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Ann Thorac Surg 1997;63:1556-1560
© 1997 The Society of Thoracic Surgeons

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Original Article: General Thoracic

Ex Vivo Adenoviral-Mediated Gene Transfer to Lung Isografts During Cold Preservation

Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri

	Abstract

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Background. Although whole-organ gene transfer has been reported in heart and liver transplant models, it has not been well characterized in lung grafts. The aim of this study was to determine the feasibility of ex vivo gene transfer to rat lung isografts during cold preservation using an adenoviral vector.

Methods. F344 rats, divided into four groups, underwent orthotopic left lung transplantation. In group I, lung grafts were flushed with adenovirus carrying the ß-galactosidase gene. After storage at 10°C, grafts were implanted in recipient animals. Group II underwent the same procedure but graft storage was at 4°C. Groups III (10°C) and IV (4°C) served as controls. On postoperative day 5, recipients were sacrificed, and native and transplanted lungs were examined.

Results. In group I, all animals showed successful, albeit patchy, gene expression. This occurred in 2 of 4 animals in group II, the other 2 showing no expression. Transduced cells were consistent morphologically with endothelial cells and pneumocytes. A minimal mononuclear inflammatory infiltrate was present. Control groups showed no transduction.

Conclusions. It is feasible to perform ex vivo adenoviral-mediated gene transfer to rat lung isografts during cold preservation.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
See also page 1561.

Recent advances in gene therapy techniques have made possible the introduction of recombinant genes into mammalian somatic cells, thereby opening the possibility of treating diseases at the genetic level. Currently, 106 human gene transfer clinical trials have been approved by the Recombinant DNA Advisory Committee of the National Institutes of Health. The majority of these studies (103) are classified as phase I, and are being tested in malignant tumors, human immunodeficiency virus infection, and inherited diseases such as adenosine deaminase deficiency, familial hypercholesterolemia, and cystic fibrosis [1, 2].

See also pages 1527 and 1562.

To achieve successful gene expression, an efficient vector system is necessary. Several different delivery systems have been used, including cationic lipids, retroviruses, and adenoviruses. Viruses are considered the most efficient vectors, partly because they have evolved excellent mechanisms for cellular attachment, penetration, and avoidance of intracellular lysosomal degradation [3]. Retroviruses require dividing cells for gene expression, and therefore are impractical for use in heart or lung tissue. In contrast, adenoviruses do not require replicating cells to introduce their expression plasmids and can also infect a wide variety of somatic cells [4]. As a result of site-specific deletion of the E1 adenoviral genomic locus, they are incapable of replication, which minimizes their pathogenicity.

In organ transplantation, gene transfer offers the opportunity to modify the donor organ before implantation. This opens the possibility of targeting problems specific to transplantation, namely, ischemia–reperfusion injury, infection, and acute and chronic rejection. Recombinant gene expression in the donor may alter major histocompatibility antigen expression in the graft or lead to the synthesis of antiinflammatory cytokines, possibly resulting in modifications in immunosuppressive regimens, and decreases in the adverse effects of systemic immunosuppression. It has been recently demonstrated in rat hepatic allografts that the expression of viral interleukin-10 resulted in a significant suppression of the in vitro alloreactivity of peripheral blood lymphocytes [5]. Likewise, in a mouse model, the expression of murine transforming growth factor-ß1 in donor heart allografts resulted in a significant prolongation of graft survival [6].

In the transplant setting, where multiple cadaveric organs are usually harvested from a single donor, the ex vivo technique of gene transfer should result in gene expression only in the organ of interest; therefore, other harvested tissues do not concomitantly receive a gene that may be unnecessary or undesirable.

To achieve efficient recombinant gene expression that is specific to transplanted lung tissue, we tested the feasibility of ex vivo adenoviral-mediated gene transfer to harvested lung isografts during cold preservation in a rat model. Replication-deficient adenovirus carrying the prokaryotic reporter gene encoding for ß-galactosidase was used. The tissue distribution of transgene expression and the host inflammatory response were evaluated.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Animals
Inbred male F344 rats (Harlan Sprague Dawley Inc, Indianapolis, IN), weighing 270 to 300 g, were used in all experiments. All animal protocols were approved by the Animal Studies Committee at Washington University. Animals received humane care in compliance with "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85–23, revised 1985).

