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Ann Thorac Surg 2001;71:1817-1823
© 2001 The Society of Thoracic Surgeons
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, 3108 Queeny Tower, One Barnes-Jewish Hospital Plaza, St. Louis, MO 63110
Presented at the Poster Session of the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Abstract |
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 Top Abstract IntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Methods. F344 rats underwent left main bronchus instillation of pCF1-CAT (chloramphenicol acetyl transferase) (130 µg) ± pCF1-ß-Gal (ß-galactosidase) (130 µg) in saline. Part Ia: 4°C preservation versus cotransfection. Lung isografts (4 groups, n = 8) were stored after transfection for 1 (2 groups: one received only pCF1-CAT), 6, and 18 hours. Recipient sacrifice was after 48 hours. Part Ib: 4°C preservation versus transgene expression. Rats were sacrificed 48 hours after transfection in a nontransplant setting (2 groups, n = 8; one received only pCF1-CAT). In a third group (n = 8) lungs were harvested 24 hours after transfection, stored for 18 hours, and recipients were sacrificed after 24 hours. The CAT and ß-Gal enzymatic-linked immunosorbent assays were performed. Part II: Lung/plasmid exposure time. In three groups (n = 6) after pCF1-CAT transfection the left main bronchus was not clamped, clamped for 10 minutes, or clamped for 1 hour. Sacrifice was after 48 hours.
Results. Part Ia: Lung CAT protein was (in picograms per 100 µg of total protein): median, 42 (range, 25 to 95) after 1 hour (only CAT); 67 (19 to 296) after 1 hour, 32 (6 to 157) after 6 hours; and 9 (5 to 243) after 18 hours. Lung ß-Gal protein was (in picograms per 100 µg of total protein): median, 20 (range, 5 to 353) after 1 hour; 17 (6 to 157) after 6 hours; 4 (1 to 74) after 18 hours (1 hour versus 18 hours, p = 0.04 for both proteins). CAT and ß-Gal production were significantly correlated (p = 0.0001, r = 0.924). Part Ib: Lung CAT protein was (in picograms per 100 µg of total protein): median, 2 (range, 0.6 to 10) no transplant, only CAT; 7 (0.3 to 13) no transplant; 3 (0.9 to 14) transplant. Part II: Left lung CAT protein was (in picograms per 100 µg of total protein): median, 31 (range, 6 to 83) no clamp; 74 (25 to 430) 10 minutes of clamp; 111 (30 to 263) 1 hour of clamp. Right lung CAT protein was (in picograms per 100 µg of total protein): median, 0.06 (range, 0 to 0.9) no clamp; 1 (0 to 6) 10 minutes of clamp; 1 (0 to 18) 1 hour of clamp.
Conclusions. Efficient lung isograft endobronchial cotransfection results from using naked plasmid. Cold preservation affects transfection efficiency but not transgene expression. Lung parenchyma/naked plasmid exposure time determines transfection efficiency.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Gene therapy technology has enabled successful reporter and functional transgene expression in experimental lung grafts [3–7]. In vivo and ex vivo transfer of single genes such as heat shock protein-70, endothelial constitutive nitric oxide synthase, transforming growth factor-ß1 using both viral and nonviral vectors has been shown to decrease lung graft ischemia–reperfusion injury and rejection [3–7].
Rather than using single gene transfer to impact on isolated events, multiple gene transfer by cotransfection may permit effective gene therapy through simultaneous modulation of concurrent biochemical pathways involved in ischemia reperfusion injury and rejection [3]. It could also be possible that there may be a synergistic response with a lower transfection dose/response ratio.
In a previous study we documented effective rat lung allograft transfection by naked plasmid encoding transforming growth factor-ß1 delivered through endobronchial instillation. In this model reduction of acute rejection was observed 5 days after transplant [7]. Thus, effective use of the simplest and less toxic vector for gene transfer, the naked plasmid, was documented for the first time within lung transplantation [7].
