- Email: [email protected]
Gene Transfer Into the Donor Heart During Cold Preservation for Heart Transplantation
Gene Transfer Into the Donor Heart During Cold Preservation for Heart Transplantation Satoshi Gojo, MD, Kazuo Niwaya, MD, Shigeki Taniguchi, MD, Kazuhiko Nishizaki, MD, and Soichiro Kitamura, MD Department of Surgery III, Nara Medical University, Nara, Japan
Background. Ex vivo gene transfer to heart grafts may hold promise as a means of changing alloreactivity or xenoreactivity after transplantation. However, it remains to be determined how effectively gene transfer can be accomplished within a short time in cold-stored grafts that are ready to be transplanted. Methods. We performed an experimental study using a replication-defective adenovirus (Adex1CALacZ) encoding the Escherichia coli b-galactosidase (b-gal) gene to perform gene transfer to heart grafts awaiting transplantation. Thirty hearts of Wistar rats were removed and their coronary arteries were perfused with University of Wisconsin solution containing 1 3 109, 1 3 1010, or 1 3 1011 plaque-forming units of the recombinant adenovirus at 4°C for 60 minutes. As a control, other hearts were perfused with University of Wisconsin solution with an adenoviral vector that did not contain the b-gal gene (Adex1w1) for the same period. After perfusion, the grafts were implanted in the necks of syngeneic adult rats. The grafts were removed each week after transplantation and their expression of b-gal was assessed by 5-bromo-4-chloro-3-indoyl-b-D-galactoside staining. Results. Successful gene transfer and expression of the
b-gal gene were demonstrated in adenovirus-perfused hearts. Gene transfer occurred preferentially in the cardiomyocytes over the endothelial cells and smooth muscle cells of the coronary vessels. In hearts perfused with 1 3 109 plaque-forming units of the adenovirus, gene expression persisted for 4 weeks after transfer, but it diminished gradually and was minimal by day 28. Histologic analyses revealed slight inflammatory reactions in the myocardium. In hearts perfused with 1 3 1010 and 1 3 1011 plaque-forming units of the adenovirus, b-gal diminished 3 weeks after transplantation and a prominent infiltration of leukocytes was recognized in the myocardium. Conclusions. This study demonstrated that the cardiomyocytes of heart grafts express an exogenous gene product after adenovirus-mediated gene transfer under hypothermic preservation conditions. However, immune or inflammatory reactions to recombinant adenoviruses must be taken into account when a large number of adenoviruses are injected into the coronary arteries.
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virus of Japan liposome-mediated transfection improve the efficacy of gene transduction in the transplant setting [8, 9]. A previous report from our laboratory using a recombinant adenovirus demonstrated that virtually all cardiomyocytes are infected, even in 10 multiplicity of infection in vitro [10]. It also has been reported that intracoronary gene transfer to transplanted murine [4] and rat [11–13] cardiac grafts is feasible at the time of harvest using a recombinant adenovirus. The study reported here examined the quantitative effects of the adenovirus and the safety of our transcoronary infusion protocol for gene transfer.
he development of technology for transferring genetic material into the heart and lung has opened new frontiers in the study of the pathogenesis and treatment of cardiovascular and pulmonary diseases [1, 2]. The application of gene transfer to the field of transplantation is uniquely appealing because of access to the donor organ at the time of harvest [3, 4]. Gene transfer to heart grafts, leading to the expression of proteins capable of inhibiting the immune response to alloantigens or xenoantigens, may result in local modulation of immunity after grafting [5]. First, we must establish a system of efficient gene delivery to the heart grafts. Although several studies have demonstrated successful gene transfer into the ventricular myocardium using direct injection of plasmid DNA, the number of myocytes transfected with this method appears to be too small for successful application to gene therapy [6, 7]. Liposome- and hemagglutinating Accepted for publication Aug 28, 1997. Address reprint requests to Dr Gojo, Department of Surgery III, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634, Japan (e-mail: [email protected]).
© 1998 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1998;65:647–52) © 1998 by The Society of Thoracic Surgeons
Material and Methods Adenoviral Vector The replication-deficient recombinant adenovirus, Adex1CALacZ, was constructed by in vivo homologous recombination in 293 cells between the expression cosmid cassette and the parental virus genome. The expression cosmid cassette was constructed by inserting the 0003-4975/98/$19.00 PII S0003-4975(97)01295-2
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expression unit, composed of the cytomegalovirus enhancer plus chicken b-actin promoter, a complementary DNA coding sequence of a b-galactosidase (b-gal) gene, and the rabbit b-globin polyadenylate signal sequence, into the SwaI site of pAdex1w, which was a 42-kilobase cosmid containing a 31-kilobase adenovirus type 5 genome lacking E1A, E1B, and E3 genes [10]. The recombinant viruses subsequently were propagated within 293 cells (ATCC; CRL1573) and viral solution was stored at 280°C. To prepare the adenovirus stock, a Bioruptor 200 (CosmoBio, Tokyo, Japan) was used for disrupting the 293 infected cells. The titers of the viral stocks were measured by an end-point cytopathic effect assay [14]. In this experiment, the titer of recombinant adenovirus was 1.0 3 1010 plaque-forming units per milliliter (pfu/mL). As a control vector, we used an adenoviral vector that did not contain an expression unit (Adex1w1), the titer of which was 4.3 3 108 pfu/mL.
