USE OF AMPHOTROPIC RETROVIRAL VECTORS FOR GENE-TRANSFER IN HUMAN COLON-CARCINOMA CELLS

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ec otropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somati c gene therapy. The first experiment assessed the viability of amphotr opic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees 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-acetylcystei ne, which is likely to be used as a preparatory agent for mucus remova l. To determine whether human intestinal cells are transducible by the se vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the repor ter gene was highest in the HT-29 cells. Subsequent studies using thes e cells showed that with regular stocks of vector, gene transfer peake d at a stock dilution of 1/10 and declined at full strength. This prob lem could be partially overcome by centrifugal concentration of the re troviral stocks. With this approach, gene transfer increased with incr easing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that ampho tropic retroviral vectors may eventually be used for in vivo gene tran sfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.