La maladie de Huntington (MH) est causée par une expansion de répétitions CAG sur l’exon 1 du gène huntingtine (htt), codant pour une protéine mutée. Il a été montré que la diminution d’expression de cette protéine est une piste thérapeutique très prometteuse. Dans ce projet, nous avons étudié et comparé trois approches dites «antisens» : une stratégie allèle non-spécifique, visant à diminuer de manière générale l’expression de htt ; une stratégie allèle spécifique ciblant les répétitions CAG afin d’impacter préférentiellement l’allèle muté ; et enfin une stratégie de saut d’exon permettant d’enlever des sites de clivage à l’origine d’une forme raccourcie et toxique de la protéine htt. Nous avons évalué ces approches grâce à deux outils différents : les tricyclo-DNA (TcDNA), qui sont une nouvelle classe d’oligonucléotides antisens (AON) plus performante que les chimies précédentes, et le système U7snRNA vectorisé, permettant d’induire une expression stable des séquences antisens. Dans un premier temps, ces différentes molécules ont été évaluées in vitro dans des lignées de fibroblastes de patients en quantifiant le niveau d’ARNm et de protéines htt par RTqPCR et Western blot respectivement. Par la suite, les séquences les plus efficaces in vitro ont été sélectionnées et les AON et AAV-U7snRNA correspondants ont été injectés en intracérébroventriculaire (ICV) dans un modèle murin de la MH (souris YAC128). Les résultats les plus encourageants ont été obtenus avec le TcDNA-NS (pour allèle Non Spécifique), permettant une diminution significative de l’expression de htt dans le cortex, l’hippocampe et le striatum 2 et 6 semaines après une injection ICV. Ces résultats prometteurs suggèrent le potentiel des TcDNA comme nouvel outil thérapeutique pour la MH. / Huntington’s disease (HD) is caused by a CAG repeat expansion in the exon 1 of huntingtin gene (htt), encoding for a mutant protein. It has been shown that the silencing/down regulation of huntingtin protein is a promising therapeutic lead. In this project, I have explored and compared three strategies using the antisense approach: a non-allele specific strategy, aiming to silence the global expression of htt; an allele specific strategy targeting CAG repeats to silence preferentially the mutant allele; and an exon-skipping strategy in order to remove cleavage sites which originally cause a shorter and toxic form of the htt protein. These strategies have been evaluated using two different tools: tricyclo-DNA (TcDNA), a new class of antisense oligonucleotides (AON) more efficient than the previous chemistries, and a vectorized approach using U7snRNA system allowing a stable expression of antisense sequences. Firstly, these different molecules have been assessed in vitro in HD fibroblasts quantifying mRNA and htt protein levels with RTqPCR and Western blot respectively. Subsequently, the most efficient sequences have been selected and intracerebroventricular (ICV) injections have been performed with corresponding AON and AAV-U7snRNA in a HD mouse model (YAC128). The most encouraging results have been obtained with the TcDNA-NS (for Non Specific allele), allowing a significant decrease of htt expression in cortex, hippocampus and striatum 2 and 6 weeks after ICV injection. These promising results suggest the potential of TcDNA as a new therapeutic tool for HD.

This thesis was focused on DNA analysis. The fluorescently labelled oligonucleotides were at first hybridised and subsequently analysed by HRM analysis to determine the melting temperatures of the oligonucleotides depending on the environment. This thesis describes the change of melting temperature of oligonucleotides in environments containing mono and bivalent ions and the influence of protein binding on the stability of these DNA structures. From determined melting points, it was specified whether the ion/protein stabilised or destabilised the oligonucleotide. Furthermore, plasmids were isolated and analyzed by atomic force microscopy.

