Loss of Binding and Entry of Liposome-DNA Complexes Decreases Transfection Efficiency in Differentiated Airway Epithelial Cells

Hirotoshi Matsui,Larry G Johnson,Scott H Randell,Richard C Boucher

doi:10.1074/jbc.272.2.1117

Hirotoshi Matsui, Larry G Johnson + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.2.1117

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Abstract

The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like "islands" and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVbeta:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-microm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.

Highlights

Inefficiency of gene transfer is one of the major problems confronting cationic liposomes as vectors for cystic fibrosis (CF)1 gene therapy
Day 5 human bronchial epithelial cells and Rat Tracheal Epithelial (RTE) cells were transfected with pCMV␤-LipofectACE® (1:5, w/w) complexes, and 48 h later they were stained with X-gal
Transfection efficiency must be increased before cationic liposomes become practical vectors for in vivo gene therapy of CF lung disease

Summary

Introduction

Inefficiency of gene transfer is one of the major problems confronting cationic liposomes as vectors for cystic fibrosis (CF) gene therapy. Cationic liposomes deliver genes efficiently into some cell lines and primary culture cells of certain tissues [1,2,3,4,5,6], the transfection efficiency in well differentiated airway cells in vivo is limited [7], paralleling observations with adenoviral vectors (8 –10). To identify the mechanisms that may account for differentiation-dependent differences in the efficiency of lipid-mediated gene transfer in airway epithelial cells, we used in vitro cell culture systems that simultaneously contain populations of both differentiated and dedifferentiated airway cells. We subsequently focused on the rat system to identify cellular factors that may regulate the relatively efficient lipid-mediated gene delivery to poorly differentiated (edge) airway epithelial cells as compared with redifferentiated (central) airway epithelial cells

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