Ann Thorac Surg 1998;66:903-907
© 1998 The Society of Thoracic Surgeons
Original articles: General Thoracic
Isolated lung liposome-mediated gene transfer produces organ-specific transgenic expression
Richard Lee, MDa,
Carlos H.R. Boasquevisque, MDa,
Mariano M. Boglione, MDa,
Masafumi Hiratsuka, MDa,
Ronald K. Scheule, PhDa,
Joel D. Cooper, MDa,
G. Alexander Patterson, MDa
a Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, One Barnes Hospital Pl, 3108 Queeny Tower, St. Louis, MO 63110
Presented at the Poster Session of the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.
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Abstract
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Background. Gene therapy is a promising strategy for the treatment of inoperable pulmonary tumors and rejection after lung transplantation. However, unlike ex vivo administration, intravenous in vivo transfection lacks organ specificity and has a limited duration of expression. The objectives of this study were to limit transfection to a single lung and to increase the duration of gene expression in vivo.
Methods. Sixteen male Fisher rats were anesthetized and divided into two groups. Animals in group I (n = 7) received an intrajugular administration of 1,320 µg of chloramphenicol acetyl transferase (CAT) complementary DNA complexed with cationic liposomes. Animals in group II (n = 9) received 660 µg of CAT complementary DNA complexed with cationic liposomes into the pulmonary artery of an isolated left lung over 10 minutes. After 40 minutes of incubation, the lung was flushed with 10 mL of normal saline solution, and the perfusate was suctioned through a left pulmonary venotomy. The circulation to the left lung was then restored. After 48 hours, the animals were divided into subgroups (a and b) and CAT activity was assessed in the lungs, hearts, livers, and kidneys of groups Ia (n = 3) and IIa (n = 5). After 21 days, CAT activity was assessed in the left lungs of groups Ib (n = 4) and IIb (n = 4).
Results. After 48 hours, animals that had received intravenous administration of CAT cDNA showed strong expression in the lungs and hearts and negligible expression in the livers and kidneys. In contrast, animals in group IIa, which had received isolated left lung perfusion of CAT cDNA showed expression only in the left lung. After 21 days, the left lungs of animals in group Ib, which had received intravenous administration of CAT complementary DNA, showed no CAT expression, but the left lungs of animals in group IIb, which had received isolated left lung perfusion of CAT complementary DNA, exhibited strong CAT expression.
Conclusions. Compared with intravenous administration, isolated lung liposome-mediated gene transfer provides prolonged organ-specific gene expression. This provides a useful model to study the effects of gene therapy on pulmonary tumors, which may have further application when gene therapy is used in clinical practice.
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Introduction
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The advancement of techniques to introduce exogenous DNA into mammalian somatic cells has laid a foundation to treat inherited and acquired diseases at the genetic level. Gene therapy appears to be especially promising for the treatment of inoperable cancer. Several in vitro studies have shown the efficacy of gene therapy in the treatment of human small cell lung carcinoma and non–small cell lung carcinoma [1], human malignant mesothelioma [2], sarcoma [3, 4], and hepatocellular carcinoma [5]. On the basis of these results, the use of gene therapy has already been incorporated into a phase I clinical trial for the treatment of lung cancer [6].
However, deficiencies in the specificity of transfection and duration of expression have limited the in vivo application of gene therapy. Intravenous administration results in transfection of multiple organs and has a limited duration of expression [7]. In contrast, ex vivo models of gene transfection in lung transplantation provide organ-specific gene transfection with an increased duration of expression [8].
The use of isolated lung perfusion provides a model for in vivo transfection, which mimics an ex vivo approach. In vivo experimental models of isolated lung perfusion with chemotherapeutic agents are well established [9] and have already demonstrated a benefit in the treatment of metastatic colorectal adenocarcinoma [10] and sarcoma [11].
We hypothesized that, when compared with intravenous administration, a model of isolated lung perfusion as a route of gene transfection would result in a prolonged organ-specific gene expression in vivo.
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Material and methods
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Plasmid expression vector
The plasmid pCF1-CAT (Genzyme Corporation, Framinghan, MA) consists of human cytomegalovirus immediate early gene promoter/enhancer, a hybrid intron, the chloramphenicol acetyltransferase (CAT) complementary DNA (cDNA), the bovine growth hormone polyadenylation signal sequence, and a kanamycin resistance gene; this was constructed as previously described [12].
Preparation of cationic lipid: DOPE:DNA complexes
Lipid #67 (Genzyme) is an amphiphile consisting of a hydrophobic lipid anchor (cholesterol) linked to a spermine headgroup in a T-shape configuration. Lipid #67:DOPE (dioleoylphosphatidylethanolamine), in a molar ratio of 1:2, was supplied as dried films and prepared as previously described [12]. Before use, the films were hydrated with sterile water, vortexed until opaque, incubated on ice for 10 minutes, and then vortexed again for 2 minutes. Equal volumes of lipid #67:DOPE and CAT cDNA were mixed and incubated at room temperature for a minimum of 30 minutes. Final lipid and DNA concentrations were 1 mmol/L and 4 mmol/L, respectively.
