Ann Thorac Surg 2000;70:1332-1337
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
In vivo gene gun–mediated transduction into rat heart with Epstein-Barr virus-based episomal vectors
Kazuhiko Nishizaki, MDa,c,d,
Osam Mazda, MD, PhDa,c,d,
Yoshiko Dohi, PhDb,c,d,
Tetsuji Kawata, MDa,c,d,
Kazumi Mizuguchi, MDa,c,d,
Soichiro Kitamura, MDa,c,d,
Shigeki Taniguchi, MDa,c,d
a Department of Surgery III, Nara Medical University, Nara, Japan
b Department of Public Health, Nara Medical University, Nara, Japan
c Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan
d Division of Cardiovascular Surgery, National Cardiovascular Center, Osaka, Japan
Address reprint requests to Dr Nishizaki, Department of Surgery III, Nara Medical University, Kashihara, Nara 634–8522, Japan
e-mail: nszk{at}naramed-u.ac.jp
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Abstract
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Background. Gene guns have been used to transfer genes into various organs, but there has been no report of successful gene gun–mediated gene transfer into the heart. In this study, we assessed the possibility of gene therapy using a gene gun and an episomal plasmid vector.
Methods. Gene transfer was performed using two sizes of gold particles and two plasmids (an episomal vector and a conventional plasmid vector). From the first to eighth week after the bombardment, rats were sacrificed. The excised hearts were subjected to X-gal staining and histologic examination. To ensure that plasmid was not distributed to organs other than the heart, the presence of the ß-gal sequence was examined by polymerase chain reaction analyses.
Results. Gene expression persisted for 6 weeks. The episomal vector apparently contributed to long-lasting expression. Infiltration of monocytes or leukocytes was very faint. The ß-gal DNA was detected in bombarded hearts but not other organs.
Conclusions. Gene gun–mediated transfer of the episomal vector into beating heart may provide a simple, efficient, and useful strategy for gene therapy.
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Introduction
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In vivo gene transfer into the beating heart is an attractive strategy for gene therapy for cardiovascular diseases. Many techniques for gene transfer have been reported, including direct injection of naked plasmid DNA and infusion of various viral or nonviral vectors. In this study, we tested the possibility of direct in vivo transfer of plasmid DNA into heart by a gene gun.
In a gene gun system, micron-sized gold particles are coated with plasmid DNA and then accelerated at high velocity toward target cells or tissues. Cells penetrated by these particles have a high likelihood of being transfected by the DNA thus introduced. Gold and tungsten particles are commonly used as the carrier of plasmid DNA. Because of the high specific gravity and small diameter, these particles easily penetrate into cells. Also, they are not cytotoxic. The gene gun was first devised to transfect plant cells, the walls of which act as a physical barrier to conventional transfection techniques [1]. More recently, it was demonstrated that gene transfer into various mammalian tissues could also be successfully achieved by the gene gun. These tissues include liver, skin, skeletal muscle [2], and pancreas [3]. To our knowledge, however, heart has not yet been targeted.
We employed an Epstein-Barr virus (EBV)-based episomal vector to obtain long-lasting transgene expression in vivo. The EBV-based episomal vector is a plasmid vector carrying oriP and the EBV nuclear antigen 1 (EBNA1) gene from EBV. The EBNA1 gene facilitates the maintenance of the episomes through binding to oriP. After being transfected into human cells, the plasmid persists extrachromosomally at low copy numbers owing to replication and nuclear retention of plasmid DNA [4].
We show here that gene gun–mediated transfer of the EBV-based episomal vector into rat heart results in long-lasting expression of a marker gene.
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Material and methods
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Animals and surgery
Male Wistar rats were used for this study. All rats were between 10 and 12 weeks old. They received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (National Institutes of Health publication 86 to 23, revised 1985). After being anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection), rats were intubated and ventilated with room air. An anterolateral thoracotomy was performed at the location of most pronounced cardiac pulsation and beating hearts were exposed. The pericardium was stripped, and the right chest and abdomen were pressed to push the heart out of the thoracic cavity. After bombardment in the anterior wall of left ventricular near the apex, a chest tube was placed to drain air and fluids, and the wound was closed in two layers.
