Aurore Fraix

Highly efficient gene transfer into hepatocyte-like HepaRG cells: new means for drug metabolism and toxicity studies

HepaRG progenitor cells are capable of differentiating into hepatocyte‐like cells that express a large set of liver‐specific functions. These cells, however, only express small amounts of an important cytochrome P450, the CYP2E1, which limits their use for toxicological studies of drugs metabolized by this pathway. Our aim was to establish an efficient transfection protocol to increase CYP2E1 expression in HepaRG cells. Transfection protocols of the green fluorescent protein (GFP) reporter gene were evaluated using electroporation and cationic lipids belonging to the lipophosphonates, lipophosphoramidates and lipids derived from glycine betaine. Following optimization of the charge ratios, plasmid DNA and formulations with neutral co‐lipids, the lipophosphoramidate compounds KLN47 and BSV10, allowed expression of the GFP in ∼50% of adherent progenitor HepaRG cells, while electroporation targeted GFP expression in ∼85% of both progenitor and differentiated cells in suspension. Transient enforced expression of active CYP2E1 was also achieved in progenitors and/or differentiated HepaRG cells using the electroporation and the lipophosphoramidate compound BSV10. Importantly, in electroporated cells, CYP2E1 expression level was correlated with a significant increase in CYP2E1‐specific enzymatic activity, which opens new perspectives for this CYP‐dependent drug metabolism and toxicity studies using HepaRG cells.

The gene transfection properties of a lipophosphoramidate derivative with two phytanyl chains

Development of efficient and non-toxic gene delivery systems is among the most challenging requirements for successful gene therapy. Cationic lipophosphoramidates constitute a class of cationic lipids we have already shown to be efficient for in vivo gene transfer. Herein, we report the synthesis of a cationic lipophosphoramidate bearing two phytanyl chains (BSV18) as hydrophobic domain, and studied its gene transfection properties. In vitro, BSV18 exhibited a high transfection efficacy associated with a low cytotoxicity. (31)P NMR studies of various cationic lipophosphoramidates in water solution suggested that the phytanyl chains may favor the formation of an inverted hexagonal phase, a supramolecular arrangement which is presumed to enhance the endosomal escape and consequently increase the transfection efficiency. In vivo, systemic delivery of BSV18-based lipoplexes allowed a high efficiency of gene transfection into the mouse lung. With a view to clinical application, we evaluated not only the efficiency of lung transfection but also the eventual in vivo side-effects. Thus, in addition to monitoring the in vivo transfection efficiency by bioluminescent imaging and identifying by immunohistochemistry the cell types transfected, we also assessed in living animals the potential liver reaction as well as the inflammatory and immune responses induced by BSV18-mediated transfection. All those adverse effects were actually highly transient. Thus, taken together, these results indicate that lipophosphoramidates equipped with two phytanyl chains may have great potential for lung gene therapy, in particular for Cystic Fibrosis.

Cationic lipophosphoramidates with two disulfide motifs: synthesis, behaviour in reductive media and gene transfection activity

Lipophosphoramidates have previously been identified as efficient vectors for gene delivery. The incorporation of functional groups that respond to a physiological stimulus is hypothesised to further improve the efficacy of this type of vector and eventually reduce its cytotoxicity. In the present work, we report the effects of the incorporation of two disulfide motifs into the hydrophobic domain, close to the phosphoramidate group. Three cationic vectors possessing such a red/ox sensitive function were synthesised. The capability of one of them (5b) to compact DNA is reported jointly with its ability to release that DNA in the presence of a reducing agent. Finally, compound 5b was tested as a vector for gene delivery into human cells in vitro and its cytotoxicity was also evaluated.

Arsonium‐Containing Lipophosphoramides, Poly‐Functional Nano‐Carriers for Simultaneous Antibacterial Action and Eukaryotic Cell Transfection

Gene therapy of diseases like cystic fibrosis (CF) would consist of delivering a gene medicine towards the lungs via the respiratory tract into the target epithelial cells. Accordingly, poly-functional nano-carriers are required in order to overcome the various successive barriers of such a complex environment, such as airway colonization with bacterial strains. In this work, the antibacterial effectiveness of a series of cationic lipids is investigated before evaluating its compatibility with gene transfer into human bronchial epithelial cells. Among the various compounds considered, some bearing a trimethyl-arsonium headgroup demonstrate very potent biocide effects towards clinically relevant bacterial strains. In contrast to cationic lipids exhibiting no or insufficient antibacterial potency, arsonium-containing lipophosphoramides can simultaneously inhibit bacteria while delivering DNA into eukaryotic cells, as efficiently and safely as in absence of bacteria. Moreover, such vectors can demonstrate antibacterial activity in vitro while retaining high gene transfection efficiency to the nasal epithelium as well as to the lungs in mice in vivo. Arsonium-containing amphiphiles are the first synthetic compounds shown to achieve efficient gene delivery in the presence of bacteria, a property particularly suitable for gene therapy strategies under infected conditions such as within the airways of CF patients.

Synthesis of Aryl-Thiophosphonates via A [1,2] or [1,3] Phospho-Fries Rearrangement

Abstract The synthesis of O,O-diethyl arylthiophosphonates is achieved following a base-induced [1,2] or [1,3] phospho-Fries rearrangement that makes use of O,O-diethyl O-aryl thiophosphates as substrates. The coordination properties of O,O-diethyl arylthiophosphonates are also discussed.

New Lipo-Phosphoramidates for Gene Delivery

Abstract Lipo-phosphoramidates constitute an interesting class of synthetic vectors for gene delivery. A structure–activity relationship study points out that the incorporation of the linoleyl polyunsaturated lipid chains produces efficient vectors for gene delivery in the lung. In addition, neutral lipo-phosphoramidates possessing an imidazole motif in the polar head region have been successfully employed in association with a cationic lipophosphoramide to improve in vitro transfection efficiencies. Our recent achievements on this topic are briefly summarized here.

Study of the Stability of O,O-Dialkyl-O-Arylthiophosphate: Evidence of the Formation of O,S-Dialkyl-O-Arylphosphate

GRAPHICAL ABSTRACT Abstract During the synthesis of cationic lipids that included thiophosphoramidate moiety or pincer ligands based on O,O-diethyl-thiophosphonate as coordination group, the rearrangement of O,O-diethyl-thiophosphoryl in O,S-diethylthiophosphoryl group was observed. This rearrangement, which does not occur by a simple heating at 130°C, is catalyzed by nucleophilic amine like N,N-dimethylaminopyridine. However, at some occasion, dealkylation of the O,S-diethylthiophosphoryl compound is observed.