 0–2 in another (Group Tb-3, n=4). In a third group of SD rats (Group Tx, n=5), an orthotopic left lung transplantation was performed after the donor lung was flushed with an HVJ-liposome complex solution and preserved for 1h. Gene expression and distribution in lung tissue was then quantified by counting the X-gal stained cells. Results: Both the transtracheal and transplantation approaches resulted in low levels of transfection in the vascular endothelial cells (0.2±0.1 and 4.0±1.8%), respectively, but a moderate degree of transfection to the airway (11.0±7.1 and 28.0±20.7%) and alveolar cells (3.0±1.8 and 6.0±3.6%). Three repetitive injections via the airway increased gene expression in airway epithelial cells of 41.0±12.0% compared with the single administration of 11.0±4.3%. Conclusions: Our results suggest that the repeated transtracheal gene transfection using HVJ-liposome may have benefits for treatment of problems after lung transplantation. In addition, gene transfer using a flushing solution during harvest may provide an opportunity for gene manipulation in the setting of lung transplantation.

Key Words: Gene transfer • HVJ-liposome • Hybrid vector • Lung transplantation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Recent advancements in molecular biology and genetics have enabled gene delivery in the field of thoracic organ transplantation, which is useful as a therapeutic tool and for analyzing the role of target molecules [1,2]. During transplantation, the opportunity to treat the donor organ at the time of harvest using genetic manipulation may ameliorate early graft failure. In addition, repeated transbronchial gene therapy is an alternative in the setting of lung transplantation.

However, the potential of gene therapy is severely limited by host immune response in case of re-transfection with an adenovirus, low efficiency in transfection, long incubation times, and a time lag for synthesis of the target protein following gene transfection [3–8]. The hemagglutinating virus of Japan (HVJ)-liposome gene transfer system [9] has shown such benefits as high transfection efficiency, short incubation time, no cell division requirement, no toxicity, and low antigenicity. Using this novel system, foreign genes have been successfully introduced into livers, kidneys, hearts, and lungs [1,10,11]. In the present study, we attempted to determine the effect of repeated administrations of the HVJ-liposome system via a transtracheal approach, and evaluate lung toxicity and optimize the concentration of the HVJ-liposome system in a flushing solution with reference to the transplantation setting. Further, we compared the distribution of gene expression in the transtracheal and trans-pulmonary artery (PA) in a lung transplantation setting.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals
Adult Sprague–Dawley (SD) rats (Charles River Japan, Yokohama, Japan) weighing from 250 to 300g were used in all of the experiments. The animals were anesthetized by an intraperitoneal injection of pentobarbital (20mg/kg) followed by inhalation anesthesia. After orotracheal intubation, the animals were ventilated with room air, with a tidal volume of 2.5–3.0ml and respiratory rate of 90–100breaths/min. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH, publication 85-23, revised 1985).

2.2. Preparation of HVJ-liposomes and construction of plasmids
HVJ-liposomes were prepared as described previously [12]. Briefly, 10mg of mixed lipids (ePC:DOPE:eSph:Chol:PS=13.3:13.3:13.3:50:10, molar ratio) was dried by reversed phase evaporation. Cholesterol (Chol), egg yolk phosphatidylcholine (ePC), egg yolk sphingomyelin (eSph), and dioleoylphos-phatidylethanolamine (DOPE) were purchased from Sigma (St Louis, MO). The dried lipids were hydrated in 200µl of balanced salt solution (BSS, 137mM NaCl/5.4mM KCl/10mM Tris–HCl, pH 7.6) containing ß-gal genes. Liposomes were prepared by shaking and sonication. Purified HVJ (Z strain) was inactivated by UV irradiation (110ergs/mm2 per s) for 3min just prior to use. The liposome suspension (0.5ml, containing 10mg of lipids) was mixed with HVJ [15,000 hemagglutinating units (HAU)] in a total volume of 4ml of BSS. The mixture was incubated at 4°C for 5min, and then for 60min with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation, after which the top layer of the sucrose gradient was collected. This HVJ-liposome solution was collected as a pellet by centrifugation at 19,350xg at 4°C for 30min, re-suspended with 2ml of BSS, and used for gene transfer with the addition of CaCl2 (final concentration; 1mmol).

