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(Circulation. 1995;92:479-482.)
© 1995 American Heart Association, Inc.

Articles

Efficiency of In Vivo Gene Transfection Into Transplanted Rat Heart by Coronary Infusion of HVJ Liposome

Yoshiki Sawa, MD; Ken Suzuki, MD; Hong-Zhi Bai, MD; Ryota Shirakura, MD; Ryuichi Morishita, MD; Yasufumi Kaneda, MD; Hikaru Matsuda, MD

From the First Department of Surgery (Y.S., K.S., H.-Z.B., R.S., H.M.), the Department of Geriatric Medicine (R.M.), and the Department of the Institute for Molecular and Cellular Biology (Y.K.), Osaka University Medical School, Osaka, Japan.

Correspondence to Hikaru Matsuda, MD, First Department of Surgery, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.

	Abstract

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Background Current methods of in vivo gene transfer into myocardium are limited by low efficiency. To improve in vivo gene transfer, a gene transfer method using hemagglutinating virus of Japan (HVJ) as a viral vector can be an alternative.

Methods and Results In vivo gene transfection of FITC-labeled oligonucleotide (F-ODN) and cDNA of ß-galactosidase (ß-gal) was examined with use of the HVJ liposome (H group) or without it (C group). In the H group, F-ODN or cDNA of ß-gal were complexed with liposomes, DNA binding nuclear protein (HMG1), and the viral protein coat of HVJ. After the harvest of donor rat hearts arrested by cardioplegia, the coronary artery was infused with the liposome gene complex. The hearts were transplanted into the abdomens of recipient rats and harvested 3 days after transplantation. Regarding F-ODN, the H group clearly showed FITC staining in the nuclei of the myocytes and endothelial cells in almost all layers of the myocardium as compared with the C group. Regarding the expression of ß-gal, the H group showed a clear expression of ß-gal on myocytes, whereas very low expression of ß-gal was seen in the C group.

Conclusions The donor hearts were transfected with F-ODN and ß-gal gene in almost all layers of the myocardium as a result of coronary infusion of the HVJ liposome during cardioplegic arrest. Our method is seen as a novel in vivo gene transfer technique for the heart and may provide a new tool for both research and therapy of heart transplantation.

Key Words: genes • myocardium • liposomes • transplantation

	Introduction

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The ability to express recombinant genes in cardiac myocytes in vivo holds promise for the treatment of several disorders of the cardiovascular system.^{1 2} Several groups have demonstrated recently that plasmid DNA is taken up and expressed in cardiac myocytes after direct injection into the left ventricular wall in vivo.^{3 4 5 6 7} The level of recombinant gene expression in the myocardium as a result of this technique appears to be higher than that observed in skeletal muscle after similar DNA injections.¹ However, these techniques still have several problems such as limitation to the transfected area in myocardium, the evocation of a potential inflammatory response, and a deleterious effect on myocardial function.^{1 2} These findings indicate that in vivo gene transfer in all layers of the myocardium must be modified by means of alternative techniques in an attempt to increase the efficiency of gene transfer for the entire heart and to decrease the inflammatory response.

We have established an efficient gene transfer method by using the hemagglutinating virus of Japan (HVJ) liposome, which has shown a high gene transfer activity without cytotoxicity in vivo.^{8 9 10 11 12 13 14} Functional DNA and non–histone chromosomal protein high mobility group 1 (HMG1) coencapsulated in liposome were introduced into the cytoplasm of rat liver cells by HVJ-mediated membrane fusion.⁸ The migration of HMG1 contributed to the efficiency of gene expression in hepatocytes of adult rats.^{8 9} For this reason, we believe that our method may help to improve the efficiency of gene transfer into the myocardium. In this study, we proved the hypothesis that the donor heart may be transfected with the HVJ liposome method by coronary infusion during cardioplegic arrest.

	Methods

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FITC Oligonucleotide
In the first experiment, we introduced FITC-labeled phosphorothioate oligonucleotide (F-ODN; 16 mer) into myocardium by using the HVJ liposome method to demonstrate the localization of transferred genes histochemically. F-ODN was kindly provided by Clontech Inc.^{13 14} ODN was labeled with FITC on the 3' and 5' ends of the ODN using fluorescein-ON phosphoramidite.

