Céline Largeau

Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexes.

OBJECTIVEnBlocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi)-based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA).nnnMETHODSnTwo synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA.nnnRESULTSnWeekly injections of anti-IL-1, anti-IL-6, or anti-IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex-based treatment.nnnCONCLUSIONnOur results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates.

Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic cargo. Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.

How to make siRNA lipoplexes efficient? Add a DNA cargo

BACKGROUNDnWe recently reported an efficient formulation of siRNA targeting TNF-alpha, that was able to restore immunological balance in a mouse arthritis model following intravenous injection.nnnMETHODnSince this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect.nnnRESULTSnWe showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate.nnnCONCLUSIONSnThese results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.

Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH(2)-glycopeptides already reported to act as sLe(x) mimics. The alpha and beta anomers of these CF(2)-glycopeptides have been prepared, as well as their 1-hydroxy analogues which were present in solution as an equilibrium mixture of alpha- and beta-pyranose and alpha- and beta-furanose forms. These molecules showed inhibitory activities comparable to their CH(2) counterparts with a moderate influence of the pseudo-anomeric center configuration.

Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (± DOPE) (± Lac-10-Chol) that present reproducible absolute entrapment value (1.45u2009±u20090.10%), with a size of 109u2009±u20097u2009nm and a slight positive charge (3.77u2009±u20091.59u2009mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (Pu2009>u20090.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (Pu2009<u20090.05).