Plasmids
The plasmid vectors, pSES.ß and pS.ß, were previously described [5]. Briefly, pSES.ß (Fig 1, left) is composed of the Escherichia coli ß-gal gene located between SR
promoter and the SV40 polyA additional signal, EBV oriP, EBV EBNA1 gene under control of the SR promoter, the ampicillin resistance gene, and the replication origin for E coli. The other plasmid, pS.ß (Fig 1, right), was constructed from pSES.ß by deleting EBNA1 and oriP.
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Fig 1. Plasmids used in this study. Maps of pSES.ß (left) and pS.ß (right) are shown. (Prom = promoter, polyA = SV40 polyA additional signal.)
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Bombardment
We used a gene delivery system (model TF-1) designed by Nihon Medical & Chemical Instruments Co, Ltd, (Osaka, Japan). With this device, high-pressure helium provides the motive force for gold particles. The size of gold particles influences the transduction efficiency as well as the degree of tissue damage. Although the larger particles reach the deeper layer, cell damage could be more serious. Therefore, two sizes of gold particles were examined (1.0 Au and 1.5 Au: 1.0 µm and 1.5 µm in diameter, respectively). Supercoiled plasmids were precipitated gold particles at a ratio of 20 µg DNA:5 mg gold particles. The DNA-coated particles were suspended in 500 µL ethanol, and 20 µL of the suspension was distributed on a 25-mm titanium plate (0.5-mm thickness). Each rat received two consecutive bombardments in the anterior wall of left ventricular near the apex. The size of bombarded area was about 0.5 to 1.0 cm2. The helium pressure was set at 1.05 MPa, and the distance between the surface of the heart and the titanium plate was 2 cm. As a control group, 10 rats (1.0 Au, n = 5; 1.5 Au, n = 5) were bombarded with uncoated gold particles.
Histologic analysis
For histologic examination, animals were sacrificed 1, 2, 3, 4, 6, or 8 weeks after bombardment. An 18-gauge catheter was inserted into the abdominal aorta and a median sternotomy was performed. After the pulmonary artery and inferior vena cava were cut, the heart was perfused with 50 mL iced PBS followed by 20 mL 4% paraformaldehyde (PFA) in a retrograde manner using this catheter. The hearts were excised, sliced (500 µm thick) by a vibratome slicer (DOSAKA, Kyoto, Japan), and refixed in 4% PFA for 1 hour. After washing three times in PBS at room temperature, all sections were incubated overnight at 37°C in X-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indoyl-ß-D-galactosidase [X-gal], 3 mmol/L K4[Fe(CN)6], 3 mmol/L K3[Fe(CN)6], 1 mmol/L MgCl2, and 0.1% Triton X-100 in PBS). After being washed in PBS, the samples were refixed overnight with 4% PFA at 4°C, dehydrated in ethanol, and embedded in paraffin. We judged whether the bombarded portion of the heart stained blue by microscopic observations.
Polymerase chain reaction
For polymerase chain reaction (PCR) testing, DNA was isolated from the lung, brain, liver, spleen, kidney and heart of animals 7 days after bombardment. Synthetic oligodeoxynucleotide primers were prepared corresponding to the ß-gal gene sequence (sense primer: 5'-GCC GAC CGC ACG CCG CAT CCA GC-3', antisense primer: 5'-CGC CGC GCC ACT GGT GTG GGC C-3') [6]. The reaction mixture consisted of 1 mg DNA, 1.5 mmol/L MgCl2, 2 mmol/L dNTP, 0.5 µmol/L of each oligonucleotide primer, and 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech Ltd, Uppsala, Sweden). As positive controls, 5 ng pS.ß and pSES.ß were also tested. The reaction was performed by Gene Amp 2400 (Perkin Elmer, Norwalk, CT). The amplification profile consisted of 25 cycles of denaturing at 98°C for 15 seconds, annealing at 65°C for 2 seconds, and extension at 74°C for 30 seconds. PCR products were analyzed by polyacrylamide gel electrophoresis.
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Results
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Bombardment did not affect rat survival
We used 133 rats in this study. All rats survived surgery and bombardment procedures, with the exception of 3 rats in early experiments that died of respiratory failure within a few hours after surgery. The remaining rats were maintained on a normal diet until subsequent steps in experiments.