Transfection to Heart Grafts and Heterotopic Heart Transplantation Wistar rats aged 8 to 10 weeks and weighing 200 to 300 g were used as donors and recipients. 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). The donor hearts were removed after the caval and pulmonary veins had been ligated. A cannula (16-gauge, Angiocath; Terumo) was introduced and tightened into the aorta, and the pulmonary artery was left open. After total vascular occlusion of the heart, perfusion of the heart was accomplished through the aorta using 50 mL of University of Wisconsin solution containing 1 3 109 (n 5 15), 1 3 1010 (n 5 15), or 1 3 1011 (n 5 15) pfu of Adex1CALacZ over 60 minutes at 4°C. Control hearts were perfused in an identical manner with University of Wisconsin solution containing 1 3 109 (n 5 15) pfu of Adex1w1. After perfusion to infect the heart, the grafts were washed out with 20 mL of University of Wisconsin solution and transplanted into syngeneic rats (aged 10 to 12 weeks) using a non–suture cuff technique [15].
5-Bromo-4-chloro-3-indoyl-b-d-galactoside Staining and Histologic Analysis Three mice were sacrificed each week after heterotopic heart transplantation. Histologic examination of the donor hearts was performed every 7 days after transplantation for 5 weeks. The donor hearts were dissected and fixed in 4% paraformaldehyde for 1 hour. The organs were sliced (500 mm thick) using a vibratome slicer (Oxford Co), refixed in the same solution for 2 hours, and washed in phosphate-buffered saline solution. They then were overlaid with 1 mg/mL of 5-bromo-4-chloro-3indoyl-b-d-galactoside (X-gal), 15 mmol/L of potassium ferricyanide, 15 mmol/L of potassium ferrocyanide, and 2 mmol/L of magnesium chloride in phosphate-buffered saline and incubated for 6 hours at 37°C. Slices positive for b-gal activity were embedded in paraffin, and sections (4 mm) were stained with hematoxylin and eosin to reveal any inflammatory reaction.
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Polymerase Chain Reaction A polymerase chain reaction technique was used to detect which other tissues of the host possessed the b-gal gene that was delivered by the recombinant adenovirus. A polymerase chain reaction was performed on DNA isolated from the lung, brain, liver, spleen, kidney, ovary (we used female rats as recipients), and donor heart of the experimental animals 7 days after transplantation. Synthetic oligodeoxynucleotide primer sequences were chosen from separate exons of the genes. For example, the complementary DNA product detected the Escherichia coli b-gal sequences (59-GCCGACCGCACGCCGCATCCAGC-39 and 59-CGCCGCGCCACTGGTGTGGGCC-39) [16]. Tubes with 1 mg of sample DNA and reaction mixture were placed in a Perkin-Elmer/Cetus thermal cycler. The reaction mixture consisted of 6 mmol/L of (NH4)2SO4, 10 mmol/L of KCl, 120 mmol/L of Tris-HCl (pH 8.0), 1% Triton X-100, 0.01% BSA, 1 mmol/L of MgCl2, 200 mmol/L of dNTP, 50 pmol/L of each oligonucleotide primer, and 2.5 U of KOD DNA polymerase (Toyobo Co, Ltd, Japan). 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. Samples were analyzed by acrylamide gel electrophoresis.
Results Survival The total ischemic time of the donor hearts ranged from 100 to 120 minutes. All animals to be grafted survived until they were sacrificed. The grafts that were perfused with 1 3 109 pfu of the adenoviruses (Adex1CALacZ or Adex1w1) had normal contractility when the animals were sacrificed. In contrast, those that were perfused with 1 3 1010 or 1 3 1011 pfu of the adenoviruses had bradycardia and an irregular rhythm beginning 14 days after transplantation, but they did not arrest, even 28 days after transplantation.
Gene Expression To characterize the distribution of adenovirus-mediated gene expression on the cellular level, donor hearts were stained with X-gal. 5-Bromo-4-chloro-3-indoyl- b-dgalactoside is hydrolyzed by b-gal to generate galactose and soluble indoxyl molecules that in turn are converted to insoluble indigo. The substance shows blue color macroscopically. The hearts that had been perfused with 1 3 109 pfu of Adex1CALacZ or Adex1w1 were examined initially by X-gal staining after heterotopic heart transplantation. On the seventh day after transplantation, the blue spots were seen mainly in both the ventricles and the atria of the hearts that had been perfused with Adex1CALacZ (Fig 1). Histologic examination demonstrated that the gene-transferred cells were predominantly cardiomyocytes of the ventricles and atria (Figs 2A, 2B). In the coronary arteries and veins, cells positive for b-gal were sparse compared with cardiomyocytes. The total absence of blue-stained spots in the hearts that
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had been perfused with Adex1w1 demonstrated that the brief, 6-hour incubation in the X-gal chromogen precluded the possibility of false-positive results from endogenous b-gal activity (Fig 2C). Although gene expression persisted for 4 weeks, the stained cells gradually decreased and, 5 weeks later, no stained cells were detected.