Animals
Sixteen inbred male Fisher rats weighing 270 to 300 g (Charles River Laboratories, Wilmington, MA) were used in all experiments. All animals received humane treatment in accordance with the "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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).
All animals were premedicated with subcutaneous ketamine (25 mg/kg) and atropine (0.25 mg/kg) injection, endotracheally intubated with a 14-gauge catheter and ventilated with 0.5% halothane and 99.5% oxygen. The animals were then divided into group I (n = 7) and group II (n = 9) (Fig 1 ).
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Fig 1. Summary of experimental design. Animals in group I received lipid #67:DOPE:CAT cDNA intravenously into the external jugular vein. Animals in group II received lipid #67:DOPE:CAT cDNA into the pulmonary artery of an isolated left lung. Afterward both groups were subdivided into groups a and b, for animals surviving 48 hours and 21 days, respectively.
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Experimental design
Animals in group I received an injection of 1,320 µg of lipid #67:DOPE:CAT cDNA into the left external jugular vein. Animals in group II received 660 µg of lipid #67:DOPE:CAT cDNA into the left lung by an isolated lung perfusion technique as previously described [9] (Fig 2 ). Briefly, the left side of the chest was entered through the fourth intercostal space. The lung was retracted with a cotton-tipped applicator and the left pulmonary vessels were dissected free. The pulmonary artery and vein were clamped proximally and distally, respectively, with microclips. Under an operating microscope a small incision was created in the pulmonary artery and vein.
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Fig 2. Illustration of isolated lung perfusion. The lung was retracted from the thoracic cavity with a cotton-tipped applicator. The pulmonary artery and vein were clamped proximally and distally, respectively, with microclips. The arterial catheter was removed after completion of perfusion. The arteriotomy was then repaired with a single 9-0 nylon suture.
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A PE-10 catheter (0.28 mm inner diameter, 0.61 mm outer diameter; Becton Dickenson & Co, Franklin Lakes, NJ) was inserted into the pulmonary artery for infusion. Six hundred sixty micrograms of lipid #67:DOPE:CAT cDNA in 5 mL of normal saline solution was infused over 10 minutes. The effluent was collected by a suction catheter next to the venotomy. After 40 minutes of incubation, the pulmonary artery was perfused with 10 mL of normal saline solution and the effluent was collected by suction.
After completion of perfusion, the catheters were removed and the pulmonary artery was repaired with 9-0 nylon suture. The microclips were removed, and bleeding from the venotomy was controlled by direct pressure with a cotton-tipped applicator. After hemostasis had been confirmed, the thorax was closed in three layers. A 16-gauge catheter connected to a sterile syringe was introduced into the left chest through a separate puncture wound to facilitate lung reexpansion. When animals began to breathe spontaneously, their chest and endotracheal tubes were removed.
After 48 hours, the animals were divided into subgroups (a and b). The animals in groups Ia (n = 3) and IIa (n = 5) were sacrificed and the lungs, hearts, livers, and kidneys were assessed for CAT activity. The animals in groups Ib (n = 4) and IIb (n = 4) were survived for 21 days. At that time, the animals were sacrificed and the left lungs were assessed for CAT activity.
CAT activity assay
Transgene expression was detected by a CAT activity assay as described elsewhere [13]. Briefly, after tissue homogenization and dilution in Tris-EDTA, three consecutive freeze-thaw cycles were performed. After incubation at 65°C, samples were centrifuged at 10,000 rpm, the supernatant recovered, and a quantitative spectrophotometric protein analysis performed. Three hundred micrograms of protein extract were incubated overnight at 37°C with 40 µL of acetyl coenzyme A (5 mg/mL) and 8 µL of 14C-chloramphenicol. Ethyl acetate was added, the samples vortexed, centrifuged at 14,000 rpm, and the organic supernatant recovered. This supernatant was nitrogen-dried and then resuspended in ethyl acetate. Thin-layer chromatography was followed by overnight autoradiography. In the presence of functional CAT enzyme, both monoacetyled and diacetyled forms of chloramphenicol are produced, which by thin-layer chromatography are distinct from the nonacetyled chloramphenicol. When gene transfer occurs and the enzyme is functional, a distinct pattern appears after thin-layer chromatography and autoradiography, which is absent if transfection has not occurred.
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Results
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Seventeen animals received administration of lipid #67:DOPE:CAT cDNA. All animals in group I (n = 7) recovered without complication. One animal in group II (n = 10) died during pulmonary artery repair because of a technical failure and was excluded from the study.
Organ specificity
After 48 hours, all animals in group Ia (n = 3), which received intravenous CAT cDNA administration, exhibited strong CAT expression in the lungs and heart (Fig 3 ). There was negligible expression in the livers and the kidneys. In contrast, animals in group IIa (n = 5), which received isolated lung perfusion of CAT cDNA, displayed strong expression in the left lung without any expression in the heart, liver, or kidneys (Fig 4 ). There was minimal expression in the right lung of 1 animal in group IIa. This was attributed to a defective microclip, which was detected and replaced after liposome infusion began. No other animals in group IIa expressed CAT activity in the right lung.
