No description available.

Le cancer est la principale cause de mortalité au Canada. Les taxanes (e.g. le paclitaxel et le docétaxel (DCTX)) constituent des remèdes efficaces contre une série de tumeurs solides telles que les cancers du sein, du poumon et de l’ovaire. Par ailleurs, des acides nucléiques (e.g. les oligonucléotides antisens (AON) ou les petits ARN interférents (siRNAs)), capables de supprimer sélectivement certains oncogènes impliqués dans la carcinogénèse, sont actuellement étudiés pour traiter une large gamme de cancers. Bien que l’activité des taxanes et des acides nucléiques soit bien établie sur des modèles humains et/ou animaux, plusieurs aspects physico-chimiques et cliniques restent encore à améliorer. Leur solubilité limitée (pour les taxanes), leur dégradation rapide dans le sang (pour les acides nucléiques), leur élimination précoce, leur absence de sélectivité et leur toxicité envers les tissus sains sont les principaux facteurs limitant leur efficacité. C’est pourquoi de nombreux efforts ont porté sur l’élaboration de systèmes de vectorisation ciblés à base de polymères, dans le but de surmonter les problèmes associés aux thérapies actuelles. Dans cette thèse, deux types de micelles polymères ont été développés pour la vectorisation de DCTX et d’acides nucléiques. D’une part, des micelles de poly(oxyde d’éthylène)-bloc-poly(oxyde de butylène/styrène) ont été étudiées pour la première fois pour solubiliser le DCTX et le protéger de l’hydrolyse. Ces polymères se sont révélés moins toxiques que le surfactant utilisé commercialement pour solubiliser le DCTX (i.e. polysorbate 80) et ont permis une libération prolongée du principe actif. D’autre part, deux systèmes différents de micelles polyioniques (PICM) ont été mis au point pour la vectorisation d’acides nucléiques. De nouveaux conjugués de poly(éthylène glycol) (PEG)-oligonucléotide ont été proposés pour la protection et la libération contrôlée d’AON. Lorsque ces conjugués ont été formulés avec des dendrimères de poly(amidoamine) (PAMAM), des complexes de taille homogène ont été obtenus. Ces PICM ont permis de prolonger la libération de l’AON et de le protéger efficacement contre la dégradation enzymatique. De plus, des polymères de poly(oxyde d’éthylène)-bloc-poly(méthacrylate de propyle-co-acide méthacrylique) ont été incorporés afin de conférer des propriétés acido-sensibles aux PICM. Dans ces micelles, formées de ce dernier polymère formulé avec le dendrimère PAMAM, des oligonucléotides (AON et siRNA) ciblant l’oncogène Bcl-2 ont été encapsulés. L’internalisation cellulaire fut assurée par un fragment d’anticorps monoclonal (Fab’) situé à l’extrémité de la couronne de PEG. Après l’internalisation cellulaire et la protonation des unités d’acide méthacrylique sous l’effet de l’acidification des endosomes, les micelles se sont affranchies de leur couronne. Elles ont ainsi exposé leur cœur composé d’acide nucléique et de dendrimère PAMAM, qui possède une charge positive et des propriétés endosomolytiques. En effet, ces PICM acido-sensibles ciblées ont permis d’augmenter la biodisponibilité des acides nucléiques vectorisés et se sont avérées plus efficaces pour silencer l’oncoprotéine Bcl-2 que les micelles non ciblées ou que le dendrimère de PAMAM commercial seul. Finalement, les nanovecteurs polymères présentés dans cette thèse se révèlent être des systèmes prometteurs pour la vectorisation des anticancéreux et des acides nucléiques. / Cancer is considered as the leading cause of premature death in Canada. Taxanes (e.g. paclitaxel and docetaxel (DCTX)) are effective against a range of solid tumors including breast, lung, and ovarian malignancies. In addition, nucleic acids (e.g. antisense oligonucleotides (AON) and short interfering RNA (siRNA)) which are capable of selectively suppressing oncogenes involved in carcinogenesis are currently being investigated for the treatment of a wide variety of cancers. Although the activity of taxanes and nucleic acid drugs is well-established in human and/or animal models, several physicochemical and clinical