In VivoandIn VitroGene Transfer and Expression in Rat Intestinal Epithelial Cells by E1-Deleted Adenoviral Vector

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Abstract
The intestine is proposed to be an attractive target site for somatic gene therapy due to a large mass of proliferating tissue and stem cells in the crypts. Previous studies using a retroviral vector have shown that a reporter gene, bacterial β-galactosidase (β-Gal), can be transferred and expressed in the small intestinal epithelial cell. However, transduction efficiency is relatively low in rat and mice intestines. In the present study, we employed an E1-deleted adenoviral vector (which encodes the β-Gal gene) to investigate the feasibility of gene transfer into rat small intestinal epithelial cell lines and small intestines in male Sprague-Dawley rats. In in vitro studies, expression of AdCMVβgal was quantitatively measured in IEC-6 and IEC-18 cell cultures using X-Gal histochemistry and chemiluminescent reporter gene assays. The results indicate that AdCMVβgal can be efficiently transferred into intestinal epithelial cell lines and transgene expression is virus concentration dependent In in vivo studies, a 5F intestinal feeding tube was used to deliver the vector to the duodenal segment of the rat. Expression of AdCMVβgal was primarily localized to the epithelium of the intestinal tract. Transduction efficiency of the transgene was seen in the duodenum, jejunum, ileum, and, to a lesser extent, the colon. Moreover, following a single or secondary administration of recombinant adenovirus, efficient expression of AdCMVβgal in the intestinal tract peaked at 3 days and decreased by 7 and 14 days. No antiadenoviral antibody response was detected in the serum after a single or secondary challenge with this virus. These findings demonstrate that an E1-deleted adenoviral vector, when administered through an oral-duodenal tube, transfers genetic material more successfully in the intestinal epithelium in the small intestine when compared to the large intestine. A single or secondary challenge with adenoviral vector does not cause enhanced host immune responses to this virus. It suggests that successful gene transduction by the repeat administration of the adenoviral vector makes it an alternative candidate for gene therapy applications in intestinal diseases and metabolic deficiencies. Previous studies have shown that the efficiency of gene transfer by recombinant retrovirus is relatively low in animal models and could not satisfy most gene therapy strategies in humans. In the present study, we found that using an oral-duodenal tube, an E1-deleted adenoviral vector (which encodes β-galactosidase gene) can be efficiently transferred into the small intestinal tract and expression of the transgene is primarily localized to the intestinal epithelium of individual segments of the rat. Moreover, following a single or secondary challenge with recombinant adenovirus, expression of AdCMVβgal was more efficient in the epithelium of the small intestine than the large intestine. Expression of AdCMVβgal in the intestinal tract peaked at 3 days and decreased by 7 and 14 days. Moreover, a single or secondary challenge with AdCMVβgal did not result in a detectable antiadenoviral antibody response to the virus. Thus, utilizing an oral-duodenal tube delivery system, repeat administration with an E1-deleted adenoviral vector may be useful for gene therapy applications in variety of intestinal and metabolic diseases.