Construction of Plasmid
In the second experiment, we introduced cDNA plasmid of ß-galactosidase (ß-gal) by using this method to demonstrate the expression of transferred genes histochemically. ß-gal was prepared as described previously.⁹ Briefly, pAct-c-myb (a gift from Dr Ishii, Institute of Physical and Chemical Research) containing the 5'-promoter region (370 bp) and the first intron (900 bp) of the chicken ß-actin gene was restricted with XhoI/BamHI and cloned into the SalI/BamHI site of pUC19. This plasmid (pUC-Act-c-myb) was restricted with NcoI/XbaI to remove c-myb and was then ligated with SalI linker (8 mer, Takara Syuzo Co). The Escherichia coli ß-gal gene (3.1 kb), isolated from pMC1871 by restriction with SalI, was cloned into this site.

Preparation of HVJ Liposome
Liposomes containing plasmid DNA and high mobility group 1 (HMG1), which contributes to the enhancement of gene mobility in the cytoplasm to the nuclei, were constituted as previously reported.^{8 9 10 11 12} Briefly, dried lipid (phosphatidylserine, phosphatidylcholine, and cholesterol combined at a weight ratio of 1:4.8:2) was mixed with plasmid DNA (200 µg) (previously incubated at 20°C for 1 hour with HMG1), shaken vigorously, and sonicated to form the liposome. Purified HVJ (Z strain) was inactivated by UV irradiation (110 erg/mm² per second) for 3 minutes just before use. The liposome suspension mixed with HVJ was incubated at 4°C for 10 minutes and at 37°C for 30 minutes. The HVJ liposome complex was collected for use after removal of free HVJ. This preparation method has been optimized to achieve maximal transfection efficiency, as reported previously.

Experimental Protocol
Adult Sprague-Dawley rats weighing 250 to 300 g were used for this experiment. After the isolation of donor rat hearts arrested by cold cardioplegia, an aortic cannula was inserted and the HVJ liposome complex or DNA plasmid was delivered through the aortic cannula into the coronary arteries. Heterotopic cardiac transplantation into the abdomen of recipient rats of the same strain then was performed according to the method of Ono and Lindsey.¹⁵ Mean time of cold ischemia (less than 15°C) was 32±12 minutes. All hearts were harvested 3 days after transplantation.

Experimental Group
Twenty rat hearts were divided into two groups; the HVJ liposome method was used for the H group but not for the control (C) group. In both groups, five rats were transfected with F-ODN and the other five with ß-gal (Table). In the H group only, F-ODN or cDNA of ß-gal was combined with liposome, DNA binding nuclear protein (HMG-1), and the viral protein coat of HVJ.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Study Groups

Morphological Analysis of FITC Oligonucleotide
The hearts were isolated, fixed with 3% paraformaldehyde, frozen at -80°C, cut into thin sections, and examined with fluorescence microscopy.

Morphological Analysis of ß-Galactosidase
The hearts were fixed with 2.5% glutaraldehyde, frozen at -80°C, and cut into thin sections stained with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-Gal) for identification of the expression of ß-gal in the myocardium.⁹

	Results

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None of the hearts showed rejection at the time of harvest. All hearts were contracting vigorously before excision. Histologically, none of the animals showed irreversible damage, not even contraction band necrosis, in any layers of the myocardium (Fig 5).

View larger version (106K): [in this window] [in a new window]   Figure 5. Photomicrograph: Expression of the ß-galactosidase gene on the interstitial tissue and myocytes of the myocardium transfected with the hemagglutinating virus of Japan liposome method (x200). No irreversible damage or contraction band necrosis is present.

Efficiency of Gene Transfer Method
The H group showed apparent and diffuse FITC staining in the nuclei of myocytes and the endothelial cells of the coronary capillaries in all layers of the myocardium. Specifically, the nuclei of myocytes both in the endocardium (Fig 1) and the epicardium (Fig 2) clearly showed FITC staining. The nuclei of endothelial cells also showed FITC staining (Fig 3). On the other hand, the C group showed very weak FITC staining (Fig 4). The nuclei showed very low FITC staining in myocytes and endothelial cells.

View larger version (52K): [in this window] [in a new window]   Figure 1. Photomicrograph: Myocytes of the endocardium in a heart transfected with the hemagglutinating virus of Japan liposome method.

View larger version (67K): [in this window] [in a new window]   Figure 2. Photomicrograph: Myocytes of the epicardium in a heart transfected with the hemagglutinating virus of Japan liposome method.

View larger version (64K): [in this window] [in a new window]   Figure 3. Photomicrograph: Endothelial cells in a heart transfected with the hemagglutinating virus of Japan liposome method.

View larger version (57K): [in this window] [in a new window]   Figure 4. Photomicrograph: Myocytes in the myocardium of the control group transfected without the hemagglutinating virus of Japan liposome method.

Expression of Recombinant ß-Galactosidase Gene in Myocardium
In the H group, all hearts clearly showed histochemical expression of ß-gal on the interstitial tissues and myocytes (Fig 5). However, very low expression of ß-gal was seen in the C group (Fig 6).