issues still need to be addressed. Low aqueous solubility (i.e. taxanes), rapid degradation in the blood (i.e. nucleic acids), fast clearance, non-selectivity and toxicity to normal tissues are limiting factors to their effectiveness. Hence, many efforts have been focused on developing targeted polymeric delivery systems to overcome the problems associated with the current therapies. In this thesis, two types of polymeric micelles have been developed for the delivery of DCTX and nucleic acids. On the one hand, poly(ethylene oxide)-block-poly(butylene oxide/styrene oxide) micelles were tested for the first time to solubilize and protect DCTX from hydrolytic degradation. The polymers showed less toxicity than the surfactant used commercially to dissolve DCTX (i.e. polysorbate 80) and released the drug in a sustained fashion. On the other hand, two different systems of polyion complex micelles (PICM) were developed for the sustained release and intracellular delivery of nucleic acids. Novel poly(ethylene glycol) (PEG)-oligonucleotide conjugates were assessed to protect AON against degradation and release them in a sustained manner. When these conjugates were mixed with poly(amidoamine) (PAMAM) dendrimers, monodisperse PICM were formed. These PICM further slowed down AON release and significantly protected it against enzymatic degradation. In addition, the incorporation of poly(ethylene oxide)-block-poly(propyl methacrylate-co-methacrylic acid) was exploited to impart pH-sensitivity to PAMAM-based PICM. This system was composed of the previous copolymer mixed with PAMAM dendrimer. Such PICM were loaded with AON or siRNA targeting the Bcl-2 oncogene. Micelles uptake by the cancer cells was mediated by a monoclonal antibody fragment (i.e. Fab') positioned at the extremity of the PEG corona. Upon cellular uptake and protonation of the methacrylic acid units in the acidic endosomal environment, the micelles lost their corona, thereby exposing their positively-charged endosomolytic PAMAM/nucleic acid core. The targeted, pH-sensitive PICM were found to increase the intracellular bioavailability of the entrapped nucleic acids and knock down the Bcl-2 oncoprotein more than either non-targeted micelles or commercial PAMAM dendrimers. The polymeric nanocarriers reported in this thesis appear to be promising vehicles for the delivery of anticancer drugs and nucleic acids.

Fluorescence correlation spectroscopy (FCS) analyzes the fluctuations in the fluorescence intensity, which is emitted from a tiny excition volume, to obtain information about the concentration, the mobility, and the molecular interactions of labeled molecules. The more advanced fluorescence cross-correlation spectroscopy (FCCS) increases the precision in the determination of fl ow velocities and binding constants compared to standard FCS. The miniaturization in biomedical and chemical engineering has been developing rapidly, propelled by the vision of a fully functional laboratory on a single chip and its use in human therapeutics, for example, as implanted drug delivery system. A key requirement to fulfill this vision is the ability to handle small fl uid volumes. Handling liquids using the electrohydrodynamical principle circumvents many of the disadvantages of other systems. The complex flow pattern in the active region of such a pump could not be resolved by common tracking techniques. In this thesis, two-focus FCCS (2f-FCCS) was used to map the flow pro file inside a micropump. The high precision of 2f-FCCS in the determination of fl ow measurements even with small fluorescent particles allowed the measurement of the flow velocities induced by electrohydrodynamic forces acting on the solvent, while excluding the effects of dielectrophoretic forces acting on larger particles. Analysis of the fl ow data indicates a fl ow pattern that consists of two vortices of different size and opposite direction of rotation. The flow pattern derived by 2f-FCCS explains the observed complex particle trajectories in the force field and the accumulation of particles in well-de fined regions