MicroRNAs (miRNAs) have been identified as a key regulator in various biological processes and different diseases including cancer, heart disease, and viral infections. In the context of virus-host interactions, previous genome wide functional screen involving overexpression and inhibition of murine miRNAs in vitro identified several miRNAs that suppressed viral replication in diverse herpesviruses including herpes simplex virus 1 (HSV-1), murine cytomegalovirus (MCMV) and murine gamma herpesvirus 68 (MHV-68) (Santhakumar, D. et al, 2010). One of the top broad-spectrum anti-viral miRNAs, miR-542-5p, also suppressed human cytomegalovirus (HCMV) as well as a Semliki Forest virus (SFV) and two subtypes of influenza A virus (H1N1 & H3N2) in vitro. Following the previous study, this thesis focuses on generation of in vivo anti-viral efficacy data using miR-542-5p against two diverse viruses: MCMV and influenza (H1N1) in a pre-clinical model (mouse). One of the key challenges for generating in vivo efficacy data with miRNAs is the choice of delivery vehicle. To address this issue the first part of the project focused on optimising delivery conditions (dose, route of administration) for miR-542-5p mimic to target the lungs of mice (as both MCMV and H1N1 replicate in lungs). Initially, delivery was optimised using two cationic polymers: linear polyethylenimine (in vivo-JetPEI) and branched polyethylenimine (25KDa bPEI) that have been widely used previously to deliver nucleic acids in mouse. In parallel, two novel delivery systems were tested as an alternative delivery vehicle for miRNA mimics: biodegradable cationic lipids (Lifectin) and exosomes, natural vesicles produced by cells that can transport RNA. Results from in vivo delivery studies indicate that nebulisation of miR-542-5p mimic complexed with bPEI (25KDa) showed a more significant increase in the level of miRNA in the lung compared to other delivery systems and did not result in an immune response. Using bPEI as the delivery vehicle, the miR-542-5p mimic was administered to mouse lungs to test its anti-viral function against H1N1 and MCMV. Delivery of the miR-542-5p mimic resulted in 4.6 fold reduction of H1N1 virus titre in lungs (averaged across multiple experiments). The miR-542-5p mimic also had a 2 fold reduction in MCMV titre in the lungs. These data confirm the broad-spectrum anti-viral effect of miR-542-5p in mouse as observed in previous in vitro studies. Preliminary microarray analysis of genes regulated by miR-542-5p in vitro suggest this miRNA may target multiple genes required by diverse viruses during their life cycles and may modulate the PI3K-Akt signalling pathway.

Très récemment, notre équipe a mis en évidence une réaction dite de ligation et coupure entre une famille de composés mésoioniques, les sydnone-imines, et les cyclooctynes. Cette réaction bioorthogonale agit selon un processus en deux étapes, une cycloaddition [3+2] suivie d’une rétro Diels-Alder, qui génère deux nouveaux composés : un produit de ligation et un produit de coupure. L’objectif de cette thèse consiste à améliorer la cinétique de réaction entre ces deux partenaires afin de pouvoir l’utiliser en tant qu’outil pour la libération contrôlée de principes actifs in vivo.Trois stratégies ont été développées lors de cette thèse afin d’optimiser ce système réactionnel : l’étude d’une relation structure/réactivité du partenaire sydnone-imine vis-à-vis de la réaction bioorthogonale; l’utilisation de micelles constituées d’amphiphiles possédant un motif sydnone-imine en tant que lien clivable entre la partie hydrophobe et la partie hydrophile de la molécule; et enfin, l’étude de l’utilisation de la reconnaissance moléculaire entre deux brins d’acides nucléiques peptidiques (ANP) complémentaires. / Recently, our laboratory has discovered a click and release reaction involving iminosydnones, a family of mesoionic compounds, and cyclooctynes. This bioorthogonal reaction occurs via a two step process: a [3+2] cycloaddition followed by a retro Diels-Alder, to give two new compounds: a click product and a release product.The main goal of this work is to improve the kinetic of the reaction between these two partners in order to use it as a powerful tool for in vivo drug delivery. Three strategies were developed during this thesis to optimize this reaction system: the study of a structure/reactivity relationship of the iminosydnone partner regarding the bioorthogonal reaction; the development of micelles built by amphiphiles containing an iminosydnone moiety as a cleavable linker, strategically located between the hydrophobic and the hydrophilic part of the compound and finally, the use of molecular recognition between two peptide nucleic acids (PNA) complementary strands.