	Rat Lung Transplantation

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
An orthotopic left lung rat transplant model (F344 to F344) was performed using a modification of the "cuff technique" [7] as described elsewhere [8]. Briefly, after adequate anesthesia, systemic heparinization, and mechanical ventilation, donor rat lungs were flushed through the main pulmonary artery with 20 mL of low-potassium dextran–1% glucose solution (LPDG). After dissection of the left lung, a silicone catheter was introduced into the left pulmonary artery and connected to an infusion pump. The adenoviral vector was then flushed into the left lung graft for 15 minutes. After adenoviral exposure, isografts were stored for 8 hours in LPDG until implantation. Recipient animals were anesthetized, intubated, and underwent a left pneumonectomy. The pulmonary vessels were anastomosed using the "cuff" technique, whereas the bronchial anastomosis was performed using a running 9-0 Prolene suture (Ethicon, Somerville, NJ). Ventilation and perfusion were restored and a temporary chest tube was placed, which was removed after recovery from anesthesia. On postoperative day 5, recipients were sacrificed, and native right and transplanted left lungs were harvested for histologic examination.

	Experimental Groups

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Animals were randomly divided into four groups (n = 4). In group I, donor lungs were flushed with 16 x 10¹¹ viral particles diluted in 2 mL of LPDG. They were stored in LPDG solution at 10°C for 8 hours until implantation. In group II, rats underwent the same procedure as in group I, but graft storage was at 4°C. Groups III and IV had the lung isografts flushed with LPDG alone without adding the adenoviral vector, and served as controls. Group III and IV grafts were stored at 10°C and 4°C, respectively.

	Adenoviral Vector

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
First-generation replication-deficient adenovirus serotype 5 carrying the Escherichia coli LacZ gene encoding for ß-galactosidase and driven by the constitutive cytomegalovirus promoter (Ad5.CMV.ß-gal) was used. The LacZ gene was chosen because ß-galactosidase activity is easily measured in situ with reliable and sensitive histochemical assays that use chromogenic substrates.

Ad5.CMV.ß-gal, kindly provided by Dr Allan Schwartz (Children's Hospital, St. Louis, MO), was grown in the human embryonic kidney 293 cell line (American Type Culture Collection, Rockville, MD) as described elsewhere [9]. The virus was purified and the number of viral particles was assessed based on the optical density at 260 nm (1 OD₂₆₀ = 5 x 10¹¹ particles/mL). Purified viral aliquots were stored at -80°C in a buffered solution of 10% glycerol, 1x TD buffer with 1 mmol/L MgCl₂. The viability of adenoviral preparations was assayed by adenoviral transduction of 293 cells plated in six-well plates, using limiting dilutions of adenovirus (10^-3 to 10^-12) followed by in situ Bluo-Gal staining 12 hours later. After an 18-hour staining period at 37°C, plates were examined using light microscopy for the presence of blue-stained cells, corresponding to transduced cells producing ß-galactosidase.

	Bluo-Gal Staining

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Bluo-Gal (5-bromo-indolyl-ß-o-galactopyranosyde; Gibco BRL, Gaithersburg, MD) is a chromogenic substance that is cleaved by ß-galactosidase to produce a blue precipitate. In organic solvents, such as alcohol, xylene, or toluene, Bluo-Gal is more stable than X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) in that the blue color of transduced cells does not fade during the staining process of paraffin-embedded histologic sections [10].

Staining of lung tissue with Bluo-Gal was done as described [10]. Briefly, both lungs were flushed through the main pulmonary artery with 20 mL of phosphate-buffered saline solution, 10 mL of 2% paraformaldehyde containing 0.2% glutaraldehyde, and 0.02% Nonidet P-40, followed by 20 mL of phosphate-buffered saline solution, then 10 mL of the Bluo-Gal staining buffer, which was prepared by mixing 1 mg/mL of Bluo-Gal, 5 mmol/L K₃Fe^III(CN)₆, 5 mmol/L K₄Fe^II (CN)₆, 2 mmol/L MgCl₂, 0.1% NP-40, and 1x phosphate-buffered saline solution. Lungs were excised and immersed in Bluo-Gal for 3 hours at 37°C.

After gross examination, lungs were fixed overnight in 4% paraformaldehyde at 4°C for paraffin embedding. Histologic sections were counterstained with nuclear fast red for a better definition of pulmonary structures. The presence of blue-stained cells confirmed transgene expression. Hematoxylin and eosin staining was also performed.

	Results

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Recombinant Gene Expression
All animals appeared healthy and survived until assessment on postoperative day 5 . In group I (grafts preserved at 10°C), all four transplanted lungs had histologic evidence of ß-galactosidase expression. Three of four lungs had patchy gene expression on gross and microscopic examination (Fig 1A), whereas gene expression could only be identified on microscopic sections in the fourth animal. Native right lungs showed no ß-galactosidase activity, consistent with the absence of gene transduction. There appeared to be no crossover of transduction from isograft to native lung.

