Cationic Phosphonolipids as Nonviral Gene Transfer Agents in the Lungs of Mice

Abstract

With the aim of developing new gene transfer tools for treating CF with gene therapy, we have synthesized a novel family of molecules named cationic phosphonolipids. The most efficient among them were selected by in vitro screening to compare their activities in vivo in mouse lungs. We used a reporter gene whose activity was measured cytofluorimetrically (FACS-Gal assay) and by means of a chemiluminescence technique. These tests allowed us to identify the percentage of transfected cells and to quantify total β-galactosidase in the lungs. This enabled us to identify two molecules, significantly efficient in comparison with DNA alone: GLB73 (p = 0.0015) and GLB253 (p = 0.007). Their use resulted in a time lag between transfection and maximum efficiency: maximum efficiency was observed 4 days after transfection with GLB73, whereas it was noticeable only on day 7 with GLB253. Moreover, from toxicity studies carried out in vivo, GLB73 seems to be nontoxic. In vivo results were correlated with in vitro results obtained with CF epithelial cell lines. Consequently, GLB73 is a potential candidate for phase I clinical trials in humans.

Overview summary

Since the cloning of the CF gene in 1989, gene therapy has become a therapeutic option for CF patients. Cationic lipids have been shown to be a possible alternative to viral vector-mediated gene delivery and much work has focused on efforts to increase the level of transfection efficiency. We have developed a novel family of molecules, named cationic phosphonolipids. Among more than 40 molecules synthesized, we have previously selected the most efficient compounds by in vitro studies. In the present study we have tested in vivo five molecules. We have shown, using a reporter gene (β-galactosidase), that two novel molecules, GLB73 and GLB253, are significantly efficient in terms of in vivo gene delivery to mouse airway. The kinetic expression of these two lipids was evaluated and showed that GLB73 displays maximal efficiency day 4 after transfection, whereas GLB253 is maximally efficient on day 7.