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Pleural Space as a Site of Ectopic Gene Delivery^*

Transfection of Pleural Mesothelial Cells With Systemic Distribution of Gene Product

^* From the Pulmonary and Critical Care Division (Mr. Devin and Dr. Lane), Vanderbilt University, Nashville, TN; and the Department of Pulmonary Medicine (Drs. Light and Lee), St. Thomas Hospital, Nashville, TN.

Correspondence to: Richard W. Light, MD, FCCP, Department of Pulmonary Medicine, St. Thomas Hospital, 4220 Harding Rd, Nashville, TN 37205; e-mail: rlight98{at}yahoo.com

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Study objectives: Successful ectopic gene therapy requires the transfection of the cells at the ectopic site, with local and systemic delivery of the gene product. This study aimed to evaluate the pleural mesothelial surface as a potential site for ectopic gene therapy.

Design: A secreted placental alkaline phosphatase (PALP) plasmid was injected bilaterally into the pleural spaces of seven rabbits via a chest tube, while an irrelevant reporter plasmid was injected into seven control rabbits. Blood was collected at baseline and at 24, 48, and 72 h after the injections. Pleural fluid was collected by lavage at 24, 48, and 72 h after the injections. The PALP level was measured by chemiluminesence.

Measurements and results: Significant expressions of PALP proteins were observed in the serum of the treatment rabbits, with a threefold increase over baseline at 24 h, a ninefold increase at 48 h, and a twofold increase at 72 h. The serum PALP levels in the control rabbits remained at baseline levels at all time points. The pleural fluid PALP levels peaked at 24 h and decreased over the next 72 h. Mimicking the in vivo pattern, pleural mesothelial cells transfected in vitro demonstrated a similar increase in PALP levels.

Conclusions: The results of the present short-term pilot study suggest that pleural mesothelial cells can be successfully transfected with plasmids, with increases in both the local and systemic levels of the gene product. The pleural space should be further evaluated for ectopic gene therapy.

Key Words: gene therapy • mesothelial cells • pleura

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Gene therapy provides an attractive alternative for disease treatment. However, the ability to correct a defective gene with a functional one is not yet possible. One novel approach is ectopic gene therapy, which involves the transfection of cells at an ectopic site that does not normally express the protein. The transfected cells then maintain the production of the gene product with subsequent systemic distribution of the protein for which an individual is deficient. There are a number of diseases that currently are treated with the repeated replacement of recombinant or purified proteins, such as diabetes mellitus,¹ hemophilia A,² pituitary dwarfism,³ and ₁-antitrypsin (₁-AT) deficiency.⁴ These diseases require long-term treatment, and the cost of these recombinant or purified proteins can be great. Ectopic gene therapy potentially could provide long-term treatment and could eliminate the need for frequent injections of recombinant or purified proteins, thereby significantly reducing the cost of treatment.

Several sites have been investigated for their potential as reservoirs for ectopic gene transfection. These included the skin,⁵ liver,⁶ bone marrow,⁷ and the peritoneum.^{8 9 10} Thus far, there has been only marginal success achieved with the transfection of these sites, warranting the investigation of additional sites.

The pleural space is a previously unexplored site with the potential for ectopic gene expression and the systemic delivery of a therapeutic protein. The pleural space is characterized by a large surface area lined by a thin layer of mesothelial cells. This property may allow the efficient uptake of the gene and the subsequent high expression of the gene product. In addition, physiologic pleural fluid is drained constantly into the systemic circulation via the parietal lymphatic system, thus offering an excellent mechanism with which to deliver gene products to the systemic circulation. Pleural malignant mesothelioma cells have been successfully transfected by suicide genes.¹¹ However, the transfection of normal pleural mesothelial cells with the aim of ectopic gene expression has not previously been attempted.

We hypothesized that it is possible to transfect pleural mesothelial cells with plasmids, and that this transfection will lead to the expression of the gene product in the pleural space with a resultant systemic distribution of the protein. The objectives of the present short-term pilot study were as follows: (1) to transfect primary pleural mesothelial cells in vitro, (2) to transfect pleural mesothelial cells in vivo, and (3) to demonstrate the systemic distribution of the gene product in a rabbit model.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
The Vanderbilt University Institutional Animal Care and Use Committee approved the study protocol.