above the microelectrode array. In the second part of this thesis, the mechanism of RNA interference (RNAi) was studied by dual-color FCCS in vivo. RNAi is an evolutionary conserved gene silencing mechanism, which uses short double-stranded RNA molecules, called short interfering RNAs (siRNAs), as effector molecules. Due to its speci city and simplicity, RNAi yields a great potential for a widespread therapeutic use. To broaden the therapeutic applications, the in vivo stability of siRNAs has to be improved by chemical modi cations, but some of these modi fications inhibit the gene silencing mechanism. The presented FCCS assays are very well suited to investigate the individual assembly steps of RNAi machinery with very high specifi city and sensitivity in real time and to study the cleavage activity of the activated RNAi machinery. A direct correlation between activity of the RNAi machinery and the results from the FCCS measurements could be shown. The in fluence of several chemical modi cations on the assembly and activity of the RNAi machinery was investigated with these assays. / Fluoreszenz-Korrelations-Spektroskopie (FCS) analysiert die Fluktuationen im Fluoreszenzsignal eines kleinen angeregten Volumens, um Informationen über die Konzentration, die Bewegung und die Interaktionen der markierten Moleküle zu erhalten. Die Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) erhöht die Genauigkeit bei der Messung von Fließgeschwindigkeiten und Bindungskonstanten im Vergleich zur Standard-FCS. Die Miniaturisierung der Biomedizin und Chemie hat sich rapide entwickelt, angetrieben von der Vision eines kompletten Labors auf einem Chip und dem Einsatz dieses in der medizinischen Therapie, zum Beispiel als implantierter Medikamentenspender. Ein Schlüsselelement zur Erfüllung dieser Vision ist der Transport von kleinsten Flüssigkeitsmengen in diesen miniaturisierten Systemen. Der Transport von Flüssigkeiten mittels des elektrohydrodynamischen Prinzips umgeht viele Nachteile von anderen Systemen, allerdings zeigt eine solche Pumpe ein kompliziertes Strömungsbild in der aktiven Region, welches sich mit herkömmlichen Methoden wie Teilchenverfolgung nicht vermessen ließ. Hier wurde Zwei-Fokus-FCCS (2f-FCCS) genutzt, um das Strömungsbild in der Pumpe zu vermessen. Die hohe Genauigkeit der 2f-FCCS bei der Bestimmung von Fließgeschwindigkeiten auch mit kleinen fluoreszierenden Teilchen ermöglichte die Messung der Fließgeschwindigkeiten, aufgrund der auf das Lösungsmittel wirkenden elektrohydrodynamischen Kräfte, unter Ausschluss der auf größere Teilchen wirkenden dielektrophoretischen Kräfte. Die Analyse der Daten ergab, dass das Strömungsbild aus zwei entgegengesetzt rotierenden unterschiedlich großen Wirbeln besteht. Dieses Strömungsbild erklärt die komplizierten Teilchenbewegungsbahnen und die Anreicherung der Teilchen in klar abgegrenzten Bereichen über den Mikroelektroden. Im zweiten Teil dieser Arbeit wurde der RNAi-Mechanismus in lebenden Zellen mittels Zwei-Farben-FCCS untersucht. RNA Interferenz (RNAi) ist ein evolutionär erhaltener Geninaktivierungsmechanismus, der kurze doppelsträngige RNA Moleküle, so genannte kurze interferierende RNAs (siRNAs), als Effektormoleküle nutzt. Die Spezifi tät und Einfachheit der RNAi hat ihr ein weites Feld in der medikamentösen Therapie geöffnet. Zur Erweiterung dieses Feldes ist es nötig die Stabilität der siRNAs im Körper mittels chemischer Modi fikationen zu erhöhen. Einige dieser Modifikationen hemmen aber den RNAi-Mechanismus. Die hier vorgestellten FCCS Experimente sind sehr gut geeignet, um die einzelnen Schritte des Zusammenbaus der RNAi Maschinerie mit hoher Empfi ndlichkeit und Spezi fität in Echtzeit zu untersuchen und die Aktivität der RNAi Maschinerie zu studieren. Es konnte ein Zusammenhang zwischen der Aktivität der RNAi Maschinerie und den Ergebnissen der FCCS Messungen hergestellt werden. Der Einfluss von verschiedenen chemischen Modikationen auf den Zusammenbau und die Aktivität der RNAi Maschinerie wurde mit diesen neuartigen Methoden untersucht.

No description available.