View larger version (89K): [in this window] [in a new window]   Figure 6. Photomicrograph: Expression of the ß-galactosidase gene in the myocardium transfected without the hemagglutinating virus of Japan liposome method (x200).

	Discussion

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Gene transfer is the introduction of foreign DNA or gene sequences into host somatic cells. Recent advances in molecular biological techniques have brought us to the threshold of a new area of efficient gene transfer into somatic cells.^{1 2} The first trial of human gene therapy has been initiated recently in patients with inherited enzyme deficiencies and malignancies.¹ The topic of cardiovascular gene transfer has developed rapidly over the past 5 years, and numerous important advances offer substantial potential clinical applications both in cardiology and cardiovascular surgery.^{1 2 3} Despite the inherent challenges in grasping and mastering the fundamentals and methodology of this field, the topic is of immense relevance and clinical importance. Acquisition of the tools of gene therapy probably will be no more difficult for surgical researchers than was the acquisition of the tools of myocardial metabolism and ventricular mechanisms. In this situation, surgical procedure may offer a unique model to the surgeon for the investigation of gene therapy as a method of myocardial protection.

In this study, harvested donor hearts were transfected with both FITC oligonucleotide and c-DNA of ß-gal during cardioplegic arrest by the in vivo gene transfer method with use of the HVJ liposome. These genes were transferred into all layers of the myocardium. The result for FITC oligonucleotide, which indicates the extent of the transferred gene, demonstrated that the gene can be transferred into the nuclei of endothelial cells and myocytes. Moreover, the result of gene transfer of ß-gal proved the efficiency of protein synthesis after the transfer of the gene into the nuclei with this method. These findings proved our hypothesis that the heart is transfected with genes in all layers of the myocardium as a result of coronary infusion of the HVJ liposome during cardioplegic arrest. Our method appears to be a novel in vivo gene transfer technique for the heart.

However in vivo gene transfer into the myocardium facilitates the possibility of gene therapy for myocardium,¹ existing methods of in vivo gene transfer are limited by low efficiency and potential toxicity. For instance, a promising vector, adenovirus, was reported as having both antigenicity and cell toxicity.¹⁴ On the other hand, monkeys transfected with retrovirus showed T cell lymphoma as a result of contamination with a wild-type retrovirus.¹⁶ Regarding cell toxicity, we have proved the nontoxicity and lack of antigenicity of HVJ in the liver and the kidney.^{9 10} Direct injection of cDNA into the myocardium has been shown to cause focal inflammatory reaction and necrosis.¹ However, no necrotic areas were detected in this study. This suggests that the coronary infusion of HVJ liposome is noninvasive for the myocardium. As for efficiency, an alternative method, the cationic liposome method, has been found to have less than 1% efficiency for in vivo gene transfer.^{17 18} In this study, the nuclei of myocytes were stained with FITC in all layers of the myocardium. This efficiency and nontoxicity appears promising for clinical application of the HVJ liposome method for in vivo gene transfer into myocardium.

A recent report indicated the possibility of a single intravenous injection of expression plasmid for systemic gene expression including the myocardium.¹⁹ However, this procedure appears to have some problems such as targeting the heart with low efficiency for myocytes. An alternative is the infusion of cDNA into the myocardium by use of a coronary catheter with an inflated balloon under the beating heart.^{20 21} However, this method is still limited to the transfected area in myocardium, when all layers of the heart need to be transfected. For diffuse delivery of genes into all layers of the myocardium, the infusion of genes through the coronary artery²² appears to be superior to direct injection into the myocardium. Moreover, the specific period of adhesion to endothelial cells for viral vectors is necessary for improvement of this technique. These technical considerations may confirm our results that the surgical procedure, in particular delivery of cardioplegic solutions, promises a unique chance to the surgeon to perform in vivo gene transfection into myocardium.

In this study, a transplantation model was used because it was very difficult to maintain the transfected rat heart long enough for protein synthesis. However, this HVJ liposome method by coronary infusion during cardioplegic arrest appears to be feasible for the cardioplegia-arrested heart during open heart surgery. In the near future, this method may facilitate gene therapy for transplantation in order to introduce therapeutic genes.

In summary, donor hearts were transfected with FITC oligonucleotide and the ß-gal gene in all layers of the myocardium by coronary infusion of the HVJ liposome during cardioplegic arrest at the time of harvest. Our method is seen as a novel in vivo gene transfer technique for the transplanted heart and may provide a new tool for both research and therapy of heart transplantation.

	References

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