In Vitro Transfection of Mesothelial Cells
Mesothelial Cell Culture:
Pleural mesothelial cells were harvested from New Zealand white rabbits that were 1.5 to 2.0 kg in size. The rabbits were anesthetized with an IM injection of 35 mg/kg ketamine (Fort Dodge Animal Health Laboratories; Fort Dodge, IA) and 5 mg/kg xylazine hydrochloride (Fermenta; Kansas City, MO), and were killed with carbon dioxide. The abdomen was opened to expose the diaphragm. Under direct vision, 10 mL Hank’s balanced salt solution (Life Technologies; Grand Island, NY) was injected into the pleural cavity of each hemithorax from beneath the diaphragm and then was removed by aspiration after 2 min. Approximately 10 mL 0.25% trypsin-ethylenediaminetetraacetic acid solution (Life Technologies) then was injected into each pleural cavity and was allowed to bathe the pleural surfaces for 10 min during which the rabbits were rotated. The solution, containing the mesothelial cells released by the trypsin, was then aspirated, collected in Dulbecco’s modified Eagle’s medium (DMEM) [Life Technologies] and was centrifuged at 3,000 rotations per minute (rpm) for 10 min at -4°C. The supernatant was discarded, and the cell pellet was resuspended in DMEM. The cells were plated in 75-cm² cell culture flasks (Costar; Cambridge, MA) and were cultured with DMEM, supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum (Life Technologies). The cells were incubated at 37°C with 95% air and 5% CO₂. The medium was changed the following day to remove nonadherant cells. The mesothelial cells were grown to confluence before the experiment. Passages 3 to 7 were used. The cells then were trypsinized and transferred to six-well tissue culture plates (Becton Dickinson; Franklin, NJ) at least 24 h prior to the cell experiments. The area of each well is 9.6 cm².

The purity of the cultured cells was verified prior to the experiments by the following methods: (1) the cells demonstrated the typical cobblestone morphology of mesothelial cells; and (2) the cells were prepared on glass slides and were stained with low-molecular-weight cytokeratin. One hundred cells were counted, and the percentage of cytokeratin-positive cells was recorded. The average result from three different high-power fields was taken. Over 95% of the cultured cells were cytokeratin-positive, confirming their epithelial origin.

Preparation of Plasmid/Liposome Complex:
A placental alkaline phosphatase (PALP) transgene driven by a cytomegalovirus (CMV) promoter was used in the study. The PALP plasmid was propagated in Escherichia coli and was isolated using a DNA purification system (Promega; Madison, WI). The isolation of the PALP plasmid was verified by cutting with the NheI restriction enzyme and by presence of the resultant 0.9-kb band. On verification, the PALP plasmid was incubated with the proprietary transfection enhancer 4T nuclear localizing sequence. Four hundred micrograms of the PALP plasmid was added to a 3 M excess of the 4T nuclear localizing sequence. The plasmid DNA and transfection enhancer were placed in a polymerase chain reaction machine and interacted. The reaction was heated to 50°C for 1 h, after which the temperature was allowed to drop by 2°C every 10 min to 21°C. The total volume then was brought to 1,750 µL using distilled water. Forty micrograms of the cationic liposome N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride-dioleoyl phosphatidylethanolamine (supplied by Dr. Xiang Gao; Vanderbilt University; Nashville, TN) was added. The plasmid-liposome complex was incubated at 37°C overnight. This was added to each of the wells along with Hank’s balanced salt solution and 10% fetal calf serum. Samples were taken prior to transfection and at 24, 48, 72, and 96 h following transfection. The first sample was used as the background comparing this to subsequent samples to determine the fold increase. The samples were centrifuged at 3000 rpm for 5 min, and the supernatant was saved. This procedure was repeated until all contaminating cells were removed. Samples were stored at -70°C until assayed.