The purpose of this current study was to further evaluate the applicability of naked plasmid gene transfer within lung grafts in conditions that mimic the clinical lung transplantation setting. In particular, by using reporter genes encoding for chloramphenicol acetyl transferase and ß-galactosidase, prokaryotic proteins not produced in mammal systems, we specifically sought to determine the feasibility of in vivo naked plasmid cotransfection of lung graft parenchyma to document the possibility of an efficient multiple gene transfer to lung grafts. Furthermore, we aimed to evaluate the influence of cold preservation and of exposure time between lung graft parenchyma and the plasmid vector, on naked plasmid transfection and transgene expression. The former intended as the action of plasmid vector entering the aimed cells and starting the expression of the encoded gene. The latter being the ongoing expression over time of the encoded gene once the plasmid has entered the cells.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Plasmids
The plasmids (pCF1) encoding chloramphenicol acetyl transferase (CAT) and ß-galactosidase (ß-Gal), kindly provided by Dr Ronald K. Schule and Dr Nelson Yew (Genzyme Corporation, Framingham, MA) consist of the human cytomegalovirus immediate-early promoter and enhancer followed by the tripartite leader from adenovirus, a hybrid intron, the CAT or the ß-Gal genes, and a polyadenylation signal from bovine growth hormone. Purity of plasmids was tested and endotoxins units were lower than 1 (EU < 1).
Gene delivery
After anesthesia and orotracheal intubation with a 14-gauge catheter, a selective cannulation of the left main bronchus was performed over a guidewire with PE50 tubing. The position of the guidewire was confirmed either by direct assessment of the right hilum or by fluoroscopy. Naked plasmid suspensions within saline solution (100 µL) were instilled. The animals were then placed in the left decubitus position and ventilated for 10 minutes. Thereafter, they were either awakened or underwent left lung harvest according to the experimental design.
Study design
This study was conducted in two parts. Part I: The effect of cold preservation on gene cotransfection (Ia) and the effect of cold preservation on transgene expression (Ib) were studied. Part II: The influence of exposure time between naked plasmid and lung parenchyma was investigated.
Part Ia: cold preservation versus gene cotransfection
Four groups of F344 rats (n = 8) underwent isogenic orthotopic left lung transplant [6] (Fig 1). In three groups the donor left lung was cotransfected according to the protocol with 130 µg of pCF1-CAT and 130 µg of pCF1-ß-Gal and underwent 4°C low potassium dextran glucose solution storage for either 1, 6, and 18 hours, respectively. In the fourth group the left lung was transfected with pCF1-CAT plasmid alone and stored for 1 hour and served as a single transfection control group. The correct position of the guidewire and of the cannula within the left main bronchus was determined by direct assessment of the right hilum. In this group recipients were sacrificed 48 hours after implantation. An enzymatic-linked immunosorbent assay was used to quantify CAT and ß-Gal protein expression in the lung isograft, as well as in the heart, liver, and kidney. Immunohistochemistry on isograft cross-sections determined the transfection pattern.
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Part II: lung parenchyma/naked plasmid exposure time
F344 rats underwent selective left lung gene transfection with 130 µg of pCF1-CAT. The correct position of the cannula within the left main bronchus was determined by direct assessment of the right hilum through a right thoracotomy. After 10 minutes of left decubitus ventilation, rats were randomized into three groups (n = 6). In two groups, the left main bronchus was clamped for 10 minutes and 1 hour, respectively, in an effort to increase left lung exposure to the plasmid. In the third group, no clamping was performed. Animals were sacrificed 48 hours after transfection. CAT protein expression was assessed in the left and the right lungs using a CAT enzymatic-linked immunosorbent assay.
Assessments
Enzymatic-linked immunosorbent assay for CAT and ß-Gal
The CAT protein was extracted by homogenizing tissue for 30 seconds in a lysis solution of 100 mmol/L potassium phosphate at pH 7.8, 0.2% Triton X-100, 0.5 mmol/L DTT, 0.2 mmol/L PMSF, 5 µg/mL leupeptin. The homogenate was then stored at room temperature for 15 minutes and centrifuged at 15000 rpm for 15 minutes and the supernatant was used to perform CAT and ß-Gal enzymatic-linked immunosorbent assay (CAT ELISA kit and ß-Gal ELISA kit; Boehringer Mannheim, Mannheim, Germany). Total protein concentration of the supernatant was calculated by using the BCA Protein Assay kit (Pierce, Rockford, IL).
Immunohistochemistry for CAT
Lungs were inflated with Histochoice (Amresco, Solon Ohio), fixed overnight, and cross-sectioned in four pieces and paraffin embedded.
