Claude Ferec

Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency.

Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.

Cationic Phosphonolipids as Nonviral Vectors: In Vitro and in Vivo Applications

Since the development of the concept of gene therapy using cationic lipids as nonviral vectors by Felgners group in 1987, numerous molecules have been synthesized. Such vectors were first proposed to avoid viral vector-induced drawbacks. But, it quickly became clear that a thorough knowledge of their physical and chemical characteristics was fundamental to use them under optima conditions. Over the last years our laboratory has developed a family of cationic lipids called phosphonolipids whose structure is based on that of natural phosphonolipids; compared with other vectors, these compounds had to be well-tolerated by biologic membranes. Some of our synthesized molecules exhibited an interesting potential for gene transfer, both in vitro and in vivo. Structural changes in the different parts (hydrophobic, hydrophilic, and intermediary domains) of these vectors were evaluated in vitro on different cell-lines; these studies led us to select some of these molecules to carry out in vivo tests. So, the plasmid/phosphonolipid complexes were first administered to mice by intratracheal and aerosol routes with a beta-galactosidase plasmid as reporter gene. In a second set of experiments, we explored the possibilities offered by intravenous injection; in these studies, we used a luciferase plasmid as reporter gene because of its high sensibility. These experiments revealed a transgene expression essentially localized in the lungs. In a further study, we compared systemic administration with local ones; we, then, observed that the optimum formulation of a given molecule depended on its route of administration.

TRANSGENE EXPRESSION KINETICS AFTER TRANSFECTION WITH CATIONIC PHOSPHONOLIPIDS IN HEMATOPOIETIC NON ADHERENT CELLS

Cationic lipids are considered to be capable of efficiently and safely mediating DNA transfer into cells, although expression is transient. A new family of cationic lipids, called phosphonolipids, has been developed, with the relationship between the hydrophobic domain of the lipid molecules and the significant enhancement of transduction efficiency in a non-adherent cell line characterised in the present study. The kinetics of transfection efficiency were also investigated. Our results demonstrate that the peak of the transient expression of these reporter genes mediated by cationic lipids occurred within 3 to 14 days, depending on the aliphatic chain length of the complex used and on its formulation in the presence or absence of DOPE. Furthermore, the kinetics of transgene expression were found to differ in adherent and non-adherent cells. These results were obtained using three different techniques: CPRG, luminescence, and FACS-gal, and were in agreement with electron microscopy studies. We thus hypothesized that the plasma membrane composition of cells could affect the efficiency of transfection with cationic lipids. Our results suggest that phosphonolipids constitute a promising class of compounds for gene transfer protocols, and that galenic optimization should improve and modify the transfection efficiency of these DNA-lipid complexes.

Biodistribution study of phosphonolipids: a class of non-viral vectors efficient in mice lung-directed gene transfer.

A multitude of cationic lipids have been synthesized since they were first proposed for use in gene therapy. Cationic lipids are able to efficiently transfect cells both in vitro and in vivo. Whereas most research groups have focused their investigations on the toxicity of these molecules, and on the location of expression of the DNA transferred by these vectors, little has been done to determine their biodistribution and elimination pathways. Our group has developed a family of cationic lipids termed phosphonolipids. Following a large in vitro screening experiment, we have selected several molecules for in vivo testing, with some of these phosphonolipids forming lipoplexes efficient in transfecting mouse lungs. It was thus of interest to study their fate after intravenous injection.

Visualization of the transgene distribution according to the administration route allows prediction of the transfection efficacy and validation of the results obtained.

Gene transfer to the lung can be achieved via a systemic, that targets the endothelium, or local, that targets the epithelium, delivery route. In the present study, we followed the distribution of a plasmid after transfection using some of our phosphonolipids, which have previously shown their efficiency in transfecting mouse lungs. The plasmid was radiolabeled and varying combinations of plasmid/phosphonolipid were administered by intravenous injection, or by endotracheal spray. The distribution of radioactive labeling was observed over a time course using a γ-camera. These images were then correlated with the results for luciferase expression levels in the lungs. In each case, lungs were well targeted. However, whereas an intravenous injection reaches all of the lung immediately, progressive diffusion occurs when the plasmid/phosphonolipid is administered via an aerosol. Elimination of the radioactivity associated with plasmid occurs via the urinary tract after intravenous injections, and via the feces using the aerosol delivery approach. The radioactivity detected in the lungs correlated strongly with transgene expression. Thus, such an imaging technique is a powerful strategy to predict the formulation that will generate the best transfection efficiency. This study reveals that scintigraphic imaging permits both validation of the administration method and the results obtained for each animal, thereby reducing the statistical variability of in vivo experiments.

Folate-equipped nanolipoplexes mediated efficient gene transfer into human epithelial cells.

Since recombinant viral vectors have been associated with serious side effects, such as immunogenicity and oncogenicity, synthetic delivery systems represent a realistic alternative for achieving efficacy in gene therapy. A major challenge for non-viral nanocarriers is the optimization of transgene expression in the targeted cells. This goal can be achieved by fine-tuning the chemical carriers and the adding specific motifs to promote cellular penetration. Our study focuses on the development of novel folate-based complexes that contain varying quantities of folate motifs. After controlling for their physical properties, neutral folate-modified lipid formulations were compared in vitro to lipoplexes leading to comparable expression levels. In addition, no cytotoxicity was detected, unlike what was observed in the cationic controls. Mechanistically, the delivery of the transgene appeared to be, in part, due to endocytosis mediated by folate receptor targeting. This mechanism was further validated by the observation that adding free folate into the medium decreased luciferase expression by 50%. In vivo transfection with the folate-modified MM18 lipid, containing the highest amount of FA-PEG570-diether co-lipid (w:w; 90:10), at a neutral charge ratio, gave luciferase transgene expression. These studies indicate that modification of lipids with folate residues could enhance non-toxic, cell-specific gene delivery.

Novel Cationic Phosphonolipids Agents for in Vitro Gene Transfer to Epithelial Cell Lines

AbstractThe use of cationic liposomes is one of the main approaches currently investigated to introduce into a cell a gene with therapeutic properties. This study presents in vitro results obtained with a new family of gene transfer agents, the phosphonolipids. We have synthesized 37 members of this family and optimized the conditions of in vitro gene transfer targeted at a lung epithelial cell line (CFT1 cells) by using a reporter gene (β-galactosidase). Two quantitative tests, a CPRG1 (Chlorophenol red galactopyranoside) test and a Flow cytometric assay (FACS-Gal1 assay), have been used to determine the percentage of transfected cells. The cationic phosphonolipids were tested alone or formulated with 50% DOPE1 (Dioleoylphosphatidylethanolamine) (w:w). The results obtained with the CPRG test led us to select 7 compounds that were more efficient than the commercialized lipids Lipofectin, Lipofectamine and Transfectam. The transfer kinetics of the transgene were studied and showed that more than 20 % of ce...

New Phosphoramidate Dicationic Vectors for Gene Therapy

Mathieu Mével,1 François Lamarche,1 Jean-claude Clément,1 Jean-jacques Yaouanc,1 Pascale Laurent,1 Laure Burel,1 Philippe Giamarchi,1 Tristan Montier,2 Pascal Delépine,2 Pierre Lehn,2 Paul-Alain Jaffrès,1 and Claude Férec2 1CEMCA, UMR CNRS 6521, Faculté des Sciences et Techniques, Université de Bretagne Occidentale, France 2Unité INSERM 613, Institut de Synergie des Sciences et de la Santé, Université de Bretagne Occidentale, France