In Vitro PALP Assay:
PALP is heat-stable, whereas endogenous alkaline phosphatase (ALP) is inactivated when heated at 56°C for 30 min.¹² To determine the concentration of PALP, 100 µL detection buffer was added (100 mM Tris and 100 mM NaCl [pH 9.5]) to 100 µL the sample taken from each well. In addition to the samples, the background was determined by using 100 µL the media in which the samples were suspended. In our experiment, samples were heated at 65°C for 3 h to ensure adequate inactivation of the native ALP. Thus, the remaining heat-stable ALP is a product of the PALP gene used for transfection. The samples were allowed to cool, and a 1:100 dilution (in detection buffer) of 100 µL disodium 3-[4-methoxyspiro(1,2-dioxetone-3,2'-(5'-chloro)tricyclo(3.3.1.1_3.2)decan)-4-yl]phenyl phosphate (CDP star; Roche; Indianapolis, IN) was added. The samples were measured using luminometry (All Monolight 3010; BD Bioscience Pharmingen; San Diego, CA) [measuring time, 30 s], and the results were expressed in relative light units (RLU). The values from all three wells were averaged for each time point and were expressed as the fold increase over background.

In Vivo Transfection of Rabbits
Insertion of Chest Tubes:
Fourteen New Zealand white rabbits (weight, 1.5 to 2.0 kg) were divided equally into the treatment and control groups. Chest tubes were inserted into both pleural spaces of all rabbits using the same methods as described in our previous studies.^{13 14} In brief, under anesthesia, the chest was shaved and the skin was sterilized with 10% povidone iodine (Baxter; Deerfield, IL). The rabbit was placed in the lateral decubitus position, and a small (ie, < 3 cm) skin incision was made midway between the tip of the scapula and the sternum, approximately 2 cm above the costal margin. Chest tubes were made from IV solution set tubes (Baxter) with three extra openings made near the distal end of the tube to enhance drainage. The chest tubes were inserted by blunt dissection into the right and then the left pleural cavities. The chest tube was secured at the muscle layers with purse string sutures. The proximal end of the chest tube then was tunneled underneath the skin and was drawn out through the skin posteriorly and superiorly between the scapulas. The exterior end of the chest tube was sealed with a one-way valve with cap (Medexinc; Hilliard, OH) via an adapter and then was sutured to the skin. A three-way stopcock was attached to the end of the chest tube through which any aspirated air was evacuated from the pleural space.

Preparation of Plasmid/Liposome Complex:
The PALP transgene was used as the treatment plasmid. In order to provide an adequate control group, we used an irrelevant gene, transforming growth factor-ß₁ (supplied by Dr. Jeffrey Davidson; Vanderbilt University). The same CMV promoter as our PALP gene drove this control plasmid. Both plasmids were prepared and incubated with the liposome using the method described for the in vitro transfection of mesothelial cells. The final volume was brought to 2.5 mL using distilled water. The treatment plasmid was injected intrapleurally into both pleural cavities of the seven rabbits in the treatment group, via the chest tubes. The chest tube was flushed of any residual fluid with 1 mL distilled water. The same procedure was used to deliver the control plasmid to the rabbits in the control group.

Collection of Samples:
Venous blood was drawn from the marginal ear veins of all the rabbits prior to surgery, and at 24, 48, and 72 h after the intrapleural injection of the plasmids. In one of the control rabbits, cannulation of the ear vein was unsuccessful, and it was excluded from the study. Samples were centrifuged at 3,000 rpm for 5 min, and the supernatant was collected. This was repeated until all contaminating cells were removed. Samples were stored at -70°C until assayed for PALP.

To ensure that the systemic rise in PALP was a result of ectopic gene production in the pleural space, pleural lavage was performed bilaterally in three control rabbits and three treatment rabbits at 24, 48, and 72 h after the administration of each respective plasmid. Pleural lavage was performed by injecting 5 mL 0.9% saline solution intrapleurally via the chest tubes. The rabbits were rotated to ensure thorough distribution of the saline solution, and the fluid was aspirated from the chest tubes. Samples were centrifuged and stored as described above. Protein concentrations were determined on all of the pleural samples (Coomassie Plus Protein Assay; Pierce, Rockford, IL) and was measured (Multiscan; Titertek; Huntsville, AL).