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Fig 3. Comparison of CAT expression in animals that received 1,320 µg of lipid #67:DOPE:CAT cDNA 48 hours after intravenous administration (group Ia, n = 3). The comparison is labeled in sequence by organ: right or left lung, liver, heart, kidney. Each column represents one animal. The bottom row represents nonacetylated chloramphenicol. The middle and top rows represent monoacetyled and diacetylated chloramphenicol, respectively. A strong expression was observed in both the heart and lungs of all animals. The liver and kidneys demonstrated negligible expression.
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Fig 4. Comparison of CAT expression in animals that received administration of 660 µg of lipid #67:DOPE:CAT cDNA 48 hours after isolated lung perfusion by animal (group IIa, n = 5). All animals showed strong expression in the left lung without expression in the heart, liver, or kidneys. Animal number four demonstrated minimal expression in the right lung because of a technical failure at infusion. The remaining animals showed negligible expression in the right lungs.
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Duration of expression
The animals in group Ib (n = 4), which received intravenous CAT cDNA administration, displayed no CAT activity in the left lung after 21 days (Fig 5 ). In contrast, the animals in group IIb (n = 4), which received isolated lung perfusion of CAT cDNA displayed strong CAT activity in the left lung (Fig 6 ). Upon sacrifice, these lungs had a normal appearance, and did not appear to be affected by the intervention.
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Fig 5. Comparison of CAT expression in left lungs of animals that received 1,320 µg of lipid #67:DOPE:CAT cDNA 21 days after intravenous administration (group Ib, n = 4). No expression was detected in any animal.
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Fig 6. Comparison of CAT expression in left lungs of animals that received 660 µg of lipid #67:DOPE:CAT cDNA 21 days after isolated lung perfusion (group IIb, n = 4). Strong expression was detected in all animals.
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Comment
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Compared with intravenous administration, we were able to establish reproducible, prolonged, organ-specific gene expression in vivo using an isolated lung perfusion technique. These results mimic those achieved by ex vivo models of lung transplantation [8]. Thus, by temporarily isolating the lung from the pulmonary circulation, we were able to limit transfection to the target organ and maintain gene expression for at least 21 days.
By isolating the lung in this and previous studies, either by removal in the transplantation studies or by vascular occlusion in this study, we have been able to extend the duration of gene expression. The most likely explanation for this effect is attributable to the direct increase in concentration of exposure of the lung to liposome-containing gene. However, it is unknown if any metabolic advantage to gene uptake occurs during the ischemic interval.
The limitations of this study include the small sample size and the limited number of parameters measured. However, the effects were reproducibly demonstrated in all animals and comparable to studies using ex vivo transfection. In addition, it was the intention of this study to demonstrate the feasibility of intravenous in vivo organ-specific transfection using a nonfunctional marker gene. Optimal conditions of incubation and concentration of liposome–DNA and exact duration of expression will be reserved for functional genes, which may have different requirements and results.
The immediate application of this model is to study the effects of gene therapy in primary and metastatic pulmonary tumors. The treatment of inoperable pulmonary tumors remains a clinical problem for which the standard treatment of systemic chemotherapy holds little promise for long-term survival. In advanced non–small cell lung cancer, no chemotherapeutic regimen has been able to produce a significant survival improvement or change in the natural history of patients with an advanced stage of this disease over the past two decades [1]. In metastatic soft tissue sarcoma, 5-year survival after surgical resection approaches only 25% [14–16] and treatment failures are usually attributable to local micrometastatic disease [17]. In addition, host toxicity often limits effective systemic chemotherapy in the treatment of metastatic sarcoma [18] and colorectal adenocarcinoma [19]. Applying this isolated lung technique to established models of these pulmonary diseases [10, 11] will allow for the study of the efficacy of gene therapy using one lung versus its contralateral control.
This model may also find use in the treatment of rejection after lung transplantation. Several strategies of functional gene transfer to ameliorate rejection after transplantation have been proposed and are currently under investigation. If this technique can be developed into a closed chest model, it would provide the ability to locally transfect the target organ and express genes that inhibit the rejection process after the time of transplantation.
In the future, this technique of isolated lung perfusion may find use in the clinical treatment of inoperable pulmonary carcinoma. A number of gene therapy strategies for inoperable pulmonary tumors have already been studied [1–5], and may prove to be clinically useful [6]. Application of this technique will allow specific transfection of the target organ with an increased duration of expression. If this technique can be developed into a closed chest model, additional uses may be found in the treatment of rejection after lung transplantation. Further investigation is warranted.
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Acknowledgments
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We thank Katherine Grapperhaus for performing the CAT assay and William Osburn of Genzyme Corporation for amplifying the CAT cDNA. This work was supported by the National Institute of Health grants 1 R01 HL 41281 and 5 T32 HL07776.
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Abstract
Introduction
