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International audience; One of the major challenges of modern biology and medicine consists in finding means to visualize biomolecules in their natural environment with the greatest level of accuracy, so as to gain insight into their properties and behaviour in a physiological and pathological setting. This has been achieved thanks to the design of novel imaging agents, in particular to fluorescent biosensors.

International audience; The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

International audience; A major harrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80 %). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.; Le problème majeur rencontré lors du transfert de gène et/ou d'oligonucléotides dans les cellules de mammifères tient à la faible perméabilité des cellules aux acides nucléiques. Dans ce travail nous avons développé une nouvelle stratégie pour la vectorisation d'acides nucléiques, basée sur l'utilisation d'un peptide court (MPG) associant les propriétés d'une séquence de fusion dérivant de la protéine gp41 de VIH avec celle d'une séquence de signalisation nucléaire dérivant de l'antigène T de SV40. Ce vecteur peptidique diffuse rapidement dans les fibroblastes humain (HS-68) en culture, avec une localisation majoritairement nucléaire en moins de trois minutes. MPG présente une forte affinité pour l'ARNm codant pour la sous-unité de 66 kDa de la transcriptase inverse du VIH (Kd =100 nM). La formation du complexe peptide/acide nucléique se fait majoritairement par des interactions électrostatiques impliquant les résidus basiques du peptide et les charges négatives des groupements phosphates de l'ARN. Ce vecteur permet a lui seul de transférer rapidement (1 h) et avec un rendement supérieur à 80 % cet ARNm dans des fibroblastes en culture. De plus, MPG n'affecte pas la traduction de l'ARNm transféré et présente l'avantage d'être non cytotoxique aux concentrations utilisées pour le transfert d'ARN. Ce nouveau vecteur constitue un excellent outil pour développement du transfert de gênes.