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. 1999 Jun;44(6):800–807. doi: 10.1136/gut.44.6.800

Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure

S Wirtz 1, P Galle 1, M Neurath 1
PMCID: PMC1727540  PMID: 10323880

Abstract

BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.
METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. β-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.
RESULTS—Intravenous or intraperitoneal injection of AdCMVβGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVβGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVβGal caused a higher β-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMVβGal vectors.
CONCLUSIONS—Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.


Keywords: adenovirus; gene transfer; colitis; colon

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Figure 1  .

Figure 1  

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Strategy for generation and propagation of Ad5 vectors. This vector can be used to obtain expression vectors for cytokine signalling proteins (S Wirtz, P R Galle & M F Neurath, unpublished work) or reporter genes such as β-galactosidase. To obtain the AdCMVβGal vector, the entire β-galactosidase coding region was inserted into the Ad5 pBHG11 vector via homologous recombination using a shuttle vector (pΔE1sp1) and cotransfection strategies in 293 cells. After recombination, large scale amplification of AdCMVβGal was performed in 293 cells. Finally, high titre viral stocks were generated and used for in vitro and in vivo studies. CMV, cytomegalovirus; ITR, inverted terminal repeat.

Figure 2  .

Figure 2  

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β-Galactosidase activities after different routes of reporter vector administration. Six to eight week old healthy Balb/c mice were infected with 1 × 109 pfu AdCMVβGal by the indicated administration routes. After three days, organs were removed and homogenised, and luminescent β-galactosidase activity was determined. Results are expressed as mean (SD) from three independent experiments (three mice/group) and are reported as increase in enzyme activity (relative light units (RLU))/100 mg of tissue) compared with untreated control mice. * and † indicate significantly (p<0.05) increased or decreased respectively RLU values compared with the other two administration routes. nd, not determined.

Figure 3  .

Figure 3  

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Time course of β-galactosidase expression in the colon of Balb/c mice. Six to eight week old Balb/c mice were infected at days 0 and 3 with 1 × 109 pfu AdCMVβGal via the rectum. The colon was removed at the indicated time points, and luminescent β-galactosidase activity was determined in colonic homogenates. Results are expressed as mean (SD) from three independent experiments (three mice/group) and represent an increase in enzyme activity (relative light units (RLU)) compared with uninfected control mice.

Figure 4  .

Figure 4  

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β-Galactosidase activities in the colon of mice with experimental colitis. Six to eight week old Balb/c mice with or without trinitrobenzenesulphonic acid (TNBS) induced colitis were infected with 1 × 109 pfu AdCMVβGal via the rectum. After three days the colon was removed and homogenised. In addition, epithelial and lamina propria cells were isolated and luminescent β-galactosidase activity was determined. Results are expressed as mean (SD) from three independent experiments (three mice/group) and represent an increase in enzyme activity (relative light units (RLU)) compared with untreated control mice. Asterisks indicate significantly (p<0.05) increased RLU values compared with the control group.

Figure 5  .

Figure 5  

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Histological analysis of the small bowel (A, E), liver (B, F), lung (C, G), and kidney (D, H) of mice with experimental colitis given AdCMVβGal or phosphate buffered saline (PBS). Eight week old Balb/c mice with trinitrobenzenesulphonic acid (TNBS) induced colitis were infected with 1 × 109 pfu AdCMVβGal via the rectum. After seven days, organs from AdCMVβGal treated (lower panel) and PBS treated control (upper panel) mice were removed. Cryosections were made and stained with haematoxylin/eosin. No inflammatory reaction was noted upon adenoviral gene delivery. Additional experiments after a second administration of AdCMVβGal after 14 days showed similar results (not shown). Magnification × 200. 

Figure 6  .

Figure 6  

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β-Galactosidase activities in infected lamina propria cells (LPMCs). LPMCs from healthy Balb/c mice or mice with trinitrobenzenesulphonic acid (TNBS) induced colitis were isolated. Then 1 × 106 unstimulated or phorbol ester/phytohaemagglutinin (PMA/PHA) stimulated cells were infected with AdCMVβGal at a multiplicity of infection of 1000. After three days the cells were lysed and luminescent β-galactosidase activity was determined. Results are expressed as mean (SD) from three independent experiments (three mice/group) and represent an increase in enzyme activity (relative light units (RLU)) compared with untreated control mice. Asterisks indicate significantly (p<0.05) increased RLU values compared with the control group.

Figure 7  .

Figure 7  

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Time course of reporter gene expression in colitic mice with or without daily treatment with cyclosporin A (CsA). Mice with trinitrobenzenesulphonic acid (TNBS) induced colitis were infected with 1 × 109 pfu AdCMVβGal and treated with CsA as indicated. Mice were killed at the indicated time points, and luminescent β-galactosidase activity in homogenised colonic specimens was determined as specified in Methods. Results represent mean (SD) from three independent experiments (three mice/group) and represent an increase in enzyme activity (relative light units (RLU))/100 mg tissue compared with untreated control mice.

Figure 8  .

Figure 8  

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Adenoviral gene delivery to colonic lamina propria mononuclear cells (LPMCs) and spleen CD4 T cells using viruses with normal or modified fibre structure. β-Galactosidase activities in AdCMVβGal or AdZ.F(pk7) infected spleen CD4 T cells from healthy control mice and LPMCs from mice with trinitrobenzenesulphonic acid (TNBS) induced colitis were measured. A total of 1 × 106 cells were infected with AdCMVβGal at a multiplicity of infection of 1000. After three days the cells were lysed, and luminescent β-galactosidase activity was determined. Results represent mean (SD) from three independent experiments.

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