Jean-Claude Clement

Synthesis and Transfection Activity of New Cationic Phosphoramidate Lipids: High Efficiency of an Imidazolium Derivative

In an effort to enhance the gene‐transfer efficiencies of cationic lipids and to decrease their toxicities, a series of new phosphoramidate lipids with chemical similarity to cell membrane phospholipids was synthesised. These lipids contained various cationic headgroups, such as arginine methyl ester, lysine methyl ester, homoarginine methyl ester, ethylenediamine, diaminopropane, guanidinium and imidazolium. Their transfection abilities, either alone or with the co‐lipid DOPE, were evaluated in HEK293–T7 cells. We found that imidazolium lipophosphoramidate 7 a/DOPE lipoplexes gave the most efficient transfection with low toxicity (15 %). The luciferase activity was 100 times higher than that obtained with DOTAP/DOPE lipoplexes. The size, ζ potential, pDNA–liposome interactions and cellular uptakes of the lipoplexes were determined. No definitive correlation between the ζ potential values and the transfection efficiencies could be established, but the uptake of lipoplexes by the cells was correlated with their final transfection efficiencies. Our results show that imidazolium phosphoramidate lipids constitute a potential new class of cationic lipids for gene transfer.

Cationic Phosphonolipids Containing Quaternary Phosphonium and Arsonium Groups for DNA Transfection with Good Efficiency and Low Cellular Toxicity

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.

GENERAL ROUTE FOR THE SYNTHESIS OF MONO N-ALKYLATED DERIVATIVES OF TETRAAZAMACROCYCLES

Abstract The selective synthesis of mono N-substituted derivatives of tetraazamacrocycles can be achieved using a new boron triprotection easily removed after alkylation.

Mono N-alkylation and N-acylation of cyclen and cyclam via their metaltricarbonyl complexes (M = Cr, Mo)

Abstract Reaction of metaltricarbonyl complexes of cyclen and cyclam with enolisable aldehydes or acid chlorides yields, after removal of the protecting M(CO) 3 moiety, selectively mono N-functionalized derivatives.

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.

Phosphonolipids as non-viral vectors for gene therapy

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.

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.

Mono N-functionalization of cyclic and linear tetraamines via their tridentate tricarbonylchromium complexes

The fac-LCr(CO)3 tridentate complexes of 1,4,7,10-tetraazacyclododecane 1, 1,4,8,11-tetraazacyclotetradecane 2, 1,4,7,10-tetraazadecane 3 and 1,5,8,12-tetraazadodecane 4 have been selectively alkylated in high yield at the uncomplexed nitrogen atom, giving rise to mono N-functionalized tetraamines and bis-macrocyclic compounds.

CFTR transgene expression in primary ΔF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids

Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population. The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet.In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents. Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects. Consequently, all of these drawbacks brought forth the development of nonviral transfection systems. Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome. Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo.In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a ΔF508 homozygous patient. The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex. A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection.