Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells.

Nathan Gabrielson,Daniel Pack

doi:10.1038/npre.2008.2262.1

Abstract

AbstractMost in vivo gene therapies will require cell-specific targeting. Although vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing. In this study, we examine the impact of targeting two distinct endocytic routes-the caveolar and clathrin pathways-on polyethylenimine-mediated gene delivery in HeLa cells. Targeting complexes to the caveolar pathway with folic acid and the clathrin pathway with transferrin yields enhanced gene delivery relative to unmodified polyethylenimine. Colocalization studies with caveolin-1 and clathrin heavy chain indicate that the ligands successfully deliver their cargo to the intended pathways. However, inhibition of only the caveolar pathway-whether through the use of small molecule drugs or RNA interference-reduces gene delivery efficiency, suggesting that successful polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. Transfections in the presence of chloroquine and pH tracking studies suggest that a contributing factor to the success of the caveolar pathway is avoidance of lysosomes. Collectively, these data demonstrate that uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials.

Highlights

The most effective gene delivery strategy for a given application is often dictated by particular disease characteristics
Vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing
We examine the impact of targeting two distinct endocytic routes—the caveolar and clathrin pathways—on polyethylenimine-mediated gene delivery in HeLa cells

Summary

Introduction

The most effective gene delivery strategy for a given application is often dictated by particular disease characteristics. For systemic diseases like hemophilia, treating any of a number of tissues or cell types may be adequate provided that sufficient levels of the transgene product are expressed and have access to the circulatory system.[1] for localized disorders like cancer or cystic fibrosis, it is often beneficial and sometimes critical to target gene delivery vectors to specific diseased cell types.[2,3] It has been reported that the “receptors” for polycationic gene delivery vectors are clusters of negatively charged proteoglycans on the cell surface These “receptors” do not afford cell specificity. While such targeting methods have met with success in generating particles that are capable of specific cellular interactions, little attention has been given to the possible effects on the intracellular trafficking of targeted gene delivery complexes

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Discussion

Conclusion