Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, s... more Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.
Drug targeting: Anti-HSV-1 activity of mannosylated polymer-bound 9-(2-phosphonylmethoxyethyl)adenine
Biochemical and Biophysical Research Communications, 1990
The antiviral drug, 9-(2-phosphonylmethoxyethyl) adenine (PMEA) was linked to a synthetic and neu... more The antiviral drug, 9-(2-phosphonylmethoxyethyl) adenine (PMEA) was linked to a synthetic and neutral polymer bearing mannosyl residues to allow its internalization by macrophages via membrane lectins. PMEA bound to the mannosylated polymer was more efficient in vitro than free PMEA in preventing lysis of human macrophages by herpes virus.
DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/catio... more DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3-4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery.
Positively charged microbubbles to target nucleic acid delivery with ultrasound
NOVEL NUCLEIC ACID/POLYMER COMPLEXES, METHOD FOR PREPARING SAME AND USE THEREOF FOR CELL TRANSFECTION
Polymeric complexes for the transfection of nucleic acids, with residues causing the destabilisation of cell membranes
Chemical vectors for gene delivery: uptake and intracellular trafficking
Current Opinion in Biotechnology, Oct 1, 2010
Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced w... more Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced with various range of macromolecules suitable to mimic some viral functions required for gene transfer. Many efforts have been undertaken these past years to identify cellular barriers that have to be passed for this issue. Here, we summarize the current status of knowledge on the uptake mechanism of DNA nanoparticles made with polymers and liposomes, their endosomal escape, cytosolic diffusion, and nuclear import of pDNA. Studies reported these past years regarding pDNA nanoparticles endocytosis indicated that there is no clear evident relationship between the ways of entry and the transfection efficiency. By contrast, the sequestration of pDNA in intracellular vesicles and the low number of pDNA close to the nuclear envelop are identified as the major intracellular barriers. So, intensive investigations to increase the cytosolic delivery of pDNA and its migration toward nuclear pores make sense to bring the transfection efficiency closer to that of viruses.
Membrane lectins on human monocytesMaturation-dependent modulation of 6-phosphomannose and mannose receptors
Febs Lett, 1985
Freshly isolated human monocytes, which do not contain cell-surface mannose-specific receptors, b... more Freshly isolated human monocytes, which do not contain cell-surface mannose-specific receptors, bind mannose 6-phosphate and actively endocytose mannose 6-phosphate-bearing neoglycoproteins (6-P-Man-F-BSA). Three days after isolation, human monocytes endocytose very actively 6-P-Man-F-BSA as well as Man-F-BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar-substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6-phosphate receptors are already present on freshly isolated human monocytes.
Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the n... more Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized iB motif to favour NFiB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Fö rster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was $1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFiB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFiB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the geneexpression efficiency.
MEMBRANE PERMEABILIZATION BY ALPHA -HELICAL PEPTIDES : A FLOW CYTOMETRY STUDY
Biochimica Et Biophysica Acta Biomembranes, Nov 1, 1995
The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by f... more The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED50 = 0.5 microM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED50 = 18 microM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED50 = 11 microM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that alpha-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.
Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polym... more Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly[Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio)propionyl residues in order to have each amino group of poly[Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond. As evidenced by agarose gel electrophoresis and ethidium bromide/pDNA fluorescence restoration, poly[Lys-(AEDTP)] polyplexes were decondensed and the plasmid released upon treatment with either dithiothreitol, glutathione in the presence of glutathione reductase, or the thioredoxin reductase. Electron microscopy showed that polyplexes exhibiting spherical particles of a mean size at about 100 nm were decondensed in the presence of glutathione and exhibited filamentous aggregates. Finally, we found that the transfection of 293T7 and HepG2 cells was 10-and 50-fold more efficient with poly[Lys-(AEDTP)] polyplexes, respectively, than with poly[Lys] polyplexes. These results indicate that disulfide-containing cationic polymers must be borne in mind for developping polymer-base gene delivery systems. * Corresponding author. Phone: +33(0)2 38 25 55 95. Fax: +33 (0)2 38 25 78 07.
Transfert d'acides nucléiques par des systèmes non viraux. Molecular strategy Functionnal stu... more Transfert d'acides nucléiques par des systèmes non viraux. Molecular strategy Functionnal studies: validation of molecular tools (HEK293 cells) Figure 1: Schematic representation of the microRNA detection strategy A) in the presence of the microRNA of interest, the system is switched to mode « on » generating a positive bioluminescence signal. B) in the absence of the microRNA, the system is switched to mode « off ». Detection and inhibition of endogenous microRNA expression (4T1 cells) MicroRNA expression in lungs metastasis Figure 3: A) Detection of endogenous microRNA expression in 4T1 cells. B) Relative expression of microRNA (quantitative PCR). C) Inhibition of microRNA expression with antisense oligonucleotides. To be continued…. Figure 4: A) Relative luciferase expression in lungs (quantitative PCR) B) MicroRNA expression in lungs metastasis (quantitative PCR). Conclusions A) B) We report a novel imaging system (RILES) able to generate positive bioluminescence signals i...
Sugar Specific Delivery of Drugs, Oligonucleotides and Genes
Targeting of Drugs 4, 1994
Gene delivery systems : current status and challenges
Proceedings of the Interntional Congress on Ultrasonics, 2007
8B-3 Transmembrane Extraction of Fluorescent Proteins with Ultrasound and Microbubbles
2007 IEEE Ultrasonics Symposium Proceedings, 2007
Recent studies have shown that microbubbles under ultrasound (US) activation permeabilize plasma ... more Recent studies have shown that microbubbles under ultrasound (US) activation permeabilize plasma membrane. But the mechanism of permeabilization is not completely understood. The recurrent hypothesis assumes the formation of pores in the cell membrane allowing molecule delivery into cells. The aim of our study is to comfort this assumption by investigating whether US and microbubbles facilitate outward transport of molecules across the plasma membrane. Stably transfected Hela-GFP cells (GFP + cells) and propidium iodide (PI) were used to follow the US- microbubbles mediated permeabilization. To correlate these observations with molecular incorporation, US waves were generated from a 1 MHz unfocused transducer with pressures from 200 kPa to 600 kPa and duty cycles of 40% and 75%. Exposure time was 2 min with or without BR14 microbubbles (Bracco Research, Geneva). Immediately after insonation, the cells were immersed in a 4degC bath to interrupt cell activity. The percentage of GFP positive cells and the mean cell fluorescence intensity (FI) were measured by flow cytometry to evaluate the GFP release. We observed a significant decrease of FI and the percentage of GFP+ cells using US in presence of BR14 microbubbles. These effects were maximal at 400 kPa, 75% of duty cycle and 2 minutes insonation compared to control conditions. In parallel, the number of GFP+ cells that incorporated PI was higher than 50% for the same acoustic parameters. US alone had a slight impact on cell membrane permeability. These observations were also correlated to an increase of FITC-dextran incorporation that varied also vs acoustic parameters in presence of BR14 microbubbles. This result demonstrates the possibility to incorporate extracellular molecules into Hela cells throw probably pore formation. All observations would be explained by pore formation occurring under activation of microbubbles with ultrasound. This phenomenon leads to probably gradient dependant transport through pore ope- ning during insonation.
Uploads
Papers by Patrick Midoux