Improved Cationic Lipid Formulations for In Vivo Gene Therapy

Abstract

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

β-Galactosidase staining following intracoronary infusion of cationic liposomes in the in vivo rabbit heart is produced by microinfarction rather than effective gene transfer: a cautionary tale

“…33 Several factors favour macro-aggregate formation such as the impurity of the DNA, the composition of the suspending vehicle and the ratio of cationic liposome to DNA. 36 We hypothesise that it was these macro-aggregates that were retarding microcirculatory flow and causing infarction. In previous experiments there has been a more prolonged time period for uptake of the liposome-DNA complexes, possibly decreasing the effect of macro-aggregate formation.…”

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

“…[1][2][3][4][5][6] Despite these clear advantages, nonviral transfection systems result in substantially less gene expression than that observed with viral systems. Most cationic lipid/DNA-based systems are taken up efficiently though an endocytosis pathway, [7][8][9][10] but the exact mechanisms of DNA release from the endosomes and the subsequent translocation to the nucleus are not clear.…”

IGF-I gene transfer in thermally injured rats

“…2 The most common viruses used for transfection have been the retroviruses, adenoviruses and adeno-associated viruses. [1][2][3] Viral infection-associated toxicity, immunologic compromise, and possible mutagenic or carcinogenic effects, however, make this approach potentially dangerous. 1 The use of naked DNA not encapsulated or plasmid DNA constructs alone, without viral genes present, have been used topically or delivered with a pneumatic 'gene gun'.…”