Development and Characterization of Cationic Liposomes Conjugated with HVJ (Sendai Virus): Reciprocal Effect of Cationic Lipid for In Vitro and In Vivo Gene Transfer

Abstract

Today, nonviral gene transfer vectors attract more attention as a therapeutic strategy for human diseases, because viral vectors such as adenoviral and herpes viral vectors have been proven to have problems, especially in immunogenicity and cytotoxicity. However, the main limitation of nonviral vectors has been low efficiency of gene expression. To overcome this defect, we have developed a new class of transfection vehicles, HVJ–cationic liposomes. The use of the cationic lipid DC-cholesterol facilitates efficient entrapment of negatively charged macromolecules (plasmid DNA, oligodeoxynucleotides, and proteins) and efficient interaction with negatively charged plasma membranes of cultured cells in vitro. Moreover, the fusogenic envelope proteins of hemagglutinating virus of Japan (HVJ) enhance delivery of the enclosed materials into cells. Using firefly luciferase as a marker, we optimized the liposome formula. As a result, we have succeeded in obtaining 100–800 times higher gene expression in vitro than with the conventional HVJ–anionic liposomes. However, in vivo gene transfer experiments have revealed that the use of cationic lipid instead of anionic lipid reduced the transgene expression dramatically in organs such as muscle and liver. We further discovered that the use of anionic liposomes with a viral-mimic king lipid composition increased transfection efficiency by several times in vivo. We conclude that the alternative usage of transfer vectors, for example, HVJ–anionic liposomes for in vivo delivery to extended areas of organs and HVJ–cationic liposomes for in vitro delivery (and possibly for in vivo delivery to a restricted area of organs), is of significance.

Overview summary

The hemagglutinating virus of Japan (HVJ)–liposomes method is a liposome-based gene transfer method that enables us to deliver the contents of liposomes directly into the living cells by means of the virus–cell fusion machinery. In this study, we examined both the in vitro and in vivo gene transfer efficiency of this method using variety of liposomes with different lipid compositions. As a result, we found that the liposomes with a similar lipid composition to those of viral envelopes showed optimal gene expression both in in vitro and in vivo experiments. However, adding cationic lipid to liposomes showed a reciprocal effect in these experiments. Use of positively charged liposomes enhanced gene expression dramatically in vitro, while it reduced gene expression to great extent in vivo.