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. 1997 Oct;41(4):549–556. doi: 10.1136/gut.41.4.549

Liposome mediated gene transfer into the rat oesophagus

R Schmid 1, H Weidenbach 1, G Draenert 1, S Liptay 1, H Luhrs 1, G Adler 1
PMCID: PMC1891537  PMID: 9391258

Abstract

Background—Cancer of the oesophagus has so far eluded every attempt at pharmacological treatment. The recent advent of somatic gene therapy offers a new therapeutic approach to malignant tumours. 
Aim—To investigate whether and how gene transfer into the oesophagus can be achieved. 
Methods—A LacZ reporter gene was used as marker and transferred into the oesophagus of rats using cationic liposomes. Gene transfer was achieved by either luminal instillation into a closed segment using a double balloon catheter, or by intramural injection through a needle. Expression of β-galactosidase was monitored in the oesophagus and various control tissues by histochemistry, polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blotting. 
Results—Up to 100 days after in vivo gene transfer β-galactosidase activity could be demonstrated in the oesophagus. Following luminal application, the transgene was expressed in epithelial cells whereas intramural injection induced preferential expression in fibroblasts. 
Conclusion—In vivo gene transfer into the oesophagus is feasible and safe, and the route of administration largely determines cell type specificity. This novel approach will enable in vivo studies of growth, differentiation, and malignant transformation in the oesophagus, and may open new avenues to the confinement of oesophageal malignancies. 



Keywords: oesophagus; gene transfer; gene therapy; liposomes; balloon catheter

Full Text

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Figure 1 .

Figure 1

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: Method of introducing DNA-liposome complexes with a double balloon catheter into the oesophageal epithelium. 


Figure 2 .

Figure 2

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: Microscopic view of sections from transduced oesophageal epithelium using the double balloon catheter system. (A) Representative area positively stained for β-galactosidase activity (original magnification ×50); (B) an oesophageal section three days after luminal gene transfer, indicating transfected epithelial layers (original magnification ×250); (C) an oesophageal section with an intact keratin layer which acts as a barrier for gene transfer (original magnification ×100). Sections were counterstained with eosin. 


Figure 3 .

Figure 3

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: Microscopic view of sections from oesophagus transduced by injection. β-Galactosidase activity was detectable in the submucosa (A and B) and in the connective tissue of the muscular layer (B). Occasionally epithelial cells were positive (C). Original magnification ×100 (A), ×100 (B), ×250 (C). All sections were counterstained with eosin (A) or eosin and haematoxylin (B and C). 


Figure 4 .

Figure 4

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: Microscopic view of sections from oesophagus transduced by injection. Sections were incubated with a monoclonal anti-β-galactosidase antibody followed by a secondary biotin conjugated rabbit antimouse antibody and streptavidin-peroxidase complexes with DAB. β-Galactosidase activity is presented as a blue precipitate, whereas detection by the anti-β-galactosidase antibody is displayed as brown precipitate throughout the cytoplasm. The section was counterstained with haematoxylin. Original magnification ×250. 


Figure 5 .

Figure 5

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: Microscopic view of a section from the oesophagus injected with 200 µl DNA-liposomes. Three days following gene transfer, signs of inflammation, necrosis, and bleeding were visible at the area where the injection took place. Sections were fixed in 4% paraformaldehyde and stained with eosin (A) (original magnification ×50). Twenty eight days after gene transfer no signs of trauma, necrosis, or major inflammation were visible (B) (original magnification ×6.25). In these areas a local increase in small lymphoid aggregates remained in the submucosa (C) (original magnification ×50). Sections were counterstained with eosin (A) or with eosin in combination with haematoxylin (B and C). 


Figure 6 .

Figure 6

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: Detection of plasmid DNA in oesophageal tissue three days after direct gene transfer with DNA- liposome complexes by PCR. A specific band of 318 bp was detected after transduction with RSV-LacZ using direct injection (RSV-LacZ lanes 1-3), but not in controls, where Bluescript SK with liposomes was injected (control lanes 1-3). The 1 kB ladder (Gibco) was used as a marker. 


Figure 7 .

Figure 7

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: Detection of plasmid DNA in tissue samples after gene transfer. Three days after injection of oesophageal segments with RSV-LacZ DNA-liposomes genomic DNA was isolated. Genomic DNA (1 µg) was subjected to PCR analysis; 1 ng RSV-LacZ or 293 cells transfected with 5 µg RSV-LacZ were used as positive controls. One tenth of the PCR reaction was electrophoresed on a 1.6% agarose gel, stained with ethidium bromide, and photographed. Recombinant RSV-LacZ DNA was not detected as a specific 318 bp band in all tissues except in the oesophagus (A). Positive and negative controls are shown along with DNA markers. To enhance the signal Southern blot analysis was performed (B). The gel was blotted onto nitrocellulose and hybridised to a LacZ specific probe. No signals were detected in the negative control or in any tissue except the oesophagus. 


Figure 8 .

Figure 8

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: Expression of β-galactosidase mRNA in the oesophagus in vivo. A specific band of 318 bp was detected in RNA from oesophageal segments when reverse transcriptase was used (+) (A). The 1 kB ladder (Gibco) was used as a marker and 1 ng RSV-LacZ as a positive control. 


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