Les lipides « switch » - bascules - appartiennent à la famille des matériaux sensibles à un stimulus. Quand ces lipides bascules sont incorporés aux nanoparticules lipidiques (LNP), ils permettent la délivrance contrôlée grâce à un changement de conformation activé par une baisse de pH. Des expériences précédentes avaient démontré que les LNP bascules ont transfecté le petits ARN interférents (siRNA) in vitro et in vivo, silençant la protéine fluorescente verte (GFP) et la protéine hépatique Facteur VII, respectivement. La double administration de micro ARN (miRNA) et d'agent anticancéreux melphalan a également été réalisée par les LNP bascule sur un modèle de rétinoblastome murin. Ces résultats prometteurs nous ont encouragé à élargir les applications de LNP bascules en tant que vecteur de siRNA. De plus, le mécanisme par lequel les LNP bascules induisent la déstabilisation de la membrane et la libération de matériaux encapsulé au milleu acide reste obscur. La compréhension de ce mécanisme est cruciale pour cerner les avantages et les limites des LNP bascules, pour proposer des futures applications et pour prévenir leur toxicité. Dans ce mémoire, nous avons comme objectif d’évaluer le potentiel des LNP bascules pour le traitement du cancer. Nous avons évalué les LNP bascules comme vecteur de livraison du siRNA ciblant l'une des protéines cancéreuses les plus spécifiques découvertes à ce jour, la survivine. En parallèle, nous avons étudié le comportement biophysique des membranes contenant des lipides bascules dans des vésicules de taille micromètrique. Dans la première étude, nous avons démontré que les LNP bascules ont permis le silençage de la survivine dans une gamme de lignées cellulaires cancéreuses (poumon, cervical, ovaire, sein, côlon, rétinoblastome). Dans les cellules du rétinoblastome humain (Y79), nous avons examiné plusieurs agents cytotoxiques utilisés en clinique quant à leur synergie avec le silençage de la survivine: melphalan, topotécan, téniposide et carboplatine. Le prétraitement avec les LNP chargées de siRNA-survivine a amélioré de manière synergique la cytotoxicité du carboplatine et du melphalan mais dans une moindre mesure celle du topotécan et du téniposide. Cet effet était spécifique aux cellules cancéreuses car les cellules saines (ARPE.19) n'exprimaient pas de survivine. L'inhibition de la survivine par silençage de siRNA s'est révélée plus spécifique et moins dommageable pour les cellules saines (ARPE.19) que le YM155, un inhibiteur moléculaire de la survivine. Dans la deuxième étude, nous avons observé par microscopie confocale que les lipides bascules induisaient rapidement le stress, la fission et une courbure positive dans les membranes des vésicules unilamellaires géantes lorsqu'elles étaient exposées à des conditions acides. La dynamique de la membrane a été confirmée par des expériences de diffusion dynamique de la lumière (DLS) et de fuite de calcéine. Ces phénomènes ont également été observés lorsque des lipides bascules ont été incorporés dans une membrane hybride polymère/lipide, fournissant des propriétés sensibles au pH aux vésicules hybrides. À notre connaissance, c'est la première fois qu'une vésicule hybride sensible au pH est reportée. Nos résultats corroborent l'applicabilité des LNP bascules en tant qu'agents de vectorisation des siRNA pour le traitement du cancer grâce au silençage de la survivine, en particulier comme adjuvant à la chimiothérapie. L'investigation biophysique a révélé que les lipides bascules agissent sur la fluidité de la membrane, en particulier à pH acide. Cette sélectivité en pH garantit leur biocompatibilité à pH neutre ainsi que la libération efficace et rapide de leur cargo à pH acide. La compatibilité avec les vésicules hybrides polymère/lipide ouvre de nouvelles applications au niveau de vésicules biomimétiques et l'administration de médicaments. / Cationic switchable lipids belong to the class of stimuli-responsive materials. When incorporated in lipid nanoparticles (LNP), switchable LNP promote pH-triggered delivery of payload based on a molecular switch mechanism. Previous studies have demonstrated that switchable LNP successfully delivered small interferring RNA (siRNA) in vitro and in vivo, promoting the silencing of a reporter Green Fluorescencen Protein (GFP) protein and liver-produced factor VII, respectively. Dual delivery of micro RNA (miRNA) and anticancer agent melphalan was also achieved through switchable LNP in a retinoblastoma rat model. These promising results encouraged us to enlarge the applications of switchable LNP as siRNA carrier. Moreover, the mechanism whereby switchable LNP mediate acid-triggered membrane destabilization and, thus, payload release remains elusive. Understanding this mechanism is crucial to draw the advantages and limitations of switchable LNP, and to tailor their future applications and prevent their potential toxicity. In