Paraffin sections (5 µm) were deparaffinized and underwent a target retrieval steam treatment in a caplin jar for 30 minutes (DAKO target retrieval solution; DAKO Corp, Carpinteria, CA). Slides were washed three times in phosphate-buffered saline for 3 minutes at a time. Nonspecific sites were blocked by bathing the slides for 30 minutes in full strength Super Block Blocking Buffer in TBS (Super Block, Pierce, Rockford, IL). Slides were then incubated for 1 hour at room temperature with the primary unconjugated rabbit antibody to CAT (5 Prime –3 Prime, Inc, Boulder, CO) at 1:700 dilution in 10% normal goat serum, 10% Tris (hydroxymethyl) amino methane Buffered Solution (TBS) (Super Block) and TBS, 0.2% Triton X-100. Slides were then washed three times in TBS, 0.2% Triton X-100 for 5 minutes at a time. Secondary Super Sensitive Rabbit Link biotinylated goat antirabbit immunoglobulin for mouse/rat tissue (BioGenex, San Ramon, CA) was incubated for 30 minutes. Slides were washed three times in TBS, 0.2% Triton X-100 for 5 minutes at time and once in TBS for 5 minutes. Slides were incubated with streptavidin alkaline phosphatase conjugated (DAKO Corp) for 30 minutes. Slides were then washed in TBS for 3 minutes and in 2 mol/L urea with gentle shaking for 30 minutes, again three times in TBS for 3 minutes and placed in detection buffer at pH 9.5 for 4 minutes. Slides were developed for 15 minutes with an alkaline phosphatase substrate kit IV BCIP/NBT SK-5400 (Vector Laboratories, Inc, Burlingame, CA). Bathing the slides in Tris (hydroxy methyl) amino methane-HCl-Ethylenediamine-tetraacetic Acid (TE) at pH 8.0 stopped the reaction. Slides were counterstained with nuclear fast red H-3403 (Vector Laboratories) for 8 minutes and mounted.
Statistical analysis
Parametric data were analyzed after logarithmic correction by one-way analysis of variance. Multiple comparisons were made with Fisher’s test. Correlation analysis was by using Fisher’s r to z test. Differences were considered significant when the p value was less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The genes encoding the CAT and ß-Gal proteins were delivered using the same plasmid, pCF1, ensuring the same gene transfer potentials for both reporter genes. Efficient transfection of both CAT and ß-Gal was documented in all lungs based on the quantitative evidence by enzymatic-linked immunosorbent assay. A strongly significant correlation of the levels of protein expression of the two proteins documented the efficient cotransfection.
No adverse effect on gene transfer was observed by performing cotransfection compared to single gene transfection as observed in the two groups that underwent 1 hour of cold preservation within Part Ia of the study (Fig 3). The lower levels of ß-Gal protein expression compared to the CAT protein expression in all three groups are a result of the smaller number of ß-Gal plasmids present within 130 µg of DNA compared to CAT plasmids. The pCF1-ß-Gal plasmid is approximately 1.6-fold larger than the pCF1-CAT plasmid.
In this set of experiments we investigated the influence of cold preservation applied immediately after naked plasmid delivery, thus when the cellular uptake of the DNA is thought to be initiated. Reduced transfection efficiency was observed after prolonged cold preservation as measured by quantifying the subsequent levels of protein expression. This may have resulted from the severe ischemia reperfusion injury after prolonged cold storage, from the possible DNA degradation during the preservation period or from the negative influence of cold temperature on the not well-known cellular uptake mechanism, whether active or not, of the naked plasmid [4, 8, 9].
The organ selectivity of the proposed transfection method was confirmed as previously suggested with no CAT protein expression documented within hearts, livers, or kidney [7]. To study the effect of prolonged cold preservation on transgene expression in Part Ib, a group of lungs were harvested 24 hours after transfection, when transgene expression after plasmid transfection is known to be ongoing [10]. Hypothermic storage was for 18 hours and recipients were sacrificed 24 hours after implantation. Thus, these lungs were subjected to 24 + 24 hours of physiologic temperature to match the experimental conditions used for lungs within the other two groups. In addition they were also submitted to an interval of 18 hours of hypothermic ischemia at which enzymatic activity is known to be significantly reduced [11]. No significant impact on transgene expression was noted after prolonged 4°C preservation taking also in consideration the severe ischemia reperfusion injury. Furthermore, no significant additional CAT expression was obtained during the 18 hours of cold preservation.
The CAT protein levels within lung grafts that underwent 1 hour of cold nonventilated storage after gene delivery performed immediately before harvest (Part Ia) were in a range of 10- to 20-fold greater compared to those when delivery was performed without harvest, thus, with ongoing ventilation (Part Ib). Therefore, in part II we sought to investigate the influence of the exposure time between naked plasmid and lung parenchyma on subsequent transfection efficiency. We created a model that permitted such analysis with the exclusion of the cold preservation.
