Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

Développement de stratégies de couplage polysaccharides-liposomes en vue de l'élaboration de vaccins synthétiques

Les polysaccharides sont des composés ubiquitaires présents à la surface de nombreux organismes pathogènes (bactéries, champignons, levures, parasites ) menaçant la santé de l homme. Ils constituent des éléments importants pour le développement de vaccin dans le cadre de la lutte contre les maladies infectieuses. L objectif de ce travail est de développer des constructions vaccinales polysaccharidiques, chimiquement définies, utilisant un vecteur liposomal et capables d induire une réponse humorale T-dépendante anamnestique. Dans ce dessein, nous avons adopté une stratégie biépitope qui consiste à coupler covalemment à la surface de petits liposomes unilamellaires (diamètre ~ 100 nm) un épitope B saccharidique et un épitope Th peptidique ("T-universel") porté par une ancre lipopeptidique (Pam3CAG-Mal) aux propriétés adjuvantes (ligand des TLR2).La première partie de notre stratégie consistait à développer une méthode chimiosélective, compatible avec un milieu aqueux et permettant de fonctionnaliser le sucre non-protégé au niveau du carbone anomérique. Dans ce but, nous avons synthétisé différents réactifs hétérobifonctionnels de couplage comportant d un côté, une fonction N-méthylhydroxylamine et de l autre côté, une fonction thiol protégée qui permet de greffer le polysaccharide ainsi dérivatisé à la surface de liposomes préformés incorporant une ancre réactive au thiol. Malgré la séduction de cette stratégie, cette chimie de bioconjugaison utilisant la fonction N-méthylhydroxylamine s est révélée inapplicable au développement de vaccins à base de polysaccharides en raison i) d une instabilité intrinsèque de la plupart des réactifs hétérobifonctionnels ii) d un manque de stabilité suffisante des conjugués obtenus avec les saccharides.Nous avons également développé une nouvelle approche de bioconjugaison basée sur la click chemistry et qui permet de coupler des ligands à la surface externe de liposomes en utilisant la réaction de cycloaddition 1,3 dipolaire de Huisgen catalysée par du cuivre (I). Bien que cette méthode se soit avérée incompatible avec des ligands saccharidiques fonctionnalisés par la N-méthylhydroxylamine, elle reste aisément transposable à une large variété de ligands.Parallèlement à la mise au point de la chimie de bioconjugaison, nous avons validé in vivo notre modèle vaccinal biépitope polysaccharide/peptide en utilisant des oligosaccharides synthétiques représentatifs de l unité répétitve de l antigène O de Shigella flexneri 2a. Les constructions liposomales induisent une réponse immune chez des souris BALB/c à caractère humoral T-dépendant de type Th-2 attestée par la production des IgG1.STRASBOURG ILLKIRCH-Pharmacie (672182101) / SudocSudocFranceF

Rational design and immunogenicity of liposome-based diepitope constructs: application to synthetic oligosaccharides mimicking the Shigella flexneri 2a O-antigen.

International audienceWe have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307-319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam(3)CAG, i.e. a TLR2 ligand known for its adjuvant properties, anchored in preformed vesicles. The synthetic construct containing a pentadecasaccharide corresponding to three O-antigen repeating units triggered T-dependent anti-oligosaccharide and anti-S. flexneri 2a LPS antibody responses when administered i.m. to BALB/c mice. Moreover, the long-lasting anti-LPS antibody response afforded protection against a S. flexneri 2a challenge. These results show that liposome diepitope constructs could be attractive alternatives in the development of synthetic carbohydrate-based vaccines

Low incidence of SARS-CoV-2, risk factors of mortality and the course of illness in the French national cohort of dialysis patients

International audienceThe aim of this study was to estimate the incidence of COVID-19 disease in the French national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. Our study included all patients on dialysis recorded in the French REIN Registry in April 2020. Clinical characteristics at last follow-up and the evolution of COVID-19 illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for SARS-CoV-2. A total of 1,621 infected patients were reported on the REIN registry from March 16th, 2020 to May 4th, 2020. Of these, 344 died. The prevalence of COVID-19 patients varied from less than 1% to 10% between regions. The probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. Dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. Mortality in diagnosed cases (21%) was associated with the same causes as in the general population. Higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. Being treated at a selfcare unit was associated with a lower risk. Thus, our study showed a relatively low frequency of COVID-19 among dialysis patients contrary to what might have been assumed