A ß-galactosidase gene expression vector was obtained commercially (Promega Corporation, Madison, WI) and driven by an SV 40 promoter.

2.3. Experimental protocol
2.3.1. Experiment 1
Assessment of graft function in transplantation setting.

Pulmonary toxicity of the HVJ-liposome complex was first evaluated by assessing pulmonary gas exchange and histology to optimize the dose for therapy. In the present experiment, orthotopic left lung transplantations were performed according to a method previously reported [13]. All isografts were flushed with 20ml of a PBS solution and preserved at 4°C for 1h. Animals in Group H1 received an HVJ-liposome complex (1.0ml) containing the ß-gal gene (10µg with HVJ 750 HUS), which was added to the flushing solution just before harvesting. Those in Groups H2 and H3 received a higher dose of HVJ-liposome complex (1.0ml) containing the ß-gal gene at 50µg with HVJ 3750 HUS or at 200µg with HVJ 15,000 HUS, respectively. Control animals (Group C) received a 20ml PBS solution without containing HVJ-liposome in the same fashion. In all groups, the graft function was assessed by clamping the right (contralateral) hilum under ventilation with 100% oxygen (tidal volume 1.5ml, respiratory rate 100/min, positive end-expiratory pressure 0.5cmH2O).

2.3.2. Experiment 2
Transfection efficiency in transtracheal (single and 3 repeated administrations) and trans-pulmonary artery.

Plasmid DNA of ß-gal was co-encapsulated in liposomes with high mobility group 1 (HMG1) protein and introduced into lung tissues using HVJ-mediated membrane fusion. One group of rats received intratracheal instillation of 0.3ml of HVJ-liposome solution containing 10µg of the ß-gal gene once on Day 0 (Group Tb-1, n=4) and another the same administration 3 times on Days 0–2 (Group Tb-3, n=4). In another group that utilized a transplant setting (Group Tx, n=5), an orthotopic left lung transplantation was performed. All isografts were flushed with 20ml of PBS solution and preserved at 4°C for 1h. An HVJ-liposome complex (1.0ml) containing the ß-gal gene (10µg) was added to the flushing solution just before harvesting. The respective controls received HVJ-liposomes with empty gene cassettes in a corresponding fashion.

2.4. Histopathology
Two days after administration of the ß-gal gene, the animals were killed by an overdose of pentobarbitrate, after which the lungs were expanded and fixed by intratracheal instillation of 4% paraformaldehyde at a constant hydrostatic pressure of 10cmH2O. The specimens were then frozen at –80°C, and cut and stained with 5-bromo-4 chloro-3-indolyl-ß-D-galactoside (X-Gal; Sigma, St Louis, MO). Three-micrometer thick paraffin embedded sections from other specimens were stained with hematoxylin/eosin (H/E). Gene expression and localization in the lung tissues were quantified by counting the X-Gal stained cells. We evaluated 100 randomized areas (magnification 200x) at the proximal bronchial level, as well as in the terminal bronchiole and peripheral lung parenchyma with micro-vessels. The pathologists were blinded to the groups. Results are expressed as a mean±standard deviation.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Graft function and optimal concentration of HVJ-liposome
No significant difference in arterial oxygenation was observed in the Group 1 animals, who received an HVJ-liposome complex (1.0ml) containing the ß-gal gene (10µg with HVJ 750 HUS) in the flushing solution, as compared to the control PBS solution. However, graft function was deteriorated in Groups H2 and H3, which received an HVJ-liposome complex (1.0ml) containing 50µg of the ß-gal gene (HVJ 3750 HUS) or 200µg of the ß-gal gene (HVJ 15,000 HUS), respectively (Fig. 1).