A significant increase in transfection efficiency was documented when the left main bronchus was clamped for 1 hour after plasmid delivery. A three- to four-fold increase in CAT protein expression was observed in comparison to lungs that were not clamped. Quantification of CAT protein in the right lung documented that spillover of the plasmid suspension had occurred, but not to a level that would account for the difference revealed in the left lungs. In common practice in molecular biology, naked plasmid used in vitro in the absence of any chemical or liposomal adjunct will not produce any cell transfection.
Griesenbach [12] and Yoshimura [13] and their colleagues demonstrated very low levels of lung transfection after naked plasmid delivery by intratracheal instillation. They advocated the use of a liposomal vector to accomplish transfection through the airway route. However, the present results and our previous observation [7] indicate that under appropriate conditions naked plasmid delivered through the airway can accomplish pulmonary parenchymal transfection. Meyer [8], Zabner [14], and Tsan [15] and their associates have reported similar conclusions supporting naked plasmid as an efficient vector for gene transfection of the airway epithelium.
In other organs, given the right conditions, naked plasmid has been demonstrated to be efficient for in vivo transfection [9, 16, 17]. Moreover, it has no adverse effects, being neither immunogenic as viral vectors, nor proinflammatory as liposomes [18, 19]. Furthermore, liposome–plasmid complex transfection within lung parenchyma may be inhibited by the presence of both surfactant and mucus [20–22].
As previously suggested by Meyer and colleagues [8] and based on data previously gathered in our laboratory and in the present study, efficient lung transfection using naked plasmids is obtained by optimizing the method of gene delivery and, most important, by extending the contact and exposure time between plasmids and the airway epithelium. Song and coworkers [10] have suggested similar conclusions. They demonstrated that by halting the passage of naked plasmid through the capillaries to optimize contact with the endothelium and thus prolong the exposure time produces significant transfection efficiency.
The variability of transfection observed for each group in all experiments may be due either to biological variability or to a limit of the endobronchial instillation delivery method. Certainly this is a constant finding from our laboratory and others, either when testing reporter genes or when investigating functional genes, independently from the type of vector used whether viral or nonviral, or the delivery method. Nevertheless, the effectiveness of gene therapy has been repeatedly documented [4–7].
A limit of our transfection method is the nonhomogeneous distribution of transfected cells throughout the entire lung. The CAT immunohistochemistry has documented a gradient of transfection from the caudal to the cranial portion of the lung. This observation explains the resulting higher levels of CAT protein documented in our previous article compared to the levels in the present study. In our previous study [7], we had not yet developed a method of assessing the transfection distribution by immunohistochemistry and quantified the CAT protein expression within the caudal half of the lung.
Our study documents that lung parenchyma transfection by endobronchial naked plasmid delivery is feasible by optimizing the conditions of administration. This is a novel adjunct that may be of interest in the application of gene therapy toward lung disease, whether consequence of lung transplantation or not. Naked plasmid is well-known not to be the most efficient vector, at least as tested in vitro [9]. Although, it is the least injurious vector, with a temporal limitation toward its expression, as well as most of the vectors described in literature, but it has the potential of a safe repeat applicability.
In vivo cotransfection, as demonstrated in this study by using reporter genes, suggests the future application of gene therapy by concomitant delivery of different functional genes. Each one could be aimed at different biochemical pathways of a single injurious event such as ischemia reperfusion injury or rejection within the lung transplant setting or may be aimed at different injurious events contemporarily.
In conclusion, under optimum conditions of plasmid/graft exposure time and temperature efficient cotransfection of the lung parenchyma is feasible. The most important condition for optimal applicability of the naked plasmid vector to obtain best transfection of the lung parenchyma is prolonging the exposure time between the plasmids and the airway epithelium. On the basis of our results, gene therapy by naked plasmids either for ischemia reperfusion injury or for rejection should not be administered at the time of harvest. No transgene expression has been observed during cold preservation. Furthermore, prolonged cold preservation reduces the transfection efficiency. Therefore, when aiming gene therapy at ischemia reperfusion injury, it should be administered in advance of the time of harvest. This will provide the best transfection efficiency and ongoing transgene expression at the time of harvest; thus, the eventual functional transgene protein could be ready to act at the time of warm reperfusion.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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