Inflammatory Reactions in Donor Heart and Effects on Other Organs To investigate the adverse effects of ex vivo adenovirusmediated gene transfer, we evaluated the amount of recombinant adenovirus that was present. Hematoxylin and eosin staining indicated that there was slight infiltration of monocytes and leukocytes in all sections of the hearts that had been perfused with 1 3 109 pfu of Adex1CALacZ (the low-dose group), whereas those that had been perfused with 1 3 1010 or 1 3 1011 pfu (the high-dose groups) showed leukocyte infiltration 7 days after transplantation (Figs 3A, 3B). Most of the infiltration was seen around gene-transferred cells. Gene expression of the hearts that had been perfused with high doses of adenovirus particles diminished and the infiltration was distributed over the myocardium 21 days after transplantation (Figs 3C, 3D). To ensure that no virus had spread to other organs under this experimental protocol, we used a polymerase chain reaction to look for the sequence coding b-gal in DNA prepared from the lung, brain, liver, spleen, kidney, and ovary of the graft recipients. The b-gal sequences were present in donor heart DNA but were absent in DNA prepared from other tissues (Fig 4).
Comment The findings of this study can be summarized as follows. The expression of exogenous genes after coronary perfusion of the recombinant adenovirus was possible during 60 minutes of cold storage at 4°C. The cells that became infected appeared to be predominantly cardiomyocytes. In the hearts that had been perfused with a low dose of
Fig 2. (A and B) X-gal and hematoxylin and eosin staining of donor hearts perfused with 1 3 109 pfu of the recombinant adenovirus Adex1CALacZ on postoperative day 7. Blue-stained cardiomyocytes are the cells expressing b-gal. Leukocyte infiltration is slightly detectable within the myocardium. (C) Histologic photograph of the adenovirus-negative control hearts. Blue cells are absent. (Magnification, 3200 before 35% reduction.)
Fig 1. Macroscopic photograph of an ex vivo gene-transferred heart on postoperative day 7. In this photograph, cells positive for b-gal activity were recognized in the ventricles as blue spots. The bar indicates 1 mm of length.
adenovirus, b-gal expression persisted for at least 4 weeks, whereas in those that had been perfused with high doses, it diminished 3 weeks after transplantation. High doses of adenovirus produced an inflammatory
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Fig 3. Microscopic photographs of hearts perfused with 1 3 1010 (A) and 1 3 1011 (B) pfu of adenovirus Adex1CALacZ 7 days after transplantation. Numerous leukocytes are present around the gene-transferred cells. (Magnification 3200.) On postoperative day 21, gene-transferred cells are absent within the myocardium. The perfusion of 1 3 1010 (C) and 1 3 1011 pfu (D) of adenovirus produced inflammatory reactions throughout the myocardium. (Magnification, 3100 before 35% reduction.)
response. The coronary perfusion procedure did not produce any significant infection of the recombinant adenovirus in other organs. The infection rate of cold-preserved hepatocytes, which were exposed to a recombinant adenovirus encoding the b-gal gene at 10 multiplicity of infection, was 50% to 70% using X-gal staining [16]. It has been reported that cold-preserved rat liver grafts can be infected effectively with replication-defective adenovirus, resulting in efficient gene transfer to 5% to 30% of liver cells [17]. The infection rate of the adenoviral vector is more efficient than that of the retroviral vector or the plasmid vector with lepofectin or hemagglutinating virus of Japan liposome. We examined the infectivity of primary cultured cardiomyocytes under the same conditions and confirmed rates of infection similar to those of hepatocytes in vitro. We used a recombinant adenovirus to deliver an exogenous gene to donor hearts because it had the highest gene transfer efficacy among the available gene transfer vectors. Although X-gal staining is an efficient means of identifying cells that express the lacZ reporter gene, it does not provide a quantitative assessment of recombinant
gene expression or an accurate estimate of the percentage of cells transfected in the entire organ (unless it includes an exhaustive quantitative analysis of serial sections) [18]. We used the b-gal gene not to quantify the efficacy of gene transfer, but to characterize the identity and distribution of adenovirus-infected cells after transplantation. The identification and distribution of gene-transferred cells in our study is in accordance with the results obtained by Lee and associates [4]. The cells that were positive for b-gal activity were distributed over the myocardium in both the ventricles and the atria. Cardiomyocytes were infected preferentially over endothelial cells or smooth muscles of coronary vessels. The initial target of the immune response to allogeneic and xenogeneic organs is the endothelial cells of the vessels. This study suggested that adenoviruses were adequate for local delivery of exogenous soluble proteins to induce an immunologic tolerance [19], but not for genetic modifications of the surface of the endothelial cells. Gene expression was temporary after infection. Many earlier studies have reported that expression of the transduced gene products disappears 4 weeks after adenovirus-mediated gene transfer [20, 21]. In the low-dose
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Fig 4. DNA prepared from the lung (lane 3), brain (lane 4), liver (lane 5), spleen (lane 6), kidney (lane 7), ovary (lane 8), and donor heart (lane 2). Lane 1 contains molecular weight markers (500 – base pair [bp] ladder; top: 4 kilobase pairs, bottom: 500 bp). The primer pair used in this study detects the part (1,036 bp) of the Escherichia coli b-gal gene. From lane 3 to lane 8, the b-gal gene is not detectable. In lane 2, the band of b-gal is shown. This indicates that the heart was transfected successfully by the recombinant adenovirus.
