Chest
Volume 123, Issue 1, January 2003, Pages 202-208
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Laboratory and Animal Investigations
Pleural Space as a Site of Ectopic Gene Delivery: Transfection of Pleural Mesothelial Cells With Systemic Distribution of Gene Product

https://doi.org/10.1378/chest.123.1.202Get rights and content

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.

Section snippets

Materials and Methods

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

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.

Pleural Lavage Fluid Levels of PALP in Treatment Rabbits vs Control Rabbits

PALP was recovered in significantly higher amounts in the pleural lavage fluid from rabbits in the treatment group than from those in the control

Discussion

The objective of the present short-term study was to assess the pleural space as a site for ectopic gene expression and for the systemic distribution of transgene products. We have successfully demonstrated the following: (1) the transfection of primary pleural mesothelial cells in vitro with plasmids, (2) the transfection of pleural mesothelial cells in vivo with the expression of the gene product in the pleural space, and (3) the subsequent systemic distribution of the gene product.

Ectopic

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

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