In Vivo PALP Assay:
The blood and pleural lavage fluid samples were assayed for PALP levels using the same method as described for the in vitro PALP assay. The lavage fluid from each pleural space was analyzed separately. The levels of PALP were expressed in RLU and were standardized to the amount of protein. The systemic levels of PALP were presented as the fold increase over background, with background representing the baseline value of PALP.

Statistical Analysis
The differences between groups were compared with the Student t test (for parametric data) and the Mann-Whitney rank sum test (for nonparametric data). One-way analysis of variance was used to compare the results in the various time points within each group. Two-way analysis of variance was used to compare the differences of the serum levels between the treatment and control groups. Results were expressed as the mean ± SD unless otherwise stated. All statistical analyses were performed using a computer program (SigmaStat, version 2.03; SPSS Science; Chicago, IL). A p value of < 0.05 was considered to be significant.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Transfection of Mesothelial Cells
The PALP plasmid successfully transfected primary mesothelial cells in vitro (Fig 1 ). There was no detectable level of PALP prior to transfection. By 24 and 48 h after transfection, significant increases (16-fold and 17-fold, respectively) of PALP production were seen over the baseline levels, followed by a gradual decline at the later time points.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. PALP production after the transfection of rabbit pleural mesothelial cells with a cationic liposome-delivered PALP transgene.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Pleural fluid PALP levels following the intrapleural administration of the PALP transgene in the treatment group vs intrapleural administration of a control transgene in the control group. The vertical axis represents the amount of PALP adjusted for protein. The horizontal axis represents the time after transfection. The data represent the mean of six treatment pleural spaces and six control pleural spaces.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Serum PALP levels in rabbits following the intrapleural administration of the PALP transgene in the treatment group and the intrapleural administration of a control transgene in the control group. PALP is expressed as the fold increase over the baseline, as determined by dividing the baseline level of PALP for a given rabbit into its own respective time points. The data represent the mean increase from seven treatment rabbits and six control rabbits.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

Ectopic gene expression holds promise for the local and systemic delivery of therapeutic proteins. The ideal ectopic site for gene expression should possess several essential qualities. It should allow efficient uptake of the gene, the transfected cells should have secretory properties making it capable of producing large amounts of the gene product, and the gene product should have easy access to the systemic circulation.

The peritoneal cavity has been investigated previously^{8 9 10 11} as a potential site of ectopic gene expression. One study¹⁰ evaluated adenovirus-mediated ₁-AT complementary DNA transfer to the peritoneal mesothelium for the treatment of ₁-AT deficiency. The peak serum levels achieved were several hundred times lower than the serum levels needed to adequately correct ₁-AT deficiency.¹⁰ Other studies evaluating the peritoneum as an ectopic site also have failed to demonstrate the sustained systemic expression of the transgene product.

The pleural space is similar to the peritoneum but possesses potential advantages over the peritoneum that make it an attractive site for ectopic gene therapy. The total area of the visceral and the parietal pleurae is large (estimated as 2,000 cm² in a 70-kg man),¹⁵ and the pleural surface is lined by a monolayer of flattened mesothelial cells that is < 4 µm thick.¹⁶ This combination of a broad, smooth surface area with a single layer of cells provides a large number of target cells for successful gene uptake. In addition, the normal respiratory movement helps to distribute the pleural fluid, and its contents, throughout the pleural space. This allows the gene to gain adequate contact with the entire pleural surface.

Mesothelial cells also have excellent secretory function following transfection and can devote up to 3% of their total protein synthesis to a single secreted protein.^{17 18} In healthy humans, there is a constant production and drainage of pleural fluid that serves as a lubricant between the visceral and parietal pleurae. Proteins produced by the mesothelial cells are carried by this pleural fluid, which is drained continuously via stomas in the parietal pleura into the thoracic lymphatics and eventually empties into the systemic circulation.^{16 19} In a healthy individual, the rate of lymphatic drainage is estimated to be approximately 0.01 mL/kg/h, but the rate can be increased by at least 20 times if more pleural fluid is present.¹⁹ This effective drainage system can allow the constant delivery of the gene products to the systemic circulation. The secretory nature of mesothelial cells in combination with efficient drainage from the pleural space support the use of the pleural space for ectopic gene therapy.

