Journal
JOURNAL OF GENE MEDICINE
Volume 4, Issue 4, Pages 415-427Publisher
WILEY
DOI: 10.1002/jgm.279
Keywords
hepatocytes; cationic lipids; gene transfer; gene therapy; ex vivo
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Background The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non-viral gene therapy. Methods A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12-14-16, 22-24-26) varying in their spacer and acyl chains. Results GB12, characterized by short [(CH2)(10)] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6(TM) and Lipofectin(TM). We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12-transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene. Conclusions The non-toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright (C) 2002 John Wiley Sons, Ltd.
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