Yadi Tan

The Inhibitory Role of CpG Immunostimulatory Motifs in Cationic Lipid Vector-Mediated Transgene Expression in Vivo

We have previously reported that intravenous administration of cationic lipid-protamine-DNA complexes (LPD) induces production of large quantities of proinflammatory cytokines that are toxic and cause inhibition of transgene expression. Cytokine induction appears to be mediated by the unmethylated CpG sequences since methylation of plasmid DNA significantly decreases the cytokine levels. In this study, the inhibitory role of CpG in lipid-mediated gene transfer was further investigated using chemically well-defined, CpG-containing oligodeoxynucleotides (ODNs). Injection (intravenous) of ODNs formulated in LPD into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-alpha. The potency of CpG-containing ODNs in cytokine induction was affected by its flanking sequences and was significantly reduced when CpG was methylated. Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent injection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytokines. Finally, intraperitoneal injection of dexamethasone suppressed LPD-induced cytokine production, and led to significantly higher levels of transgene expression on both first and second injection. These studies suggest that mutation of potent CpG motifs in plasmid DNA together with the use of immune suppression agent may represent an effective approach to improve cationic lipid-mediated gene transfer to the lung.

Lyophilization of Cationic Lipid–protamine–DNA (LPD) Complexes

Cationic lipid-based gene delivery systems have shown promise in transfecting cells both in vitro and in vivo. However, these systems tend to form aggregates in liquid formulation during storage, which has limited their clinical applications. As a result, lyophilization of these systems has recently become a subject of increasing interest. In this paper, lyophilization of LPD, a novel cationic lipid-based gene delivery system, was studied. Both particle size and transfection efficiency could be preserved in the presence of sufficient amount of appropriate lyoprotectant. A series of monosaccharides and disaccharides, including dextrose, galactose, mannose, lactose, maltose, sucrose and trehalose, were evaluated for their lyoprotective effect and disaccharides showed more superior protection to monosaccharides. The effect of different freezing protocols for lyophilization was also evaluated and no significant difference was found. However, for freeze-thawing, fast freezing caused less aggregation. Finally, nonlyophilized LPD and LPD lyophilized with 10% sucrose were stored at different temperatures and their stability was followed for eight weeks. Lyophilized LPD could be stored at room temperature without significant change in particle size or loss of transfection efficiency.

Overcoming the inflammatory toxicity of cationic gene vectors

Cationic lipid- and polymer-based vectors are the most extensively studied synthetic gene vectors. These vectors have been widely used in animal models and some have been tested in clinical trials. The clinical progress of these vectors has been slowed by their inflammatory toxicity. This review summarizes the observations, the mechanisms, and various strategies employed to overcome the inflammatory toxicity of cationic gene vectors.