group in our study, the duration of gene expression was about 4 weeks, but in the high-dose groups, it was shorter than expected. Histologic examination demonstrated prominent inflammation around LacZ-positive cells in the high-dose groups. In contrast, only slight inflammation was detected in the low-dose group. It also has been reported that leukocyte infiltrates can be observed after the in vivo administration of replication-deficient adenoviral vectors, and that they are associated with regions of gene-transferred cells [22]. The results of our study and those of previous reports suggest that the transient expression of adenovirus-mediated gene transfer may be ascribed in part to the immune response involving leukocyte-mediated cytolysis. It has been demonstrated that adenoviruses that are rendered replication-deficient by deletion of the E1 region, the E3 region, or both retain the capability of expressing viral proteins in infected cells [23]. In neonatal mice, it has been reported that gene expression persists for as long as 12 months after the intravenous or intramuscular injection of recombinant adenovirus [24]. Although systemic immunosuppression or tolerization of the host to adenoviral antigens permits long-term gene expression after repeated viral injection, these methods either are invasive or would require systemic immunosuppression, creating additional problems in the clinical setting [25, 26]. An alternate approach is to engineer the adenoviral vector itself to be less immunogenic. Some new recombinant adenoviruses; insertion of the adenoviral E3 region into a recombinant viral vector [27], delta-rAD [28]; and adenovirus dodecahedron [29] have been described recently. Molecules regarding a unresponsiveness induction, such as interleukin-4, viral interleukin-10 [19], and transforming growth factor-b [30], may be candidates for ex vivo gene transduction using adenovirus. The transduction of the CTLA4-Ig gene may
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be effective in suppressing not only lymphocytemediated rejection, but also antibody-mediated rejection [31]. Islet allograft rejection is prevented with engineered myoblasts that express FasL [32]. To investigate the potential role of gene therapy in the induction of tolerance to solid organ grafts, the introduction of an allogeneic major histocompatibility complex class I gene into hematopoietic cells has been reported [33]. The absence of gene transfer to organs other than the donor heart is essential in the clinical setting. We perfused the donor hearts with University of Wisconsin solution after the perfusion of the adenovirus to remove the free adenoviral particles. No cells positive for b-gal activity were detected in the lung, brain, liver, spleen, kidney, or ovary. However, X-gal staining is insensitive for small amounts of b-gal expression. A PCR technique was used to determine which other tissues of the host possessed the exogenous gene delivered by the recombinant adenovirus. The b-gal gene sequence was not detected in DNA prepared from any organ of the host other than the heart. This finding indicates that the perfusion procedure used did not result in any significant leak of recombinant adenovirus to other organs. It also suggested that replication-competent helper virus was absent from the stocks used for perfusion and did not appear in the animals as a consequence of transplantation. In conclusion, this study demonstrated that ex vivo adenovirus-mediated gene transfer to the heart is feasible within a limited period under hypothermic preservation conditions, and that this technique can be used to modify alloimmunogenicity and xenoimmunogenicity just before transplantation while the donor heart is stored in the cold milieu. Supported in part by a grant from the Ministry of Health and Welfare, Japan.
References 1. Kitsis RN, Buttrick PM, McNally EM, Kaplan ML, Leinwand LA. Hormonal modulation of a gene injected into rat heart in vivo. Proc Natl Acad Sci USA 1991;88:4138– 42. 2. Zabner J, Couture LA, Gregory RJ, Craham SM, Smith AE, Welsh MJ. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993;75:207–16. 3. Ardehali A, Fyfe AI, Laks H, Drinkwater DC, Qiao JH, Luis AJ. Direct gene transfer into donor hearts at the time of harvest. J Thorac Cardiovasc Surg 1995;109:716–20. 4. Lee J, Laks H, Drinkwater DC, et al. Cardiac gene transfer by intracoronary infusion of adenovirus vector-mediated reporter gene in the transplanted mouse heart. J Thorac Cardiovasc Surg 1996;111:246–52. 5. Fyfe AI, Ardehali A, Laks H, Drinkwater D, Luis AJ. Biologic modification of the immune response in mouse cardiac isografts using gene transfer. J Heart Lung Transplant 1995; 14:S165–70. 6. Gal D, Weir Lleclerc G, Pickering JG, Hogan J, Isner JM. Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery. Lab Invest 1993;68:18–25. 7. Ardehali A, Fyfe A, Laks H, Drinkwater JDC, Qiao JH, Lusisi