View larger version (50K): [in this window] [in a new window]   Fig 1. . (A) A patchy transduction pattern was observed in group I, where transplanted left lungs (LL) were incubated with adenoviral vectors for 8 hours at 10°C. Recipient native right lungs (RL) showed no ß-galactosidase expression. This was confirmed by examination of paraffin sections by light microscopy (data not shown). (B) Successful gene transfer to rat lung isografts after 8 hours of storage at 4°C (group II). The histochemical staining with Bluo-Gal demonstrated a diffuse transduction pattern in the transplanted left lung. The native right lung was stained in the same manner, and did not show ß-galactosidase activity.

Microscopically, transgene expression was observed in both pneumocytes and endothelial cells. Endothelial cells were identified by their location, lining the vessels, and their elongated and protruding nuclei. Gene expression was detected in pulmonary arterioles, capillaries, and venules (Fig 2). There was no detectable ß-galactosidase activity in airway epithelial cells. Macrophages also showed ß-galactosidase activity. The significance of this is unclear, as macrophages are known to have endogenous ß-galactosidase activity in their lysosomes [11].

View larger version (141K): [in this window] [in a new window]   Fig 2. . Histologic section of a transduced isograft, stained initially using Bluo-Gal, followed by counterstaining with nuclear fast red. ß-Galactosidase gene expression was detected in the endothelium of a pulmonary vessel, albeit in a low percentage of cells (arrowheads).

	Host Inflammatory Response

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

	Comment

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
This study demonstrates the feasibility of ex vivo gene transfer to whole-lung grafts in the setting of transplantation and cold preservation using an adenoviral vector in the flush solution. The ex vivo approach offers several advantages: (1) lung specificity of gene expression is achieved and (2) the conditions in which gene transfer is performed, such as storage temperature and exposure time, can be altered independently of the donor, to optimize gene transfer efficiency.

In our experiments, the viral load was infused over a 15-minute period to prolong contact between the pulmonary endothelium and the adenoviral vector. During the infusion of the last 0.5 mL, the pulmonary vein was clamped to maximize the number of adenoviral particles in contact with lung tissue during the 8-hour cold storage period. Despite this, recombinant gene expression in this study was unpredictable. At 4°C, two of four grafts did not show transgene expression, and in another graft, transgene expression was patchy. At 10°C, all lungs showed patchy recombinant gene expression, but in one graft this could be detected by microscopic, not gross, examination, where transduction was present in only a small percentage of cells.

Similar heterogeneity in gene transfer has been observed in a rat model of liver transplantation [12]. In that study, ex vivo gene transfer was achieved by portal vein infusion of an adenoviral vector over a 15-minute period then preservation of the grafts at 4°C for 1 to 2 hours. Transgene expression was achieved in 5% to 30% of hepatocytes and was patchy. Increasing graft storage time resulted in improved recombinant gene expression. Interestingly, in vivo bolus infusion of the adenoviral vector through the portal vein did not result in transgene expression.

Other investigators have reported similar unpredictable patterns of gene transfer in lung tissue. In one study, 5 rats were injected with an adenoviral vector carrying the ß-galactosidase gene through a catheter wedged in the distal pulmonary artery [13]. No recombinant ß-galactosidase activity was noted in any of the animals using X-Gal staining. Using the same vector, and after occlusion of the pulmonary artery and vein, only 2 of 14 Sprague-Dawley rats and 3 of 11 cotton rats showed ß-galactosidase gene expression in the lung. In a similar experiment conducted in sheep [14], ß-galactosidase expression was observed in 13 of 17 animals. However, as in our model, transgene expression was patchy in the majority of cases.

The low storage temperature and rat endothelial cell susceptibility do not seem obstacles for adenoviral-mediated gene transfer, as we have efficiently transduced segments of rat left pulmonary artery ex vivo at 10°C (unpublished data). Possible explanations for this unpredictable pattern of transgene expression include (1) dilution of the adenoviral load given the large area of the rat pulmonary microvasculature, calculated at 1 m², leading to less efficient contact with target cells [13], and (2) low viral viability in some adenoviral preparations, which would alter the adenovirus to host cell ratio. A direct correlation between the virus to host cell ratio and the infection rate in cold-preserved hepatocytes infected with Ad.ß-gal has been demonstrated [15, 12]. With a multiplicity of infection of 0.1:1, corresponding to 0.1 viral particles to 1 host cell, less than 0.1% of hepatocytes were transduced. At a multiplicity of infection of 10:1, the transduction rate was 25% to 30%, and at a multiplicity of infection of 50:1, almost 100% of hepatocytes showed transgene expression, but this was associated with a 75% mortality [16].

