Use of Amphotropic Retroviral Vectors for Gene Transfer in Human Colon Carcinoma Cells

Abstract

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37°C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10× regular stock titers but was inefficient at 100×. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.

Overview summary

A variety of congenital and acquired diseases potentially could be treated by gene delivery to the epithelium of the small or large intestine. Because these epithelia are proliferating, persistence of the therapeutic gene requires integration into DNA of the respective stem cells. At present, retroviral vectors are the best choice for this purpose. For human use, amphotropic retroviruses would be appropriate, and the least invasive route of delivery would be via the intestinal lumen. The feasibility of such an approach depends on: (i) the ability of the vector to survive in the luminal environment and in the presence of preparatory agents; and (ii) the presence of the amphotropic receptor on human intestinal epithelial cells. The present report provides affirmative evidence for both of these and thus supports the feasibility of intestinal gene therapy with such vectors.