Our lab recently demonstrated that retargeting an alkylating agent, chlorambucil (Cbl), to the mitochondrion is a viable strategy to restore drug activity and overcome drug resistance in cancer. The mechanism of action of the mitochondria-targeted chlorambucil (mt-Cbl) was studied using a cervical carcinoma model. It was discovered that mt-Cbl bound to mitochondrial DNA and various mitochondrial proteins. A ρ° model revealed that the toxicity of mt-Cbl is largely dependent on its protein targets. Damage induced by mt-Cbl was found to result in the activation of several modes of caspase-independent cell death including necrosis. In contrast, Cbl was found to only activate caspase-dependent cell death that is highly sensitive to caspase inhibition. These results illustrate that the ability of mt-Cbl to activate various orthogonal cell death pathways is what allows mt-Cbl to bypass several drug resistance mechanisms, thus making mitochondrial retargeting a lucrative strategy for future anticancer drug development.

L’objectif du projet est de comprendre l’effet du peptide vecteur (R/W)9 sur les cellules tumorales EF, modèles du sarcome d’Ewing. En effet, ce peptide a la capacité de remodeler le cytosquelette d’actine dans ces cellules, ainsi que de réduire leur motilité et leur aptitude à croître en indépendance d’ancrage (Delaroche D. JBC 2010).La première étape de ce travail a été la caractérisation in vitro de l’interaction directe avec l’actine par cross-linking chimique et spectrométrie de masse (Clavier S. EuProt 2014). Ensuite, pour avancer dans la compréhension de l’effet du peptide (R/W)9, deux approches ont été développées.La première approche, basée sur du photocross-linking in cellulo ou in lysat, vise à identifier des partenaires intracellulaires du peptide vecteur. Pour cela, nous avons mis au point et validé biologiquement une version photoactivable du peptide (R/W)9. Puis, avant de passer sur cellules entières, nous nous sommes assurés de la faisabilité de la réaction de photocross-linking in vitro sur un système d’interaction modèle que nous avons également utilisé pour développer un logiciel capable d’interpréter les spectres MS/MS d’espèces photocross-linkées (Xlink-Identifier, Collaboration Dr Du X). Des expériences de purification d’affinité ont également été menées en immobilisant le peptide (R/W)9 sur des billes de streptavidine ensuite mises en présence de lysat cellulaire. Les protéines capturées ont été identifiées par spectrométrie de masse haut-débit. La seconde approche est de la protéomique différentielle avec un marquage SILAC et a pour objectif de mettre en évidence l’influence du peptide vecteur sur l’expression des protéines. / The aim of the project is to understand the effect of (R/W)9 cell penetrating peptide (CPP) on EF tumoral cells, an Ewing sarcoma model cell line. Actually, this peptide is able to remodel the actin cytoskeleton of these cells, to decrease their motility as well as their ability to grow without anchorage (Delaroche D. JBC 2010).The first step of this work was to characterize the in vitro interaction with actin using chemical cross-linking and mass spectrometry (Clavier S. Euprot 2014). Then, in order to get a deeper understanding of (R/W)9 peptide effect, two approaches were developed. The goal of the first approach based on in cellulo or in lysate photocross-linking is to identify (R/W)9 CPP’s partners. To do this, we designed and biologically validated a photoactivable version of (R/W)9 peptide. Then, before starting to work with living cells, we checked the feasibility of in vitro photocross-linking on a model interacting system that we also used to develop a software able to interpret MS/MS spectra of photocross-linked species. (Xlink-Identifier, Collaboration with Dr. X. Du.). Affinity purification experiments were also performed by incubating streptavidin magnetic beads bearing (R/W)9 peptide with cell lysates. Captured proteins were identified using high-throughput mass spectrometry. The second approach is differential proteomic with SILAC labelling and aims at assessing the influence of (R/W)9 CPP on proteins expression.

The cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and even more, carry cargoes with them. How CPPs enter cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been of particular interest and are explained in greater details. A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs like penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did only interact with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into the human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins. Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. In contrary to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively. TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNA, but not with scrambled PNA lost the action potential windup phenomenon. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was confirmed. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found to be required. Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes. In summary, different types of cargoes have been delivered by CPPs and different cargo coupling strategies have been used. CPP-mediated antisense oligonucleotide delivery has been used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

Pathogens such as <i>Listeria</i>, <i>Shigella</i>, <i>Brucella</i>, <i>Salmonella</i>, <i>Mycobacterium tuberculosis</i> and <i>methicillin-resistant Staphylococcus aureus</i> (MRSA) can traverse into mammalian cells, such as phagocytic macrophages. Once inside, these bacteria can survive and reproduce, causing chronic infections. It is of utmost importance to develop novel antibiotics with broad spectrum activity to control these deadly bacteria. Broad spectrum activity will allow for targeting of pathogens with different structures and cell membrane components.<div>This work focuses on the synthesis of a dual antibiotic agent, composed of a cationic amphiphilic polyproline helix (CAPH) possessing cell penetrating and nonmembrane lytic antimicrobial capabilities (P14LRR), and a derivative of the polymyxin B (PMX) antibacterial peptide. This dual antibiotic conjugate was created to be a tool to potentially clear intracellular pathogenic bacteria. Overall, the reduction of the disulfide bond linking the two antibiotics within the reducing environment of cells would release the individual antimicrobial agents, and could have improved cell membrane penetration and intracellular synergistic activity. Herein, the synthesis of the dual antibiotic agent, P14LRR-PMX, is discussed. </div>

The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

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No description available.

Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial. Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects. The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.

Développement et évaluation de nouvelles stratégies pour le traitement des hépatites B chroniques, dans le modèle du canard de Pékin infecté par le DHBVL’infection chronique par le HBV est la cause majeure de cirrhose hépatique et de carcinome hépatocellulaire, conduisant à plus d’un million de décès chaque année. Le faible taux de réussite des thérapies actuelles des hépatites B montre la nécessité du recours à des méthodes thérapeutiques alternatives. Ainsi, nous avons étudié une stratégie pertinente reposant sur l’utilisation de molécules antisens (PNAs) couplées à des peptides perméabilisants (CPPs). Nous avons démontré que les PNAs ciblant le signal d’encapsidation du DHBV couplés au CPP pénétraient dans les cellules et conduisait à une inhibition de la réplication virale. De plus, nous avons mis en évidence une activité antivirale du CPP (Arg)8 seul. Nous avons ensuite évaluer le mécanisme d’action antivirale du CPP in vitro et avons démontré qu’il inhibait les stades tardifs de la morphogénèse virale, conduisant à une inhibition forte de la sécrétion des particules virales. Par ailleurs, nous nous sommes intéressés à l’évaluation de stratégies immunothérapeutiques, reposant sur la vaccination génétique. Nous avons démontré les bénéfices de la co-administration de cytokines (IFNγ), avec un vaccin à ADN dirigé contre la grande protéine d’enveloppe du DHBV (preS/S), sur l’amplitude de la réponse humorale et sur le pouvoir neutralisant des anticorps induits. Enfin nous avons évalué les bénéfices d’une approche d’immunisation hétérologue « prime-boost » associant l’immunisation à ADN et un vecteur viral (AdénoCELO) recombinant, codant la protéine preS/S du DHBV et l’IFNγ. Nous avons montré que l’immunisation hétérologue induisait une réponse humorale plus forte que celle induite par l’immunisation homologue. / Development and evaluation of new strategies for treating chronic hepatitis B in the model of Peking duck infected with DHBVChronic infection with Hepatitis B virus (HBV) is the major cause of liver cirrhosis and hepatocellular carcinoma, leading to more than one million deaths each year. The low success rate of current therapies against HBV infection shows the need of alternative therapeutics. Thus, we studied a new strategy based on the use of antisense molecules (PNAs) coupled with cell penetrating peptides (CPPs). We have shown that PNAs targeting the DHBV encapsidation signal coupled to CPPs penetrated into the cells and led to an inhibition of viral replication. In addition, we have demonstrated an antiviral activity of the CCP (Arg)8 itself. We then evaluate the mechanism of antiviral action of this CPP in vitro and have shown that it inhibited the late stages of viral morphogenesis, leading to a strong inhibition of the release of viral particles. Furthermore, we were interested in evaluating immunotherapeutic strategies, based on DNA vaccination. We have demonstrated the benefits of co-administration of cytokines (IFNy), with a DNA vaccine directed against the DHBV large envelope protein (preS/S), enhancing the magnitude of humoral response and enhancing neutralizing anti-DHBV antibody response. Finally we evaluated the benefits of a heterologous immunization approach or prime-boost immunization involving DNA vaccination and a recombinant viral vector (AdenoCELO) encoding the DHBV preS/S and IFNy proteins. We have shown that heterologous immunization induced a humoral response stronger than that induced by homologous immunization. By contrast, the heterologous prime-boost strategy was less effective than homologous DNA immunization for therapy of chronic DHBV-carrier ducks.