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AJ. Direct gene transfer into donor hearts at the time of harvest. J Thorac Cardiovasc Surg 1995;109:716–20. Dalesandro J, Akimoto H, Gorman CM, et al. Gene therapy for donor hearts: ex vivo liposome-mediated transfection. J Thorac Cardiovasc Surg 1996;111:416–22. Sawa Y, Suzuki K, Bai HZ, et al. Efficiency of in vivo gene transfection into transplanted rat heart by coronary infusion of HVJ liposome. Circulation 1995;92:II479 – 82. Gojo S, Kitamura S, Hatano O, et al. Genetically modified cardiac cell transplantation. J Thorac Cardiovasc Surg 1997; 113:10– 8. Gojo S, Kitamura S, Niwaya K, Yoshida Y, Sakaguchi H, Kawachi K. Ex vivo gene transfer to transplanted heart grafts using adenoviral vector. Transplant Proc 1996;28:1818–9. Wang J, Ma Y, Knechtle SJ. Adenovirus-mediated gene transfer into rat cardiac allografts. Comparison of direct injection and perfusion. Transplantation 1996;61:1726–9. Shiraishi M, Kusano T, Hara J, et al. Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft. Surg Today 1996;26:624– 8. Precious B, Russel WC. Growth, purification and titration of adenoviruses. In: Mahy BM, ed. Virology: a practical approach, 1st ed. Oxford: IRL Press, 1985:193–205. Olausson M, Mjornstedt L, Lindholm L, Brynger H. Nonsuture organ grafting to the neck vessels in rats. Acta Chir Scand 1984;150:463–7. Csete ME, Drazan KE, Bree MV, et al. Adenovirus-mediated gene transfer in the transplant setting. I. Conditions for expression of transferred genes in cold-preserved hepatocytes. Transplantation 1994;57:1502–7. Shaked A, Csete ME, Drazan KE, et al. Adenovirus-mediated gene transfer in the transplant setting. II. Successful expression of transferred cDNA in syngeneic liver grafts. Transplantation 1994;57:1508–11. Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res 1993;73:1202–7. Suzuki T, Tahara H, Narula S, Moore KW, Robbins PD, Lotze MT. Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors. J Exp Med 1995;182:477– 86. Gerard RD, Herz J. Adenovirus-mediated low density lipoprotein receptor gene transfer accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci USA 1993;90: 2812– 6. Limarchand P, Jones M, Yamada I, Crystal RG. In vivo gene
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Background. Ex vivo gene transfer to heart grafts may hold promise as a means of changing alloreactivity or xenoreactivity after transplantation. However, it remains to be determined how effectively gene transfer can be accomplished within a short time in cold-stored grafts that are ready to be transplanted. Methods. We performed an experimental study using a replication-defective adenovirus (Adex1CALacZ) encoding the Escherichia coli b-galactosidase (b-gal) gene to perform gene transfer to heart grafts awaiting transplantation. Thirty hearts of Wistar rats were removed and their coronary arteries were perfused with University of Wisconsin solution containing 1 3 109, 1 3 1010, or 1 3 1011 plaque-forming units of the recombinant adenovirus at 4°C for 60 minutes. As a control, other hearts were perfused with University of Wisconsin solution with an adenoviral vector that did not contain the b-gal gene (Adex1w1) for the same period. After perfusion, the grafts were implanted in the necks of syngeneic adult rats. The grafts were removed each week after transplantation and their expression of b-gal was assessed by 5-bromo-4-chloro-3-indoyl-b-D-galactoside staining. Results. Successful gene transfer and expression of the
b-gal gene were demonstrated in adenovirus-perfused hearts. Gene transfer occurred preferentially in the cardiomyocytes over the endothelial cells and smooth muscle cells of the coronary vessels. In hearts perfused with 1 3 109 plaque-forming units of the adenovirus, gene expression persisted for 4 weeks after transfer, but it diminished gradually and was minimal by day 28. Histologic analyses revealed slight inflammatory reactions in the myocardium. In hearts perfused with 1 3 1010 and 1 3 1011 plaque-forming units of the adenovirus, b-gal diminished 3 weeks after transplantation and a prominent infiltration of leukocytes was recognized in the myocardium. Conclusions. This study demonstrated that the cardiomyocytes of heart grafts express an exogenous gene product after adenovirus-mediated gene transfer under hypothermic preservation conditions. However, immune or inflammatory reactions to recombinant adenoviruses must be taken into account when a large number of adenoviruses are injected into the coronary arteries.
T
virus of Japan liposome-mediated transfection improve the efficacy of gene transduction in the transplant setting [8, 9]. A previous report from our laboratory using a recombinant adenovirus demonstrated that virtually all cardiomyocytes are infected, even in 10 multiplicity of infection in vitro [10]. It also has been reported that intracoronary gene transfer to transplanted murine [4] and rat [11–13] cardiac grafts is feasible at the time of harvest using a recombinant adenovirus. The study reported here examined the quantitative effects of the adenovirus and the safety of our transcoronary infusion protocol for gene transfer.
he development of technology for transferring genetic material into the heart and lung has opened new frontiers in the study of the pathogenesis and treatment of cardiovascular and pulmonary diseases [1, 2]. The application of gene transfer to the field of transplantation is uniquely appealing because of access to the donor organ at the time of harvest [3, 4]. Gene transfer to heart grafts, leading to the expression of proteins capable of inhibiting the immune response to alloantigens or xenoantigens, may result in local modulation of immunity after grafting [5]. First, we must establish a system of efficient gene delivery to the heart grafts. Although several studies have demonstrated successful gene transfer into the ventricular myocardium using direct injection of plasmid DNA, the number of myocytes transfected with this method appears to be too small for successful application to gene therapy [6, 7]. Liposome- and hemagglutinating Accepted for publication Aug 28, 1997. Address reprint requests to Dr Gojo, Department of Surgery III, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634, Japan (e-mail: [email protected]).
© 1998 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1998;65:647–52) © 1998 by The Society of Thoracic Surgeons
Material and Methods Adenoviral Vector The replication-deficient recombinant adenovirus, Adex1CALacZ, was constructed by in vivo homologous recombination in 293 cells between the expression cosmid cassette and the parental virus genome. The expression cosmid cassette was constructed by inserting the 0003-4975/98/$19.00 PII S0003-4975(97)01295-2
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expression unit, composed of the cytomegalovirus enhancer plus chicken b-actin promoter, a complementary DNA coding sequence of a b-galactosidase (b-gal) gene, and the rabbit b-globin polyadenylate signal sequence, into the SwaI site of pAdex1w, which was a 42-kilobase cosmid containing a 31-kilobase adenovirus type 5 genome lacking E1A, E1B, and E3 genes [10]. The recombinant viruses subsequently were propagated within 293 cells (ATCC; CRL1573) and viral solution was stored at 280°C. To prepare the adenovirus stock, a Bioruptor 200 (CosmoBio, Tokyo, Japan) was used for disrupting the 293 infected cells. The titers of the viral stocks were measured by an end-point cytopathic effect assay [14]. In this experiment, the titer of recombinant adenovirus was 1.0 3 1010 plaque-forming units per milliliter (pfu/mL). As a control vector, we used an adenoviral vector that did not contain an expression unit (Adex1w1), the titer of which was 4.3 3 108 pfu/mL.
Transfection to Heart Grafts and Heterotopic Heart Transplantation Wistar rats aged 8 to 10 weeks and weighing 200 to 300 g were used as donors and recipients. 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). The donor hearts were removed after the caval and pulmonary veins had been ligated. A cannula (16-gauge, Angiocath; Terumo) was introduced and tightened into the aorta, and the pulmonary artery was left open. After total vascular occlusion of the heart, perfusion of the heart was accomplished through the aorta using 50 mL of University of Wisconsin solution containing 1 3 109 (n 5 15), 1 3 1010 (n 5 15), or 1 3 1011 (n 5 15) pfu of Adex1CALacZ over 60 minutes at 4°C. Control hearts were perfused in an identical manner with University of Wisconsin solution containing 1 3 109 (n 5 15) pfu of Adex1w1. After perfusion to infect the heart, the grafts were washed out with 20 mL of University of Wisconsin solution and transplanted into syngeneic rats (aged 10 to 12 weeks) using a non–suture cuff technique [15].
5-Bromo-4-chloro-3-indoyl-b-d-galactoside Staining and Histologic Analysis Three mice were sacrificed each week after heterotopic heart transplantation. Histologic examination of the donor hearts was performed every 7 days after transplantation for 5 weeks. The donor hearts were dissected and fixed in 4% paraformaldehyde for 1 hour. The organs were sliced (500 mm thick) using a vibratome slicer (Oxford Co), refixed in the same solution for 2 hours, and washed in phosphate-buffered saline solution. They then were overlaid with 1 mg/mL of 5-bromo-4-chloro-3indoyl-b-d-galactoside (X-gal), 15 mmol/L of potassium ferricyanide, 15 mmol/L of potassium ferrocyanide, and 2 mmol/L of magnesium chloride in phosphate-buffered saline and incubated for 6 hours at 37°C. Slices positive for b-gal activity were embedded in paraffin, and sections (4 mm) were stained with hematoxylin and eosin to reveal any inflammatory reaction.
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Polymerase Chain Reaction A polymerase chain reaction technique was used to detect which other tissues of the host possessed the b-gal gene that was delivered by the recombinant adenovirus. A polymerase chain reaction was performed on DNA isolated from the lung, brain, liver, spleen, kidney, ovary (we used female rats as recipients), and donor heart of the experimental animals 7 days after transplantation. Synthetic oligodeoxynucleotide primer sequences were chosen from separate exons of the genes. For example, the complementary DNA product detected the Escherichia coli b-gal sequences (59-GCCGACCGCACGCCGCATCCAGC-39 and 59-CGCCGCGCCACTGGTGTGGGCC-39) [16]. Tubes with 1 mg of sample DNA and reaction mixture were placed in a Perkin-Elmer/Cetus thermal cycler. The reaction mixture consisted of 6 mmol/L of (NH4)2SO4, 10 mmol/L of KCl, 120 mmol/L of Tris-HCl (pH 8.0), 1% Triton X-100, 0.01% BSA, 1 mmol/L of MgCl2, 200 mmol/L of dNTP, 50 pmol/L of each oligonucleotide primer, and 2.5 U of KOD DNA polymerase (Toyobo Co, Ltd, Japan). 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. Samples were analyzed by acrylamide gel electrophoresis.