this dissertation, we aimed to further understand the potential of switchable LNP for cancer treatment. We assessed switchable LNP as a siRNA delivery carrier by targeting one of the most specific cancer protein discovered to date, survivin. Meanwhile, we investigated the biophysical behavior of switchable-lipid containing membranes in micron-sized vesicles. In the first study, we demonstrated that switchable LNP efficiently silenced survivin in a range of cancer cell line models (lung, cervical, ovary, breast, colon, retinoblastoma). In retinoblastoma (RB) cells (Y79), several clinically used cytotoxic agents were screened for their synergy with survivin silencing: melphalan, topotecan, Teniposide, and carboplatin. Pretreatment with LNP loaded with siRNA targeted against survivin synergistically enhanced the cytotoxicity of carboplatin and melphalan but in lesser extent topotecan and teniposide. This effect was specific to cancer cells since healthy cells (ARPE.19) did not express survivin. Survivin inhibition through siRNA silencing revealed more specific and less damageable for healthy cells (ARPE.19) than a molecular approach, such as YM155. In the second study, we observed by confocal microscopy that switchable lipids rapidly induced stress, fission, and positive curvature in giant unilamellar vesicles’ membranes when submitted to acidic conditions. The membrane dynamics was confirmed by dynamic light scattering and calcein leakage experiments. Remarkably, these phenomena were also observed when switchable lipids were embedded into a hybrid polymer/lipid membrane, providing pH-sensitive properties to hybrid vesicles. To the best of our knowledge, this is the first time a pH-sensitive hybrid vesicle is reported. Our findings corroborate with the applicability of switchable LNP as siRNA delivery agents for cancer treatment through survivin silencing, especially as an adjuvant to chemotherapy. The biophysical investigation revealed that the switchable lipids act on the membrane fluidity, specifically at acidic pH. This pH selectivity guarantees their biocompatibility at neutral pH as well as its efficient and quick release of their cargo at acidic pH. Their compatibility with hybrid polymer/lipid vesicles opens new applications in biomimetic vesicles and drug delivery.

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

Polymeric monodispersed nanocarriers with controlled shape and size have been fabricated in the literature primarily using top down processes such as imprint lithography. In this dissertation, the geometric and material property limits of imprint based techniques have been studied. The resulting insight has led to the creation of new processes that significantly extend the limits of imprint processes in several ways: (i) Ability to print nanocarriers with ultra-soft biomaterials (<1MPa modulus); (ii) Sub-50nm diameter cylindrical particles with >3:1 aspect ratio with >5x enhanced wafer yield; (iii) Creation of reentrant barrel shapes that have the potential to be valuable in cellular uptake, such shapes being significant as they lead to fundamental demolding challenges in prior imprint processes; and (iv) Multi-layer nanocarriers which can potentially provide sophisticated functionality such as tailored release kinetics of one or more drugs. By understanding the requirements of bio-functional nanocarriers and related manufacturing constraints, a previously explored Bio Jet and Flash Imprint Lithography (Bio J-FIL) process was refined to perform successful imprints and improve the nanocarrier fabrication scalability. Next, two new fabrication processes have been developed. The first process is called Decoupled Functional Imprint Lithography (D-FIL) which allows fabrication of ultra-soft bio-functional materials (modulus of <1 MPa), challenging sizes (sub-50nm diameter cylinders with aspect ratio > 3:1), and reentrant barrel shapes. The second decoupled process, Dual Removable Layer Lithography (DRLL), has been developed to specifically create multi-layered cylindrical nanocarriers. Nanocarriers fabricated with D-FIL and DRLL process have been shown to chemically bind with an imaging agent, and model anti-cancer drugs. Drug (siRNA) retention (>90% over 9 days) and stimuli triggered release studies were performed on sub-100nm cylindrical PEGDA nanocarriers. It was found that these nanocarriers show accelerated triggered drug release when exposed to a hydrolase, Cathepsin B. While the exact mechanisms causing the triggered release are not fully understood, a few possible explanations are provided based on the experiments reported. Finally, the D-FIL, the DRLL, and the refined Bio J-FIL processes have been successfully demonstrated at the prototype scale as well as at the pilot scale in collaboration with an industrial partner, Molecular Imprints Inc. / text

Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.

