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 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 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-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 10x regular stock titers but was inefficient at 100x. 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.
Jacomino
M
M
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
Shukla
P
P
Henning
S J
SJ
D016475:3T3 Cells; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D018938:Caco-2 Cells; D003110:Colonic Neoplasms; D003470:Culture Media; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured
D003470:Culture Media; D000111:Acetylcysteine; D010634:Phenobarbital