Results Survival The total ischemic time of the donor hearts ranged from 100 to 120 minutes. All animals to be grafted survived until they were sacrificed. The grafts that were perfused with 1 3 109 pfu of the adenoviruses (Adex1CALacZ or Adex1w1) had normal contractility when the animals were sacrificed. In contrast, those that were perfused with 1 3 1010 or 1 3 1011 pfu of the adenoviruses had bradycardia and an irregular rhythm beginning 14 days after transplantation, but they did not arrest, even 28 days after transplantation.
Gene Expression To characterize the distribution of adenovirus-mediated gene expression on the cellular level, donor hearts were stained with X-gal. 5-Bromo-4-chloro-3-indoyl- b-dgalactoside is hydrolyzed by b-gal to generate galactose and soluble indoxyl molecules that in turn are converted to insoluble indigo. The substance shows blue color macroscopically. The hearts that had been perfused with 1 3 109 pfu of Adex1CALacZ or Adex1w1 were examined initially by X-gal staining after heterotopic heart transplantation. On the seventh day after transplantation, the blue spots were seen mainly in both the ventricles and the atria of the hearts that had been perfused with Adex1CALacZ (Fig 1). Histologic examination demonstrated that the gene-transferred cells were predominantly cardiomyocytes of the ventricles and atria (Figs 2A, 2B). In the coronary arteries and veins, cells positive for b-gal were sparse compared with cardiomyocytes. The total absence of blue-stained spots in the hearts that
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had been perfused with Adex1w1 demonstrated that the brief, 6-hour incubation in the X-gal chromogen precluded the possibility of false-positive results from endogenous b-gal activity (Fig 2C). Although gene expression persisted for 4 weeks, the stained cells gradually decreased and, 5 weeks later, no stained cells were detected.
Inflammatory Reactions in Donor Heart and Effects on Other Organs To investigate the adverse effects of ex vivo adenovirusmediated gene transfer, we evaluated the amount of recombinant adenovirus that was present. Hematoxylin and eosin staining indicated that there was slight infiltration of monocytes and leukocytes in all sections of the hearts that had been perfused with 1 3 109 pfu of Adex1CALacZ (the low-dose group), whereas those that had been perfused with 1 3 1010 or 1 3 1011 pfu (the high-dose groups) showed leukocyte infiltration 7 days after transplantation (Figs 3A, 3B). Most of the infiltration was seen around gene-transferred cells. Gene expression of the hearts that had been perfused with high doses of adenovirus particles diminished and the infiltration was distributed over the myocardium 21 days after transplantation (Figs 3C, 3D). To ensure that no virus had spread to other organs under this experimental protocol, we used a polymerase chain reaction to look for the sequence coding b-gal in DNA prepared from the lung, brain, liver, spleen, kidney, and ovary of the graft recipients. The b-gal sequences were present in donor heart DNA but were absent in DNA prepared from other tissues (Fig 4).
Comment The findings of this study can be summarized as follows. The expression of exogenous genes after coronary perfusion of the recombinant adenovirus was possible during 60 minutes of cold storage at 4°C. The cells that became infected appeared to be predominantly cardiomyocytes. In the hearts that had been perfused with a low dose of
Fig 2. (A and B) X-gal and hematoxylin and eosin staining of donor hearts perfused with 1 3 109 pfu of the recombinant adenovirus Adex1CALacZ on postoperative day 7. Blue-stained cardiomyocytes are the cells expressing b-gal. Leukocyte infiltration is slightly detectable within the myocardium. (C) Histologic photograph of the adenovirus-negative control hearts. Blue cells are absent. (Magnification, 3200 before 35% reduction.)
Fig 1. Macroscopic photograph of an ex vivo gene-transferred heart on postoperative day 7. In this photograph, cells positive for b-gal activity were recognized in the ventricles as blue spots. The bar indicates 1 mm of length.
adenovirus, b-gal expression persisted for at least 4 weeks, whereas in those that had been perfused with high doses, it diminished 3 weeks after transplantation. High doses of adenovirus produced an inflammatory
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Fig 3. Microscopic photographs of hearts perfused with 1 3 1010 (A) and 1 3 1011 (B) pfu of adenovirus Adex1CALacZ 7 days after transplantation. Numerous leukocytes are present around the gene-transferred cells. (Magnification 3200.) On postoperative day 21, gene-transferred cells are absent within the myocardium. The perfusion of 1 3 1010 (C) and 1 3 1011 pfu (D) of adenovirus produced inflammatory reactions throughout the myocardium. (Magnification, 3100 before 35% reduction.)