To assess the degree of inflammation in the grafts arising as a direct result of the adenoviral preparation, we examined hematoxylin and eosin-stained sections of transplanted lung tissues. The host inflammatory response was minimal on postoperative day 5, consisting mostly of a mononuclear infiltrate. Very few polymorphonuclear cells were present. This may be partly explained by the low percentage of cells that were transduced (<1%), resulting in an attenuated expression of adenoviral coat proteins, which are known stimulants of host inflammatory responses. In rhesus monkeys infused intratracheally with replication-deficient adenoviral preparations, a dose-dependent host inflammatory response has been demonstrated [17]. Schachtner and colleagues [13] have observed a severe inflammatory mononuclear and polymorphonuclear cellular infiltrate in rats lungs infused in vivo with Ad.ß-galactosidase after hilar occlusion, despite low ß-galactosidase gene expression. This may be attributable to the method of gene transfer used in those latter experiments, as the lungs underwent a period of warm ischemia, which may have resulted in a significant reperfusion injury.

Although adenoviral vectors are considered the most efficient gene delivery systems, some drawbacks do exist. It has been demonstrated that adenoviruses rendered replication-deficient by deletion of the E1 or E3 regions, or both, do retain the capability of expressing viral proteins in infected cells. This results in several problems: (1) the host immune response (both cellular and humoral) against adenoviral proteins leads to the destruction of those cells expressing the foreign genes and consequently to loss of gene expression. In murine lungs infected with replication-deficient adenoviruses, activation of CD4⁺ and cytotoxic CD8⁺ T lymphocytes in response to newly synthesized viral antigens has been shown, leading to the destruction of viral-infected cells [18]. (2) The resulting host inflammatory reaction may lead to functional impairment of the transplanted organ. Recently, it has been shown that infection of lung allografts with Sendai virus in immunosuppressed rats enhanced major histocompatibility complex class II antigen expression in the bronchial epithelium, resulted in infiltration of dendritic cells and CD4⁺ cells, and led to the formation of granulation tissue in small airways [19]. (3) With subsequent administrations of the same adenoviral vector, host circulating antibodies and memory T cells result in rapid destruction of these vectors, and attenuation of gene delivery and expression. Modifications of the adenoviral genome, which result in decreased production of viral coat proteins, may result in improved gene transfer efficiency. In fact, E1- and E4-deleted second-generation adenoviruses have been reported recently. The additional deletion of the adenoviral E4 genomic region resulted in reduction of adenoviral late gene expression, thereby reducing cytopathic effects and cellular immune responses [20]. A new recombinant adenovirus, delta-rAd, has been described recently, which is deleted of all viral open reading frames [21]. Although problems with production and purification of this recombinant adenovirus exist at the present time, it is hoped that this will result in a decreased host inflammatory response.

The introduction of therapeutic genes into endothelial cells may be effective in a variety of situations. Because the donor endothelium is the first structure to have contact with recipient inflammatory cells, this strategy could address two important problems in organ transplantation-ischemia-reperfusion injury and rejection. Interestingly, when the virus is infused through the pulmonary artery, pneumocytes in addition to endothelial cells are transduced, suggesting that some of the infused viruses escape from the pulmonary microvasculature into surrounding tissues.

In summary, our data demonstrate the feasibility of achieving gene transfer to rat lung isografts during cold storage, as done clinically. Other experiments are currently being conducted to determine (1) the best virus to cell ratio (multiplicity of infection) to achieve a better transduction rate; (2) onset, peak, and length of gene expression; and (3) the feasibility of using other vectors, such as cationic lipids, in the setting of lung transplantation. It is hoped that this strategy would produce an efficient system for gene transfer that would allow the expression of various functional genes in the donor organ, ultimately decreasing postoperative morbidity or mortality.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
We thank Allan Schwartz, MD, Steven Brody, MD, Xin Han, MD, Jia-J. Hui, MD, and Kathleen Grapperhaus for assistance with adenoviral preparations and ß-galactosidase histochemical assays.

This work was supported by National Institutes of Health grant 1 R01 HL41281.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 
Presented at the Forty-third Annual Meeting of the Southern Thoracic Surgical Association, Cancun, Mexico, Nov 7–9, 1996.

Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, 3108 Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110.

	References

Top Footnotes Abstract Introduction Material and Methods Rat Lung Transplantation Experimental Groups Adenoviral Vector Bluo-Gal Staining Results Host Inflammatory Response Comment Acknowledgments References

 

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