Le but de cette thèse de doctorat fut de développer des nanoparticules pour la délivrance de deux molécules hydrophobes de faible poids moléculaire, l’apigénine (AG) et un ferrocifène (FcTriOH), comme stratégie innovante pour le traitement du glioblastome(GBM). Dans un premier temps, différents types de nanoparticules, liposomes, nanocapsules lipidiques (LNC), et nanocapsules à base de polymères, furent formulés et comparés en termes de caractéristiques physico-chimiques, de libération en drogue ou encore de toxicité. Les LNCs furent ainsi sélectionnées. Dans un deuxième temps, les LNCs furent fonctionnalisées en surface par un peptide pénétrant (CPP). La concentration de peptide fut augmenté afin d’améliorer significativement l’internalisation des LNCsdans des cellules humaines de GBM. Les mécanismes de macropinocytose et d’endocytose dépendant de la clathrine et de la cavéoline furent observés. De plus, il fut montré que l’internalisation de ces LNCs fonctionnalisées était réduite dans les cellules saines humaines d’astrocyte. L’efficacité biologique des LNCs chargées en AG et chargées en FcTriOH fut évaluée et comparée : le résultat le plus prometteur fut obtenu avec les LNCs chargées en FcTriOH. Une administration intracérébrale des LNCs sur un modèle tumoral murin orthotopique montra une potentielle toxicité et un besoin d’optimiser la dose administrée. Pour finir, les études menées sur un modèle tumoral ectopique murin montrèrent des résultats prometteurs, après une administration parentérale des LNCs chargées en FcTriOH. Ainsi, cette dernière formulation pourrait ouvrir la voie au développement d’une stratégie thérapeutique alternative pour le traitement du GBM. / The aim of this thesis was to develop nanocarriers for efficient delivery of two low molecular weight hydrophobic drugs, apigenin (AG) and a ferrocifen-derivative(FcTriOH) to glioblastoma (GBM) as potential therapeutic strategies. Firstly, two liposomes, a lipid nanocapsule (LNC), and a polymer-based nanocapsule were develope dand compared by their physicochemical characteristics, drug loading capacity, storage stability, stability in biological serum, drug release profiles, complement consumption and toxicity. Due to various advantageous characteristics, the LNCs were selected for further optimization. Secondly, the LNCs were surface functionalized by adsorbing a GBM-targeting cellpenetratingpeptide (CPP). The CPP concentration increased to significantly enhance LNCinternalization in human GBM cells. The uptake mechanisms observed in U87MG cellswere : micropinocytosis, clathrin-dependent and caveolin-dependent endocytosis. Moreover, the optimized CPP-functionalized LNCs were internalized preferentially in theGBM cells compared to normal human astrocytes. Additionally, the in vitro efficacy of the AG-loaded and FcTriOH-loaded LNCs was evaluated. The FcTriOH-loaded LNC-CPP showed the most promising activity with a low IC50 of 0.5 μM against U87MG cells. Intracerebral administration of the LNCs in a murine orthotopic U87MG tumor modelshowed possible toxic effects and the need for dose optimization. Finally, studies inmurine ectopic U87MG tumor model showed promising activity after parenteral administration of the FcTriOH-loaded LNCs. Overall, these results exhibit the promising activity of FcTriOH-loaded LNCs as potential alternative GBM therapy strategy.