response. The coronary perfusion procedure did not produce any significant infection of the recombinant adenovirus in other organs. The infection rate of cold-preserved hepatocytes, which were exposed to a recombinant adenovirus encoding the b-gal gene at 10 multiplicity of infection, was 50% to 70% using X-gal staining [16]. It has been reported that cold-preserved rat liver grafts can be infected effectively with replication-defective adenovirus, resulting in efficient gene transfer to 5% to 30% of liver cells [17]. The infection rate of the adenoviral vector is more efficient than that of the retroviral vector or the plasmid vector with lepofectin or hemagglutinating virus of Japan liposome. We examined the infectivity of primary cultured cardiomyocytes under the same conditions and confirmed rates of infection similar to those of hepatocytes in vitro. We used a recombinant adenovirus to deliver an exogenous gene to donor hearts because it had the highest gene transfer efficacy among the available gene transfer vectors. Although X-gal staining is an efficient means of identifying cells that express the lacZ reporter gene, it does not provide a quantitative assessment of recombinant
gene expression or an accurate estimate of the percentage of cells transfected in the entire organ (unless it includes an exhaustive quantitative analysis of serial sections) [18]. We used the b-gal gene not to quantify the efficacy of gene transfer, but to characterize the identity and distribution of adenovirus-infected cells after transplantation. The identification and distribution of gene-transferred cells in our study is in accordance with the results obtained by Lee and associates [4]. The cells that were positive for b-gal activity were distributed over the myocardium in both the ventricles and the atria. Cardiomyocytes were infected preferentially over endothelial cells or smooth muscles of coronary vessels. The initial target of the immune response to allogeneic and xenogeneic organs is the endothelial cells of the vessels. This study suggested that adenoviruses were adequate for local delivery of exogenous soluble proteins to induce an immunologic tolerance [19], but not for genetic modifications of the surface of the endothelial cells. Gene expression was temporary after infection. Many earlier studies have reported that expression of the transduced gene products disappears 4 weeks after adenovirus-mediated gene transfer [20, 21]. In the low-dose
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Fig 4. DNA prepared from the lung (lane 3), brain (lane 4), liver (lane 5), spleen (lane 6), kidney (lane 7), ovary (lane 8), and donor heart (lane 2). Lane 1 contains molecular weight markers (500 – base pair [bp] ladder; top: 4 kilobase pairs, bottom: 500 bp). The primer pair used in this study detects the part (1,036 bp) of the Escherichia coli b-gal gene. From lane 3 to lane 8, the b-gal gene is not detectable. In lane 2, the band of b-gal is shown. This indicates that the heart was transfected successfully by the recombinant adenovirus.
group in our study, the duration of gene expression was about 4 weeks, but in the high-dose groups, it was shorter than expected. Histologic examination demonstrated prominent inflammation around LacZ-positive cells in the high-dose groups. In contrast, only slight inflammation was detected in the low-dose group. It also has been reported that leukocyte infiltrates can be observed after the in vivo administration of replication-deficient adenoviral vectors, and that they are associated with regions of gene-transferred cells [22]. The results of our study and those of previous reports suggest that the transient expression of adenovirus-mediated gene transfer may be ascribed in part to the immune response involving leukocyte-mediated cytolysis. It has been demonstrated that adenoviruses that are rendered replication-deficient by deletion of the E1 region, the E3 region, or both retain the capability of expressing viral proteins in infected cells [23]. In neonatal mice, it has been reported that gene expression persists for as long as 12 months after the intravenous or intramuscular injection of recombinant adenovirus [24]. Although systemic immunosuppression or tolerization of the host to adenoviral antigens permits long-term gene expression after repeated viral injection, these methods either are invasive or would require systemic immunosuppression, creating additional problems in the clinical setting [25, 26]. An alternate approach is to engineer the adenoviral vector itself to be less immunogenic. Some new recombinant adenoviruses; insertion of the adenoviral E3 region into a recombinant viral vector [27], delta-rAD [28]; and adenovirus dodecahedron [29] have been described recently. Molecules regarding a unresponsiveness induction, such as interleukin-4, viral interleukin-10 [19], and transforming growth factor-b [30], may be candidates for ex vivo gene transduction using adenovirus. The transduction of the CTLA4-Ig gene may
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be effective in suppressing not only lymphocytemediated rejection, but also antibody-mediated rejection [31]. Islet allograft rejection is prevented with engineered myoblasts that express FasL [32]. To investigate the potential role of gene therapy in the induction of tolerance to solid organ grafts, the introduction of an allogeneic major histocompatibility complex class I gene into hematopoietic cells has been reported [33]. The absence of gene transfer to organs other than the donor heart is essential in the clinical setting. We perfused the donor hearts with University of Wisconsin solution after the perfusion of the adenovirus to remove the free adenoviral particles. No cells positive for b-gal activity were detected in the lung, brain, liver, spleen, kidney, or ovary. However, X-gal staining is insensitive for small amounts of b-gal expression. A PCR technique was used to determine which other tissues of the host possessed the exogenous gene delivered by the recombinant adenovirus. The b-gal gene sequence was not detected in DNA prepared from any organ of the host other than the heart. This finding indicates that the perfusion procedure used did not result in any significant leak of recombinant adenovirus to other organs. It also suggested that replication-competent helper virus was absent from the stocks used for perfusion and did not appear in the animals as a consequence of transplantation. In conclusion, this study demonstrated that ex vivo adenovirus-mediated gene transfer to the heart is feasible within a limited period under hypothermic preservation conditions, and that this technique can be used to modify alloimmunogenicity and xenoimmunogenicity just before transplantation while the donor heart is stored in the cold milieu. Supported in part by a grant from the Ministry of Health and Welfare, Japan.
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