Prolonged expression of ß-gal in heart transduced with EBV-plasmid by gene gun
Two sizes of gold particles and two plasmids (pSES.ß and pS.ß) were employed (Fig 1). One hundred and twenty rats were divided into four groups, with each group of rats receiving a different combination of plasmid/gold particle (pS.ß/1.5 Au, n = 30; pSES.ß/1.5 Au, n = 30; pS.ß/1.0 Au, n = 30; and pSES.ß/1.0 Au, n = 30). Five rats of each group were sacrificed 1, 2, 3, 4, 6, and 8 weeks after bombardment, and their hearts were subjected to X-gal staining and histologic examination.
Seven days after the bombardment, cardiomyocytes in the bombarded portion in all hearts stained blue (Fig 2). The staining layer in bombarded heart was within 1 mm depth from the surface. Gene expression persisted for 3 weeks (pS.ß/1.5 Au), 4 weeks (pSES.ß/1.5 Au and pS.ß/1.0 Au), or 6 weeks (pSES.ß/1.0 Au; Table 1). The hearts of control rats (1.5 Au, n = 5; and 1.0 Au, n = 5) did not stain at all (data not shown). Transfection with an EBV-based episomal vector resulted in more sustained expression of the marker gene than that with a conventional, non-EBV plasmid vector, provided that gold particles of the same size were employed. On the other hand, when the same plasmid was transfected, 1.0 Au was more effective than 1.5 Au, in terms of longevity of gene expression. The combination of 1.0 Au and pSES.ß yielded the most prolonged expression in this study. EBV episomal vector apparently contributed to long-lasting expression of ß-gal expression.
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Fig 2. X-gal staining of the bombarded heart. Rat hearts were bombarded with 1.0 Au (A, B, E, and F) or 1.5 Au (C, D, G, and H) gold particles coated with pSES.ß (A, C, E, and G) or pS.ß (B, D, F, and H). Seven days (A–D) or 4 weeks (E–H) later, rats were sacrificed and the hearts were excised. The hearts were cut into pieces and subjected to X-gal staining as described in Material and Methods. The bar indicates 500 µm.
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On histologic examination, many cardiomyocytes in the surface layer stained blue, while few cells stained in deep layers (Fig 2, Fig 3). Probably, DNA-coated gold particles could not reach the deep layers. Some cardiomyocytes carrying DNA-coated gold particles were observed (Fig 3C and D).
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Fig 3. Microscopic observation of bombarded heart stained with X-gal. Rats hearts were bombarded with 1.0 Au (A and B) or 1.5 Au (C and D) gold particles coated with pSES.ß (A and C) or pS.ß (B and D), and 3 weeks later, rats were sacrificed and the hearts were excised. The heart sections were subjected to X-gal staining and Kernechtrot staining so that nucleus stained pink. The arrow indicates gold particle. Magnification x 400, A–C; x 200, D.
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Vector DNA detected in bombarded heart but not in other organs
To ensure that plasmid was not distributed to organs other than the heart under the experimental protocol, we performed PCR and examined the presence of the ß-gal sequence. DNA was prepared from the lung, brain, liver, spleen, kidney, and heart of rats in each group sacrificed 7 days after bombardment. The ß-gal sequence was detected in bombarded heart but not in other organs or control heart (Fig 4). These findings indicated that the plasmid DNA was present exclusively in the heart.
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Fig 4. Polymerase chain reaction (PCR) analyses of DNA from various organs. DNA was prepared from the lung (lane 1), brain (lane 2), liver (lane 3), spleen (lane 4), or kidney (lane 5) of a rat whose heart was bombarded with pSES.ß/1.0 Au. DNA was also prepared from hearts bombarded with pS.ß/1.5 Au (lane 6), pSES.ß/1.5 Au (lane 7), pS.ß/1.0 Au (lane 8), pSES.ß/1.0 Au (lane 9), or 1.0 Au alone (lane 10). As positive controls, 5 ng pS.ß (lane 11) and pSES.ß (lane 12) were also tested. PCR was performed using ß-gal specific primers as described in Material and Methods. (M = molecular weight marker.)
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Comment
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In the present study, we showed that plasmid DNA can be introduced into cardiomyocytes using a gene gun and that transfer of the EBV-based episomal vector results in long-lasting gene expression in vivo. To our knowledge, this is the first study showing that in vivo gene transfer into heart can be achieved by a gene gun.
Various gene transfer vectors and delivery methods have been devised to transfer genes into the heart, including direct injection of naked DNA into heart muscle [7], infusion of HVJ-liposomes [8] or cationic liposomes [9] into coronary artery, and injection of HVJ-liposomes into pericardium [10]. Particle-mediated gene transfer has several advantages over other methods. First, the gene gun is a nonviral vector. Side effects associated with viral vectors (eg, generation of aberrantly recombinant, replication-competent viruses) can be avoided. Second, this technique may allow expression of foreign genes in a wide variety of cell types, including terminally differentiated nondividing cells. Third, it can transfer as large a quantity of DNA as can be carried by gold particles. Using submicrogram quantities of DNA per bombardment, 1,000 to 10,000 copies of DNA can be delivered into each target cell [11]. Fourth, genes can be introduced exclusively into the bombarded region without being redistributed to other organs.
In particle-mediated transfer, micron-sized gold particles are coated with DNA and accelerated at high velocity toward target cells or tissues. The process may not depend on particular biochemical structure or physical feature of the target cell membrane like liposome/DNA complex-mediated gene transfer [11]. It may be possible to employ a gene gun to transfect cells that are relatively resistant to other delivery systems. For example, it is difficult to transfer genes into infarct areas by coronary infusion, whereas our delivery method may permit perioperative gene transfer into such areas.
In a previous study, we transfected rat cardiac graft ex vivo with replication-incompetent adenovirus vector by coronary infusion [12]. After transplantation, massive infiltration of leukocytes was observed close to the transgene-positive cells. It was difficult to avoid the immune responses against the viral vector. In the present study, however, infiltration of monocytes or leukocytes was very faint, if any (Fig 3). No cell damage or inflammatory response was demonstrated by a histologic survey. This is another advantage of gene gun–mediated gene transfer.
We consider that the present method may be useful in treating severe ischemic heart disease by transferring genes to promote angiogenesis, such as basic fibroblast growth factor (bFGF) [13], vascular endothelial growth factor (VEGF) [14], and hepatocyte growth factor (HGF) [15] genes. For patients suffering from severe ischemic heart disease resistant to conventional therapy, transmyocardial laser revascularization (TMLR) is useful. TMLR not only increases the supply of oxygenated blood to the myocardium via left ventricular transmural channels but also induces angiogenesis. If gene gun–mediated transfection of angiogenesis factor genes can be combined with TMLR, angiogenesis may be more efficiently promoted by synergistic action of two systems. Moreover, TMLR may enable gene gun to transfect cells in deeper layers. The combination may greatly contribute to the treatment of patients with various heart diseases, especially severe ischemic heart disease. On the other hand, a gene gun may be equipped on a tip of catheter. Such a device may be feasible for the gene therapy against the coronary stenosis, as well as for endocardial delivery.
We previously demonstrated high transfection efficiency with EBV-based episomal vectors into various human lymphoma cell lines [16–18], hepatocellular carcinoma cell lines [19], primary fibroblasts from skin, bone marrow cells [5], and peripheral blood CD34+ cells [20]. Recently, we have also reported that high transient expression was observed in rat heart injected with naked EBV-based plasmid DNA [21]. In the present study, we found that transfection with the EBV-based episomal vector results in more prolonged gene expression in rodent cells than that with conventional plasmid vector. This is compatible with earlier finding by Saeki and coworkers [22] who reported sustained expression of a marker gene in rat liver injected with EBV-based episomal vector by means of the hemagglutinating virus of Japan (HVJ)-liposome [22]. By our hands, the gene gun was more efficacious than naked plasmid injection. We have two reasons for this high efficiency. First, we employed an episomal plasmid. Second, the gene gun–mediated delivery system enables transfer of the plasmid-coated particles into the nucleus, whereas direct injection of plasmid allows penetration of DNA into cytoplasm but not nucleus.
Direct in vivo gene transfer into heart is an attractive strategy for gene therapy. Particle-mediated gene transfer technology provides a physical means of DNA delivery, and the EBV-based episomal vector contributes to stronger and more long-lasting expression. Our findings suggest that the combination of the gene gun and EBV-based vector may be useful for gene therapy of cardiovascular diseases.
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Acknowledgments
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This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture (09307029, 09671389 and 11470277) and the Ministry of Health and Welfare, Japan.
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Accepted for publication April 3, 2000.
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Abstract
Introduction