RNA interference (RNAi) is a post transcriptional gene silencing mechanism that allows potent and specific silencing of cognate mRNA transcripts. Selective silencing can be used to dissect complex polygenic diseases, elucidate the function of known genes and provide a tool for genetic therapy. Its use in the case of dominant inherited disorders including disorders of the central nervous system, depends on its ability to confer single nucleotide discrimination between normal and mutant gene alleles. In this thesis the ability of RNAi effector molecules to provide single nucleotide specificity was examined by targeting two dominant inherited mutations of the acetylcholine receptor that cause slow-channel syndrome. Allele-specific silencing was achieved for one mutation. The other mutation was also silenced but not in an allele specific way despite employing known techniques for increasing single-nucleotide specificity. The model used in this thesis is the congenital myasthenia slow-channel syndrome. This is a dominant inherited disorder of the neuromuscular junction which is both well-characterised and more readily accessible compared to the central nervous system, thus provides a prototype for development of allele-specific RNAi therapeutics. Here we describe a new transgenic animal model of the slow-channel syndrome and show good representation of the human disorder. The need for defining the characteristics that determine the effectiveness and the specificity of RNAi effectors at single-nucleotide level, along with the future uses of the newly described animal model are discussed.

Les oligonucléotides (ONs) antisens présentent un fort potentiel en tant qu’agents thérapeutiques. Toutefois, leurs propriétés physicochimiques limitent leur utilisation en thérapie génique. Pour pallier aux divers obstacles, des systèmes de vectorisation, tels que les micelles polyioniques (PICMs), ont été développés. Grâce à leur structure unique, les micelles protégent l’ON contre une dégradation prématurée et le couplage d’un ligand à leur surface augmente leur spécificité et leur internalisation. Dans d’autres systèmes, un polymère adjuvant aux propriétés pH-sensibles peut être ajouté pour faciliter la sortie de l’endosome et augmenter l’efficacité de l’ON. L’objectif général de ce mémoire était de mettre au point des PICMs ternaires ciblées pour l’administration d’ONs. Ces micelles assureraient à la fois l’internalisation cellulaire de leur cargaison en interagissant avec des récepteurs cellulaires et sa fuite de l’endosome grâce à un mécanisme de déstabilisation de la membrane endosomale. Pour cela, des PICMs composées d’un copolymère cationique de type poly(éthylène glycol)-bloc-poly(méthacrylate d’(alkylamino)éthyle) et d’un copolymère d’acide méthacrylique ont été préparées. Les propriétés physicochimiques de ces vecteurs ont démontré qu’ils permettaient une condensation efficace de l’acide nucléique et ce, indépendamment de la nature du polymère cationique et de l’acide nucléique. Finalement, une approche de couplage par pont disulfure a été développée afin de greffer au copolymère un fragment d’anticorps dirigé contre les récepteurs de la transferrine. En conclusion, ces travaux démontrent la versatilité et le potentiel des PICMs ternaires en tant que vecteurs d’acide nucléique, et proposent une méthodologie de couplage d’un ligand afin de formuler des PICMs ciblées. / Antisens oligonucleotides (ONs) present great potential as therapeutic agents. However, their physicochemical properties hinder their use in gene therapy. Targeting systems, such as polyion complex micelles (PICMs), have been proposed to circumvent the main hurdles related to ON delivery. Their unique core/shell structure can protect the ON against premature degradation and the coupling of a ligand on their surface can increase their specificity and internalization. In other systems, a polymer with pH-sensitive properties can be added to facilitate the release of the ON from the endosome and increase its efficiency. The present work was aimed at optimizing ternary PICMs targeted for the delivery of antisens ON. Such systems would provide both cellular internalization of cargo by interaction with receptors on the surface of cell membranes and escape from the endosome through a mechanism of destabilization of the endosomal membrane. PICMs composed of cationic copolymers of poly(ethylene glycol)-bloc-poly((alkylamino)ethyl methacrylate) with a methacrylic acid copolymer adjuvant were prepared. Their physicochemical properties suggest that efficient complexation of nucleic acids was obtained, regardless of the nature of the cationic polymer and the nature of the nucleic acid. Finally, a synthetic approach was developed for the conjugation of an antibody fragment directed against the transferrin receptor via a labile disulfide bond at the end of the cationic copolymer. In conclusion, the work presented herein displays the versatility and potential of ternary PICMs as vehicles for the delivery of ONs and also provides a method for the conjugation of a ligand to generate targeted ternary PICMs.