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The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800mul are significantly the most effective. Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.

International audience; 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. Copyright (C) 2000 Elsevier Science B.V.

Over the past years we have designed and synthesized a new class of synthetic vectors called phosphonolipids and then shown their ability to transfect lungs cells of mice with efficiency. One of them, GLB73, gave high levels of transfection. In the study reported here, we explored the potential of caecum as an alternative site for studying the feasibility of gene transfer in this site. Transfections were performed by using two reporter genes encoding for (igalactosidase and luciferase; transfection activity was assessed using two tests: chemiluminescent and cytofiuorimetric assays. The results obtained showed successful gene transfer into the caecum: up to 27% cells were LacZ+ with a mean of 11%; the maximum of efficiency was also observed 3 days after transfection which then decreased until day 7. Our lipoplex was 8-fold more efficient than the naked DNA (Mann Whitney test; p = 0.03). Moreover, we were able to visually follow the uptake of lipoplexes by enterocytes from 30 mn to 3 days post trans...

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 beta-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.

International audience; 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.

The 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...

Several phosphonates with two fatty chains and different polar heads were synthesized and evaluated for their potential to transfer DNA into epithelial (COS-7) and hematopoietic (K562) cell lines, and compared to commercially available references. In both cases, ammonium-phosphonates were particularly efficient.

International audience; Replacing the ammonium polar head in cationic lipids 1 (A = N) by a phosphonium or an arsonium group (A=P, As) improves their properties as synthetic vectors for DNA transfection. The increased volume of the cationic head is supposed to modify the interactions of the vector with the solvent and DNA.

Several studies have demonstrated that intravenous administration of DNA complexed with cationic lipid vectors induces the production of large quantities of proinflammatory cytokines. In this study we confirm these observations, using cationic lipid DOTAP and cationic phospholipid compounds. Moreover, we demonstrate that although intravenous administration of lipid-DNA complexes does not induce toxic effects in the lung, high transgene expression in lung correlates with histopathological lesions in liver, this fact being documented by high transaminase levels in serum of treated mice. We examine the contribution of various components of the lipoplexes in this observed liver toxicity, as well as in the increasing level of transaminases, and more particularly the role of nonmethylated CpG sequences of plasmid DNA. We show that blood samples from animals treated either with cationic lipid alone, or with cationic lipid complexed with methylated plasmid DNA, contain low levels of transaminases. The significant decrease in transaminase levels after injection of cationic lipid-methylated pDNA complexes leads us to believe that nonmethylated CpG sequences could play a major role in this hepatoxicity. Similar results were observed when using a vector that did not encode a transgene, demonstrating that the expression of luciferase in lung was not responsible for this liver toxicity. All these observations suggest that significant work should be devoted to understand more clearly the mechanism of cationic lipid-DNA complex toxicity, and to overcome the problems subsequent to administration of non-methylated CpG sequences of plasmid DNA.

International audience; Cationic lipids have been shown to be an interesting alternative to viral vector-mediated gene delivery into in vitro and in vivo model applications. Prior studies have demonstrated that even minor structural modifications of the lipid hydrophobic domain or of the lipid polar domain result in significant changes in gene delivery efficiency. Previously, we developed a novel class of cationic lipids called cationic phosphonolipids and described the ability of these vectors to transfer DNA into different cell lines and in vivo. Up until now, in all new cationic lipids, nitrogen atoms have always carried the cationic or polycationic charge. Recently we have developed a new series of cationic phosphonolipids characterized by a cationic charge carried by a phosphorus or arsenic atom. In a second step, we have also examined the effects of the linker length between the cation and the hydrophobic domain as regards transfection activity. Transfection activities of this library of new cationic phosphonolipids were studied in vitro in different cell lines (HeLa, CFT1, K562) and in vivo using a luciferase reporter gene. A luminescent assay was carried out to assess luciferase expression. We demonstrated that cation substitution on the polar domain of cationic phosphonolipids (N → P or As) results in significant increase in transfection activity for both in vitro and in vivo assays and decrease of cellular toxicity.

International audience; Several phosphonates with two fatty chains and different polar heads were synthesized and evaluated for their potential to transfer DNA into epithelial (COS-7) and hematopoietic (K562) cell lines, and compared to commercially available references. In both cases, ammonium-phosphonates were particularly efficient.

ABSTRACT: The ability to transfer genes into a hematopoietic stem cell and to achieve regulation of their expression in lymphoid or myeloid lineages should open many new therapeutic opportunities. Besides gene transfer mediated by virus vectors like retrovirus or adenovirus, non viral systems have the theoretical advantage of being safe and easy to manage. We developed a new family of cationic lipids called phosphonolipids, synthesized 24 new molecules, and then in a first step we tested their potential to transfer genes in human hematopoietic cell lines (K562 and TF1). A LacZ plasmid under the control of a strong viral promoter was used as a reporter gene and a FACS-Gal assay and a quantitative test CPRG assay evaluated the β gal expression. The targeted cells were analyzed 48 hours after transfection. The present work shows that seven novel molecules display a high transfer efficiency. One of them is nine-fold more efficient than the commercially available cationic lipids. The results obtained ex vivo on CD34 cells with the FACS-Gal assay show that at day 10 after transfection, 45 percent of cells are expressing gal.