ABSTRACT
BACKGROUND: 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. METHODS: The respective biodistributions of both the GLB43 phosphonolipid and plasmid DNA were investigated and compared with DNA expression sites. Using the optimal conditions determined for phosphonolipids, we followed the gene transfer agent and plasmid DNA distributions versus time by radiolabeling them with (14)C and (32)P, respectively. Otherwise, we performed imaging by radiolabeling plasmid DNA with (99m)Tc. RESULTS: The lipoplexes appear to be directly located in the lung after administration. Secondly, the plasmid is released mainly into the lungs and the phosphonolipid vector is rapidly degraded. The hydrophilic moiety of the phosphonolipid is eliminated in the urine, as is the free plasmid. CONCLUSIONS: This study reveals that there are slight differences in the observed results depending on the technique used to label the DNA; secondly, results show that the residence time of phosphonolipids in the mouse body is related to the DNA binding time.
Subject(s)
Gene Transfer Techniques , Genetic Vectors , Lung/metabolism , Phospholipids , Animals , Biological Availability , Female , Gene Expression , Gene Targeting , Genetic Therapy , Injections, Intravenous , Liposomes , Luciferases/metabolism , Mice , PlasmidsABSTRACT
Since the development of the concept of gene therapy using cationic lipids as nonviral vectors by Felgner's 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.
Subject(s)
Genetic Vectors , Phospholipids , Aerosols , Animals , Cations , Cell Line , Cells, Cultured , Drug Administration Routes , Genes, Reporter , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/chemistry , Humans , Injections, Intravenous , K562 Cells , Mice , Phospholipids/administration & dosage , Phospholipids/chemistryABSTRACT
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.
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 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.
Subject(s)
Drug Carriers , Gene Transfer Techniques , Lung/physiology , Phospholipids/chemistry , Animals , Flow Cytometry/methods , Luminescent Measurements , Lung/pathology , Mice , Phospholipids/toxicity , Structure-Activity Relationship , beta-Galactosidase/analysis , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
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.
Subject(s)
Hematopoietic System/cytology , Phospholipids , Transfection/methods , Transgenes/genetics , Cations , Cell Adhesion , Cell Separation , Chlorophenols , Flow Cytometry , Galactosides , Gene Expression , Humans , Kinetics , Leukemia, Erythroblastic, Acute , Luminescent Measurements , Microscopy, Electron , Phospholipids/chemical synthesis , Plasmids/ultrastructure , Recombinant Fusion Proteins , Tumor Cells, Cultured , beta-Galactosidase/analysis , beta-Galactosidase/geneticsABSTRACT
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 beta 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.