Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells.
BACKGROUND
Several approaches for gene therapy of cystic fibrosis using viral and non-viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO-K1 cells, since these cells have been extensively used for heterologous CFTR expression.


METHODS
An established in vitro gene transfer system based on antibiotic-mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole-cell patch-clamp recordings.


RESULTS
L. monocytogenes mediated gene transfer to CHO-K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO-K1 cells.


CONCLUSIONS
This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo.
Krusch
Stefan
S
Division of Cell Biology and Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. skr@gbf.de
Domann
Eugen
E
Frings
Monika
M
Zelmer
Andrea
A
Diener
Martin
M
Chakraborty
Trinad
T
Weiss
Siegfried
S
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008089:Listeria monocytogenes / Q000235:genetics*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D010957:Plasmids
D019005:Cystic Fibrosis Transmembrane Conductance Regulator