Selon les estimations de l'OMS, le bilan mondial du paludisme a atteint 212 millions de cas et 429 000 décèsen 2015 (OMS, 2016). Cette gravité est principalement due à Plasmodium falciparum. A l’heure actuelle, P. falciparum présente des résistances à tous les antipaludiques donnés en monothérapie.Par conséquent, pour réduire le risque d’échec thérapeutique, l'OMS a recommandé depuis 2001 l’utilisation de bithérapie, notamment d'Artemisinin Combination Therapy (ACT), comme traitement de première intention.Les ACT sont composés essentiellement d’un dérivé d’artéminisine, à demi-vie courte et un autre antipaludique à demi-vie longue, connu en monothérapie.Le parasite a également montré des signes de résistance aux ACT, principalement en Asie du Sud-est, menaçant les programmes d’éradications contre le paludisme.La découverte de nouveaux composés à activité antipaludique ou de nouvelles procédures de traitement sont urgentes.La valorisation d’anciennes molécules est également au cœur des études afin d’améliorer notamment leur biodisponibilité et réverser les mécanismes de résistance du parasite. Ainsi, des études prouvent l’intérêt de l’utilisation de nanotechnologies pour l’amélioration de l’efficacité d’antipaludiques. L’atovaquone en est un exemple, cette modification a notamment permis d’améliorer sa biodisponibilité. Notre étude a porté sur une de ces formulations, de l’atovaquone encapsulée dans une nanoémulsion cationique (NE) appelée ATQ. Une deuxième génération a ensuite été testée par association d’oligonucléotides antisens anti-ARNm de topoisomérase II(AST) de P. falciparum. En effet, des stratégies antisens thérapeutiques font leur preuve en santé humaine et présentent un intérêt croissant en parasitologie. Les NE/AST ont montré une activité anti-palustre spécifique contre P. falciparum in vitro. Leur spécificité a permis d’aboutir à l’arrêt du cycle cellulaire et une forte diminution du taux d’ARNm de la topoisomérase II. Ce phénomène a montré être dépendant de l’action de la RNase H. Un effet synergique de ces NE/AST a également été montré en association avec la chloroquine, l’atovaquone et la dihydroartémisinine sur une souche sensible de P. falciparum et des souches résistantes aux antipaludiques précédemment cités.L’ATQ a également montré une forte efficacité sur une souche résistante à l’atovaquone d’un facteur 5. En présence d’ATQ, la mitochondrie a rapidement été altérée conduisant à une mort précoce du parasite. Un traitement à l’ATQ a abouti à la guérison de souris Swiss infectée par P. berghei après deux injections en i.v. en 5 jours. Enfin, l’ATQ/AST a montré une efficacité in vitro contre P. falciparum et P. berghei in vivo. Un test de cytoadhérance des hématies parasitées a des cellules endothéliales a révélé un fort pourvoir d’inhibition de la cytoadhérance de l’ATQ/AST.Un résultat prometteur dans le cadre de traitement du neuropaludisme. / According to the estimations of theWHO, in 2015, 212million cases ofmalariahave been reported(WHO,2016). These figuresmakemalariathe most deadlyparasitic diseasein the world, with429.000deaths per year. Some treatments against Plasmodium falciparum exist. However, no really good treatment option can be found in monotherapy due to the resistance emergency. Therefore To reduce the risk of resistance, WHO has recommended since 2001 combination therapies, which is basically an Artemisinin Combined Therapy (ACT), as first-line treatment. The main problem of commercialized bi-therapy is that they are composed of two molecules with individual resistance which leaded to the emergence of resistance to the latest ACTs such as a dihydroartemisinin /piperaquine combinationmainly in South-East Asia.Thus the use of new therapeutic combination strategy that can bypass the parasites' mechanisms of resistance is urgent to effectively treat malaria. As the pathway from drug discovery to drug commercialization is both long and very expensive, it is essential to develop ways to improve existing antimalarial treatments. In the first place it’s necessary to find a new antimalarial formulation based on an already commercialized drug to modify its biodisponibility and its mechanism of action in order to revert the resistance. In the second place its necessary to associate this formulation with a novel none commercialized antimalarial strategy such as the antisens oligonucleotides already usedinhumanhealth. In our lab we have developed nanoemulsions containing atovaquone and antisense oligonucleotides anti topoisomerase II against P. falciparum.Nanoemulsionsvectoringantisens oligonucleotidesandused againstP. falciparum topoisomerase II(NE/AST) showed encouraging anti-parasite killing results.Additionalresultshave showna synergistic in vitro effectwithantimalarial drugs(chloroquine, dihydroartemisinin and atovaquone) in sensitive and resistances strains. Moreover NE/ASTrestricted Topoisomerase II gene expression and blocked the cell cycle in G2/M phase leading to parasite’s death by mitophagy.As Drug delivery systemscan improve the efficacy ofcommon antimalarial drugs by delivering the drug to its target, while protecting it from degradation in biological environment and increasing its biodisponibility, our nanoemulsions containing atovaquone (ATQ) leaded to reversion of atovaquone resistance with 5 fold decrease in its IC50. Observations made with confocal microscopy have shown mitochondrial alteration after ATQ treatment.Our novel and original bi-therapy is focused on the association ofATQ with NE/AST (ATQ/AST).We obtained an IC50 8-fold lower than atovaquone’s IC50with total inhibition of parasites’ capacity to reinfect new red blood cells. A cytoadherence test of parasitized erythrocytes to endothelial cells revealed a strong capacity of cytoadherence inhibition of ATQ / AST, a promising result in the treatment of cerebral malaria.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24192号 / 医博第4886号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 森信 暁雄, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Due to the misuse of antibiotics, multi-drug resistant (MDR) bacteria have become more rampant in our society; these MDR have given rise to diseases that are not readily curable. One such agent is the Mycobacterium tuberculosis complex, which is a causative agent of tuberculosis (TB). Timely diagnostics of the bacterial infection and detection of bacterial drug-susceptibility profiles helps to initiate the necessary treatment in a timely fashion and to limit transmission of the disease. For more affordable detection of bacterial diseases, such as TN, tag-free split aptamer probes are advantageous. This proposal aims at designing split aptamer probes for detection of point mutations in the rpoB and katG genes of M. tuberculosis that are associated with resistance to two front-line antibiotics – rifampin and isoniazid, respectively, which causes MDR-TB. The probes will be designed and tested with synthetic oligonucleotide mimics of the bacterial genes in terms of their limit of detection and selectivity in discriminating the targets with single-nucleotide substitutions.

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Affinity molecules can serve as precision tools for selective recognition and measurement of specific biomolecules in the fields of therapeutic drug monitoring and quality control in recombinant protein production. In therapeutic drug monitoring, affinity molecules can enable the accurate quantification of drug concentrations within physiological fluids, enhancing both the safety and efficacy of clinical treatments. In the realm of recombinant protein production, these molecules can allow precise isolation and measurement of desired recombinant proteins from complex mixtures by selectively targeting specific protein tags or domains, ensuring the consistency and purity of protein products. Currently, antibodies are most commonly used affinity reagents in these fields but are limited by production complexity, batch variability, high cost, and low stability. Aptamers, known as ‘chemical antibodies’ but composed of nucleotides, are considered potential next-generation affinity reagents. Aptamers are obtained via a synthetic process, termed SELEX, of iterative affinity selection and polymerase chain reaction (PCR) amplification of target-binding members from a randomized oligonucleotide library. This process is traditionally labor and resource-intensive and time-consuming. In this thesis, microfluidic technology is employed to enable time-efficient and cost-effective generation of aptamers for monoclonal antibodies and recombinant proteins toward applications in therapeutic drug monitoring and quality control of recombinant protein production. This thesis starts with a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection (called “chip-selection SELEX”), and fully integrated microfluidic affinity selection and PCR amplification (termed “full-chip SELEX”). The comparison results indicate that chip-selection SELEX offers the lowest cost and highest efficiency in aptamer isolation. We then use chip-selection SELEX to streamline the process of isolating anti-idiotype aptamers targeting human monoclonal antibodies against spike protein of SARS-CoV-2 virus. The process is completed within only 5 rounds of SELEX within two days, which represented a significant improvement when compared to conventional methods whose completion generally requires more than 10 SELEX rounds in up to a month. These anti-idiotype aptamers are combined with a graphene-based affinity nanosensor to enable rapid antibody concentration measurements to inform therapeutic decisions in a timely manner. In addition, a microfluidic dual-aptamer sandwich assay with highly efficient isolation of aptamers is developed to enable rapid and cost-effective detection of tag-fused recombinant proteins. This approach addresses both the limitations of current dual-aptamer assays and commonly encountered difficulties in the lack of aptamers available for such assays, by first using chip-selection SELEX to generate aptamers and then employing these aptamers to implement a microfluidic dual-aptamer assay for quality control during recombinant protein production. Despite the high efficiency in aptamer isolation using chip-selection SELEX, the full-chip SELEX platform is still desired for minimal manual operation and reagent consumption. The current full-chip SELEX platform has low isolation efficiency and could not offer information of affinity selection process. Herein, by introducing asymmetric PCR into the full-chip SELEX process, we improve the efficiency in aptamer isolation and can successfully monitor the selection progress. This real-time monitoring capability allows us to identify the optimal point to terminate the SELEX process, preventing the potential loss of aptamer candidates and reducing the overall consumption of time and reagents. In addition, introducing solution phase-based asymmetric PCR addresses a notable technical challenge of on-chip PCR bead replenishment, toward complete automation of the full-chip SELEX platform. Furthermore, a holder equipped with connection pins is designed to enable the reversible connection between gold electrodes and electrical wires. This design promotes the reusability of gold electrode-deposited glass substrates, resulting in a substantial reduction in chip fabrication costs. In addition to the SELEX protocol development effort, we also present efficient and cost-effective microfluidic approaches for post-SELEX aptamer characterization, including aptamer identification and kinetic aptamer-target binding measurements. To mitigate the expensive and time-consuming nature of aptamer identification from SELEX-generated target-binding sequence pools, we present an approach that is based on a cost-effective and efficient procedure to generate modified single-stranded DNA copies of the aptamer candidates and then assess the affinity of the resulting modified ssDNA strands to target molecules. The approach is applied to identify aptamers from 12 candidates with consistent results, but at a cost three times lower than that of established methods. We also present a microfluidic fluorescence assay, which exploits a synergistic combination of microfluidic technology and fluorescence microscopy, to realize cost-effective and multiplexed measurement of kinetics of aptamer-target analyte binding without requiring special-purpose equipment.

