Optimization of cationic lipid-mediated gene transfer to airway
epithelia.
Fasbender, A. J., J. Zabner, M. J. Welsh.
Howard Hughes Medical Institute, Departments of Internal Medicine and
Physiology and Biophysics, University of Iowa College of Medicine, Iowa City,
Iowa 52242
APStracts 2:0007L, 1995.
The use of cationic lipids for gene transfer to airway epithelia has shown
promise in in vitro and in vivo studies. However, previous studies have used a
wide variety of different lipid preparations and different formulations.
There have been few reports of studies designed to optimize the variables
involved in transfection and there have been no such studies focused on
airway epithelia. Therefore, we examine variables that affect cationic lipid
-mediated transfection of HeLa cells and of airway epithelial cells grown on
permeable filter supports at the air-liquid interface. To quantitate
expression of cDNA, we assayed expression of luciferase. We found that the
ratio of DNA to lipid was an important variable that determined transfection
efficiency. In both HeLa and airway epithelial cells the optimum charge ratio
of cationic lipid to anionic DNA was approximately 1.25, consistent with the
notion that a positively charged complex facilitates interaction with the
negatively charged cell membrane. After testing a series of readily available
cationic lipids, DMRIE/DOPE appeared to show good efficacy. The concentration
of DNA and cationic lipid also played an important role: in HeLa cells the
optimum concentration of cationic lipid was approximately 5 [mu]M and in
airway epithelial cells was approximately 15 [mu]M. Higher concentrations
appeared to increase toxicity in HeLa cells, but in confluent airway epithelia
the DNA:lipid complex had minimal effects on total cellular protein or on
transepithelial resistance. Expression increased as the duration of exposure
to the lipid:DNA complex increased, with prolonged times (24 hr) providing
greater expression than 4-6 hr. These studies using an in vitro model of
airway epithelium may help guide attempts to develop in vivo gene transfer for
airway disease associated with a number of diseases including cystic
fibrosis, surfactant protein B deficiency, and [alpha]-1 antitrypsin
deficiency.
Received 11 August 1994; accepted in final form 20 January 1995.
APS Manuscript Number L0230-4.
Article publication pending Am. J. Physiol. (Lung Cell. Mol. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 24 February 1995.