Foreign gene transfer into cardiac tissues is regarded as a possible approach towards gene therapy for many cardiovascular disorders, including ischemic heart disease and heart failure. Various gene transfer methods have been developed for introduction of foreign gene into the heart. 1–3 Although viral vectors or direct injection of naked plasmid DNA into the heart are tested for in vivo gene transfer in a number of experimental studies, 4,5 immunologic and cytotoxic complications associated with viral vectors and the low efficiency of naked plasmid DNA direct injection have been reported. 6–8
Particle bombardment technology using a gene gun was first developed as a method of gene transfer into plants. 9 Recent studies have extended its use to several animal tissues including skin, liver, skeletal muscle, and pancreas. 10–14 However, there has been only one report on gene transfer into the beating heart using the in chamber gene gun system. 15 To the present authors’ knowledge, the hand held gene gun system has not yet been examined for the beating heart. Compared with other methods, the hand held gene gun is considered to be more simple and practical for clinical use. Moreover, because it is safe because of nonviral vector system, viral vector related side effects can be avoided.
In this study, the authors investigated the feasibility of nonviral gene transfer into the beating heart using the hand held gene gun.
Materials and Methods
The hand held gene gun (model Helios, Bio-Rad Laboratories, Hercules, CA) was used. DNA coated gold particles in a cartridge were accelerated by pressurized helium gas to supersonic velocities for penetration through cell membranes and multiple layers of cells in tissues. The discharge of the gold particles was initiated by pressing the trigger button, which opens the main valve, and the particles in the cartridge were entrained in the helium stream.
DNA Coated Gold Particles
Gold particles were sonicated for 20 seconds in an Eppendorf tube containing 1.5 ml of 70% ethanol and were precipitated by centrifugation (10,000 rpm, 3 seconds). To 50 mg of the washed gold particles, 100 μl of 0.05 M supermidine (Sigma) was added, vortexed (3 seconds), and sonicated (3 seconds). Then 100 μg of DNA (pCAGGS/ CTLA4-EGFP) was added, and the mixture was vortexed (3 seconds). This suspension was mixed with CaCl2 solution (1 M, 100 μl) and vortexed (3 seconds). After 10 minutes, the suspension was centrifuged at 10,000 rpm, and the resulting precipitate was washed with 70% ethanol and dried. The DNA coated gold particles were suspended in 200 μl of polyvinylpyrollidone (PVP) solution (molecular weight 360,000, 0.02 mg/ml 99.5% ethanol), transferred to 15 ml tubes, and adjusted to a final volume of 6 ml with PVP solution.
Preparation of the Cartridge
Details of the method are described in the manual of the Helios Gene Gun (Bio-Rad). Briefly, a suspension of DNA coated gold particles was introduced in Tefzel tubing placed on a “tubing prep station” and dried by rotation of the tubing under nitrogen flow (0.3 ml/min) for 15 minutes. Then the tubing was cut into 1.2 cm lengths (cartridges). The gold particles and DNA required for loading was 1 mg gold particles/shot and 5 μg DNA/mg gold particles.
Construction of Nonviral DNA Vectors
The extracellular domain of mouse CTLA4 was isolated from cDNA F41F4 16 and ligated into the pBluescript vector containing the enhanced green fluorescence protein (EGFP). From this construction, a Xho I/ Not I fragment containing CTLA4-EGFP was prepared. The pCAGGS vector, containing the cytomegalovirus enhancer and chicken β-actin promoter, was modified by the insertion of oligo-linker DNA into its EcoR I site. 17 The CTLA4-EGFP fragment was then inserted into the Xho I /Not I site of the modified pCAGGS expression vector. 18,19
In Vivo Gene Gun Mediated Gene Transfer
Adult male Wistar rats (8–10 weeks old) were used. After being anesthetized with diethylethel and ventilated, a left anterolateral thoracotomy was performed. Then the beating heart was exposed and gene transfer into the beating heart was performed by using the hand held gene gun (Figure 1). Each rat received a single bombardment (5 μg DNA/mg gold particles) into the anterior wall of the left ventricle. Three sizes of gold particles (0.6, 1.0, and 1.6 μm in diameter) and three settings of helium gas pressure (200, 250, and 300 psi) were examined at various combinations (n = 6, respectively). DNA uncoated gold particles were used as a control. Transgene expressions were observed by fluorescence microscopy 3 days after bombardment.
At 3 days after bombardment, bombarded hearts were resected for the evaluation of histologic damages. The resected hearts were fixed with 10% formaldehyde, embedded in paraffin, cross-sectioned to 5 μm, and subsequently processed for light microscopy (hematoxylin-eosin staining).