DNA serves as a mode of storage for genetic information and carries essential biophysical functions which continue to gain importance for new biomolecular applications. The functionalization of proteins with nucleic acids has found several different uses in a variety of fields such as DNA origami due to the highly programmable nature of DNA. The modulation of key biomolecular properties of proteins by the conjugation of DNA nanotools, mechanical forces and potential catalytic modulators is an under discovered area of protein engineering. In this doctoral thesis work, functionalization strategies of proteins with DNA oligonucleotides, the catalytic mechanism of AdhD from Pyrococcus furiosus and effects of mechanical forces on enzymatic catalysis have been investigated through the utilization of DNA tools. In the first part of the thesis (Chapter 2), several bioconjugation strategies which can be used to attach DNA oligonucleotides to proteins have been reviewed and assessed for emerging biomolecular applications. This work provides insights about commonly used bioconjugation reactions and investigates the performance of each conjugation reaction in terms of yield, site- specificity, flexibility in conjugation position, steric hinderance, cost, reagent availability and risk of altering native protein properties. In the next section of the thesis (Chapter 3), DNA spring attachment sites are strategically modeled and created on the model enzyme for assigned bioconjugation strategies and the DNA springs are assembled on this model enzyme. The effect of the forces generated by the DNA springs during bulk catalysis is investigated and the changes in binding pockets and substrate specificity properties are demonstrated. A novel magnetic bead-based purification strategy for the separation of DNA spring conjugated enzyme is established in this work to ensure homogenous catalysis conditions. This construct allows the tuning of catalytic properties by the usage of different lengths of DNA and is shown to be reversible. As enzymatic catalysis is investigated under bulk conditions and the amino acid sequence within the active site is not altered, this strategy provides a new platform for the modulation of enzymatic trajectories without isolation and altered microenvironment limitations as well as the irreversible effects of conventional techniques such as mutagenesis and directed evolution. In the last part of the thesis (Chapter 4), the effects of different DNA constructs on AdhD are investigated. Convex DNA springs are constructed on AdhD molecules with the goal of applying compressive forces over the active site. In addition, DNA tweezers are designed to apply comparable forces to the DNA spring studied in Chapter 3, assembled on AdhD and purified using the same magnetic bead-based strategy. This construct, as discussed in Chapter 1, allows the modulation of substrate specificity profiles consistent with its mechanical design. As a continuation of the last part of the thesis, the rare earth element (REE) binding capacity of AdhD and the effect of metal binding on its enzymatic trajectory are investigated in Chapter 5. The rare earth element (REE) binding affinity of AdhD and its effect on enzymatic catalysis on both the forward and reverse reactions are demonstrated with and without the presence of a metal chelator. This discovery sheds light on the potential allosteric regulation mechanisms of AdhD and the catalytic regulator effect of REEs. The work presented in this doctoral thesis demonstrates different biomolecular approaches towards the modulation of enzymatic properties through DNA oligonucleotide conjugation, application of mechanical forces and potential catalytic regulators. In the future, the results presented in this work can be utilized to initiate in depth studies about protein-DNA conjugation and modulation of enzymatic catalysis, and discover extended applications which can be used universally across different biomolecular platforms.