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  		Fig. 1. Graft function assessment and optimal concentration of HVJ-liposomes. No significant difference in PaO2 was seen between Group H1 (10µg with HVJ 750 HUS) and the control flushing solution (418±31.7 vs. 405±8.1mmHg). However, graft function deteriorated in the animals in Groups H2 and H3, which had increased gene-containing doses (50µg with HVJ 3750 HUS and 200µg with HVJ 15,000 HUS, respectively).

 
3.2. Effects of repeated transfection via transtracheal approach
There was no gene expression found in the control animals that received the HVJ-liposome with empty gene cassettes (Fig. 2a). Fig. 2b shows photographs demonstrating gene expression in bronchial epithelium following a single administration of the ß-gal gene. Gene expression in the bronchial epithelium and alveolar macrophages following a single administration of the ß-gal genes was 11.0±7.1% in airway epithelial cells, 3.0±1.8% in alveolar epithelial cells, 22.0±8.6% in alveolar macrophages, and 0.2±0.1% in vascular endothelial cells. The repeated transfection protocol increased the stained airway epithelial cells to 41.0±12.2% (Fig. 2c), while 11.0±4.3% of the alveolar epithelial cells, 55±7.6% of the alveolar macrophages, and 1.0±1.0% of the vascular endothelial cells were stained, which represented an increase of 2–4-fold. There were no apparent inflammatory reactions in any of the groups.



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  		Fig. 2. No gene expression was found in the control animals (a). Photograph demonstrating gene expression in bronchial epithelium following a single administration of ß-gal genes (b). Three repeated administrations via the airway increased the expression in airway epithelial cells by 2–4-fold as compared to a single administration (c). All photographs shown as 100x.

 
3.3. Effect of transfection via pulmonary artery in transplantion setting
Based on the preliminary results of pulmonary toxicity caused by the HVJ-liposome solution, we chose a safe concentration of 1.0ml, which contained 10µg plasmid DNA, and added it to the flushing solution just before harvesting. Gene expression was shown in vascular endothelium (Fig. 3a), bronchial epithelium, and alveolar macrophages (Fig. 3b) as follows: 28.0±20.0% of airway epithelial cells, 6.0±3.6% of alveolar epithelial cells, 23.0±10.5% of alveolar macrophages, and 4.0±1.8% of vascular endothelial cells. Surprisingly, airway epithelial cells and alveolar macrophages were also stained. Fig. 4 summarizes transfection efficiency in the transtracheal and transplant settings.



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  		Fig. 3. Photographs of proximal bronchus, terminal bronchioli, and alveoli, demonstrating gene expression in vascular endothelium (a; 400x) and bronchial epithelium alveolar macrophages (b; 200x).

 


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  		Fig. 4. Comparison of transfection efficiency and transgene distribution in the lungs following a single transtracheal administration (Group Tb-1) and 3 times repeated administration (Group Tb-3), as well as in a transplantation setting (Group Tx).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Gene therapy is a strategy in which genetic material is transferred directly to cells within an individual patient in a fashion that permits the genetic material, either directly or through its products, to be of therapeutic benefit. In this sense, transplantation has become a target for gene manipulation during the harvest of donor organs. In a study of transplantation setting reported by Yano et al. [8], transgene expression was obtained 24h after reperfusion using 18-h cold storage models in terms of ischemia/reperfusion injury that evoked during the first 60min. In this regard, we have shown the immediate effects of HVJ-liposome-mediated oligonucleotides that act within 30–60min [14]. Kaneda et al. [9] developed a fusogenic liposome [hemagglutinating virus of Japan (HVJ)-liposome] that is a hybrid vector combining viral and non-viral technologies. Previous studies have shown that co-introduction of HMG-1 with DNA enhanced transgene expression by 3–5-fold [15,16]. HVJ-liposomes were 100 times more efficient than with a cationic liposome, and 20–50 times more efficient than with a cationic liposome-plasmid complex [15]. As compared to the studies by Boasquevisque [4,5], who used 660–1320µg of reporter plasmid DNA with 2–6hours of exposure time, we applied a low concentration of ß-gal genes (10µg) and an ischemic time of 1h, as an attempt to mimic conditions required clinically.