Examination of Biochemical Parameters and Electrocardiogram Monitoring
Blood samples were obtained before and 1, 3, 7, and 14 days after gene transfer. Biochemical parameters including the levels of creatinine phosphokinase (CPK) and MB fraction of CPK (CPK-MB) were measured by an automated serum analyzer. The electrocardiogram (ECG) was recorded before and after gene transfer under anesthetized conditions.
In this study, all rats survived, except for nine rats that died because of technical errors such as bleeding and pneumothorax after bombardment.
Expression of EGFP in the Myocardium after Gene Transfer
EGFP expressions were detected from day 1 to 3 weeks, and the highest expressions were detected at day 3 after bombardment (Figure 2). Moreover, EGFP expressions were not detected in other organs such as lung, liver, kidney, and skeletal muscle. These findings show that transgene expressions are not distributed to organs other than the bombarded heart. Whereas EGFP expressions were not detected in all hearts bombarded with DNA uncoated gold particles. Next, the present authors examined three sizes of gold particles and three settings of helium gas pressure at various combinations. At 3 days after bombardment, the most prominent expressions were detected with the combination of 1.0 μm gold particles and 300 psi helium gas pressure (Table 1). However, with this combination, transgene expressions were detected at only superficial cardiac muscle areas, most likely caused by the limited particle penetration into the cardiac tissue. The depth of the particles penetration did not exceed 100 μm (Figures 3A and 3B).
Histologic studies revealed that cardiac tissues were slightly damaged at the surface, and inflammatory response was slightly evoked at the bombarded area with various combinations of gold particles and helium gas pressure (Figures 3A and 3B). Whereas cardiac tissues around the bombarded area were severely damaged at the 400 psi of helium gas pressure.
Absence of Arrhythmia and Leakage of Muscle Specific Enzymes in the Blood
No arrhythmia was observed by ECG monitoring after gene transfer except the transient ventricular premature conduction at the time of the bombardment. No gross adverse effects on the animals’ health or behavior was noted after gene transfer. Moreover, the authors did not find any significant alterations in biochemical parameters including CPK and CPK-MB in the blood (Table 2).
Various gene transfer methods have been developed for introduction of foreign genes into the heart. 1–3In vivo gene transfer methods are classified into viral and nonviral categories. Viral vectors, including adenoviral vectors, or direct injection of naked plasmid DNA into the heart are tested for in vivo gene transfer in a number of experimental studies. 4,5 However, there is the potential for immunologic and cytotoxic complications 6,7 and the low efficiency of naked plasmid DNA direct injection may be unpractical. 8 Compared with those methods, the hand held gene gun is more simple and convenient. Moreover, because it is safe because of nonviral vector system, viral vector related side effects can be avoided.
The gene gun technology has various advantages over other methods. It is not cytotoxic and safe because of nonviral vector system. In addition, it requires only small amounts of DNA and takes only a few seconds to perform a single transfer. Because the gene gun technology is a physical method of gene delivery, it is independent of target cell type. We previously developed particle bombardment technology into skin, liver, and skeletal muscle tissues of mouse or rat in vivo. 19 Transgene expressions in epidermis and skeletal muscle were observed for 1 week, whereas it was observed in liver for 2 to 5 weeks, suggesting that gene gun mediated gene transfer induces particularly stable gene expressions in the liver.
However, the gene gun technology is rarely examined in cardiovascular field. It has been reported by Nishizaki et al. 15 that a similar technique, particle bombardment method using in chamber gene gun, can achieve the efficient transfer to the beating heart. They have reported that the deep penetration and expressions of β-gal DNA for 6 weeks were achieved by using the in chamber gene gun. In the present study, the authors achieved nonviral pCAGGS/ CTLA4-EGFP gene transfer into the beating heart using the hand held gene gun at various combinations of gold particles and helium gas pressure, and only transient expression was observed for 3 weeks in the heart. The duration of foreign gene expression depends upon the sensitivity of detection method, dose of DNA, transcriptional activity of the gene promoter in the tissue environment, and many other factors. However, there have been some reports that transient gene expression was observed for a few weeks by using a gene gun. 12,13 This may be one of the limiting functions of the gene gun system that is now available. Compared with the in chamber gene gun, because the gene gun the present authors used was hand held and bombarded to various directions, it was more simple and practical for clinical use. However, some degree of cardiac tissue damages may be occurred with the gene gun. In this study, cardiac tissue damages and inflammatory responses were slightly observed around the bombarded area, as in Nishizaki’s report. 15
Further investigations of transfer efficiency using a gene gun technique are now in progress. This technique is effective for gene transfer into the heart and may be one of the most useful methods for gene therapy for many cardiovascular diseases in the future.
In conclusion, the present authors showed that nonviral pCAGGS/ CTLA4-EGFP gene transfer into the beating heart was feasible with the hand held gene gun.
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