We demonstrated successful transfection of the pleural mesothelial cells by the PALP gene in vitro by the intrapleural route of delivery (Fig 1) . At 24 h, there was a dramatic 16-fold increase over baseline, and this was sustained for > 48 h. We further showed that transfection of the pleural mesothelial cells by the PALP gene was successful in vivo as well. The treatment group yielded significantly higher levels of PALP in the pleural fluid than did the control group (Fig 2 ). The pleural fluid levels of PALP peaked at 24 h in the treatment group and decreased over the next 48 h. Most importantly, the increase of PALP in the pleural fluid was paralleled by a rise in the systemic serum levels of PALP. Again, the serum levels of PALP were significantly higher in the treatment group than in the control group at all time points studied (Fig 3) .

Our data suggest that the peak protein level in the serum lagged the peak protein expression in the pleural space by 24 h. Interestingly, this finding is in keeping with that of Murphy and Rheinwald,⁹ who observed that peak levels of human growth hormone in the serum were obtained approximately 1 day following intraperitoneal injection of human growth hormone-transfected cells. The serum levels of PALP began to trend downward after 48 h, but at 72 h, which was our final time point, there was still a significant systemic level of PALP. When other sites are used for ectopic gene delivery, the levels of the gene product peak relatively soon and then decline. For example, when adenoviruses containing the ₁-AT gene are injected intraperitoneally, the systemic levels of ₁-AT peak at 96 h and are significantly decreased at 8 days.¹⁰ Further experiments are needed to determine the duration of the protein expression.

There are several possible explanations for the decrease in PALP expression over time. This may be a result of an immune response mounted by the rabbits to the human PALP that was injected. Alternatively, the mesothelial cells might have down-regulated the CMV promoter. The CMV promoter is known to be a potent promoter in vitro, but not always in vivo. Down-regulation of the CMV promoter is believed to be mediated via a decrease in nuclear factor-B activation²⁰ or interferon- inhibition.²¹ In addition, the decline in expression may be a result of cell turnover. The PALP gene functions in an extrachromosomal fashion, therefore, when the cell divides, the exogenous gene is not passed on to the progeny. PALP may be expressed at a constant level until the cell divides, at which point the new cell does not carry the PALP gene.

We chose to use the gene for PALP to investigate the pleural site, because the PALP produced is heat-stable, whereas endogenous ALP is inactivated when it is heated adequately.¹² This unique characteristic allows us to inactivate the endogenous ALP easily such that the remaining heat-stable ALP would be the product of the transfected PALP gene. Unlike previous studies, we used a cationic liposome instead of an adenovirus vector to deliver the gene. Recent trials using adenovirus as the vector raised concerns about its immunogenicity. Treatment-related inflammation resulting in the production of neutralizing antibodies and cytotoxic T lymphocytes might prevent its repeated administration.²² In contrast, cationic liposomes are nonimmunogenic and theoretically can be administered repeatedly without a decrease in efficacy.

Our study is the first to explore the potential use of the pleural space as a site for ectopic gene expression. Based on these interesting results, the pleural space should be further evaluated. It should be emphasized, however, that the serum levels of the gene product were evaluated only for 72 h and that by that time the levels of the gene product appeared to be decreasing. If indeed the serum levels of the gene product are decreasing that soon, additional studies are indicated to elicit the means to extend the duration of the gene expression. Additionally, practical methods to deliver the gene to the pleural space, such as ultrasound-guided delivery, need to be investigated. If these hurdles can be overcome, the pleural space may prove useful as a site for ectopic gene therapy for the replacement treatment of various protein deficiencies and may be used to deliver novel oncogene therapies for patients with malignant pleural effusions.

	Footnotes

 
Abbreviations: ₁-AT = ₁-antitrypsin; ALP = alkaline phosphatase; CMV = cytomegalovirus; DMEM = Dulbecco’s modified Eagle’s medium; PALP = placental alkaline phosphatase; RLU = relative light units; rpm = rotations per minute

This research was supported by the St. Thomas Foundation, Nashville, TN.

Received for publication February 19, 2002. Accepted for publication June 24, 2002.

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