In previous experiments using gene transfer through the pulmonary artery with a high titer of HVJ, we found lung injury despite previous successes via intracoronary gene transfer [17,18]. Therefore, we considered that the characteristics of pulmonary arteries are different from systemic arteries, while HVJ, as the name implies, acts by hemagglutinating blood. As a result, we chose a low concentration for safe use in a transplantation setting via the pulmonary artery in the present study.

A few studies that utilized gene transfer into the lung have quantitatively described transfection efficiency and transgene distribution [3,5,6] (Table 1). Tsan and White [19] and Meyer et al. [20] showed that transgene expressions of reporter genes obtained after intratracheal instillation of a naked plasmid or liposome-plasmid DNA complex were similar. Further, Zabner et al. [21] demonstrated that airway transfection using a naked plasmid or liposome-plasmid DNA complex had beneficial effects with cystic fibrosis. In a transplantation setting, D'Ovidio et al. [22] reported that endobronchial transfection of TGF-ß1 attenuated acute allograft rejection. In addition, Griesenbach et al. [23] described contrast results, which demonstrated 5- and 40-fold increases of efficiency using liposome vectors, as compared to naked plasmid DNA. However, transgene distribution and transfection efficiency have not been clearly demonstrated.


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  		Table 1. Transfection efficiency and transgene distribution in the lung in the current reports
 
In adenoviral re-transfection, even though the use of immunosuppression improved the effect of second administration, it did not reach the level of transgene expression seen with the initial administration [12]. In contrast, immune reactions were not elicited following repetitive administrations of HVJ-liposome as seen in the present experiment as well as other organs [10,24]. In addition, transgenes are distributed mainly in bronchiolar or alveolar epithelium by both transtracheal and transPA. Schmidt [3] and Jeppsson [6] reported that transgene expressions using an adenovirus were predominantly alveolar type II cells rather than bronchiolar cells (Table 1).

Regarding the trans-PA approach in the transplantation setting, HVJ-liposome-mediated gene transfer resulted in transgene expression predominantly in non-vascular bronchiolar and alveolar epithelium, as well as macrophages, which were similar to the findings of Schachter using an adenoviral vector [14,25]. The lung is an organ with a unique architecture that is composed of a branching tree airway, and pulmonary and bronchial blood supplies. Two mechanisms have been postulated: (1) The HVJ-liposomes pass through the endothelial barrier and enter the pulmonary interstitial and lung parenchyma, or (2) HVJ-liposomes reach epithelial cells through bronchial and pulmonary artery communication [25]. We have found that HVJ-liposome-mediated gene transfection with 1h of preservation via the PA is effective for gene manipulation in bronchiolar and alveolar epithelia, as well as vascular endothelium (unpublished data). We believe that the present experimental setting mimics a clinical situation, particularly with the use of an oligo-deoxynucleotide (ODN)-based gene therapy strategy, and shows the advantage of the HVJ-liposome system [15,17].

Previous studies have failed to sufficiently deliver genes into the pulmonary vasculature using adenoviral vectors [3,4,8,25] or liposomes [4,5]. To target pulmonary vascular endothelium where the cytokine cascade is evoked, a more efficient gene delivery system is needed. In the current study, we used a first generation HVJ-liposome system, though a more powerful HVJ-cationic liposome system (second generation), which has a 2–3-fold higher transfection efficiency compared to the first generation [11], is now under investigation.

In conclusion, the HVJ-liposome gene delivery system with repeated transfection via the airway increased transfection efficiency into alveolar and airway epithelial cells, suggesting that repetitive use is important for treatment. Further, successful gene transfer into alveolar epithelial cells or graft macrophages using a flushing solution may be implicated for gene manipulation to prevent early episodes following lung transplantation.


   Footnotes
 
1 Present address: Department of Thoracic Surgery, Toneyama National Hospital, Toyonaka, Osaka 560-8552, Japan. Back

2 Present address: Department of Thoracic Surgery, Dokkyo Medical College, Tochigi 321-0293, Japan. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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