Optimization of gene transfer into intestinal epithelial cells using a retroviral vector.
Somatic gene therapy has been proposed as a method of treating various metabolic diseases and conditions associated with a deficiency in secretory proteins. The intestine, because of its accessibility, large size, rapid rate of cell turnover, and known location of stem cells, is an attractive site for retroviral gene therapy. Stem cells in the intestine are known to reside in the crypts of Lieberkühn. In this study the IEC-6 cell line, derived from rat intestinal crypt cells, was used as an in vitro model to study the feasibility and dynamics of retroviral gene transfer in intestinal cells. Using a replication-deficient retrovirus, we delivered a reporter gene, bacterial beta-galactosidase (beta-gal) into NIH-3T3 fibroblasts and IEC-6 cells. Successful transduction was measured by X-gal histochemistry. Viral titers on IEC-6 cells were lower than on NIH-3T3 cells but were within the same order of magnitude. Gene transfer increased linearly with retroviral concentration up to a 1:5 dilution of retroviral supernatant. With undiluted viral medium, gene transfer was inhibited and this effect was more pronounced with the IEC-6 cells. The negative effect of the undiluted retroviral supernatant was minimized by decreasing the harvest time from the packaging cell line. An optimal plating density 12 h prior to infection was found to be approximately 10(5) cells/6-cm dish in both cell lines. The infection rate was proportionally enhanced by the use of multiple infections. In conclusion, under ideal conditions, the IEC-6 cells were infected at similar levels of efficacy as NIH-3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
R A
RA
Noel
P
P
Shukla
S J
SJ
Henning
D016475:3T3 Cells; D000818:Animals; D002452:Cell Count; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase