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Eingeschränkte Apoptose trägt zur Tumorentstehung und zur Entwicklung von Chemoresistenz bei, da die Apoptose normalerweise Zellen mit genetischen Schäden oder malignem Potential eliminiert. Dieser Prozess, der bereits für viele unterschiedlichen Tumorzellen nachgewiesen wurde, limitiert häufig die Behandelbarkeit maligner Erkrankungen und ist somit ein grosses Problem in der heutigen Krebsbehandlung. Es existieren unterschiedliche Ansätze die Auslöseschwelle für die Apoptose zu vermindern, um so Chemotherapie-resistente Tumorzellen zu eliminieren. Im ersten Teil dieser Arbeit wurde das anti-tumorale Potential des bispezifischen 4625 Antisense-Oligonukleotid in Kombination mit chemotherapeutischen Wirkstoffen in vitro und in vivo untersucht. Der zweite Teil beschreibt die Ergebnisse mit dem rekombinanten Ep-CAM spezifischen scFv Immunotoxin 4D5MOC-B-ETA in vitro und im Modell der Nacktmaus. Bcl-2 und Bcl-xL sind Inhibitoren der Apoptose, die von vielen malignen Tumorzellen überexprimiert werden. Das Herunterregulieren von Bcl-2 oder Bcl-xL erniedrigt die apoptotische Auslöseschwelle und Tumorzellen sterben durch programmierten Zelltod. Das 4625 Antisense Oligonukleotid richtet sich gegen eine Region hoher Homologie in der bcl-2/bcl-xL mRNA und hemmt simultan die Expression von Bcl-2 und Bcl-xL. Die durch das bispezifische 4625 Antisense gehemmte Expression von Bcl-2 und Bcl-xL in Tumorzellen unterschiedlicher Histologie zeigen die Ergebnisse der Immuno-Blots. Weiterhin führt 4625 zur dosisabhängigen Wachstumshemmung von Krebszellen bei Konzentrationen von 75-600 nM im MTT Assay. Für die Kombinationsbehandlung wurden Paclitaxel und 5-FU jeweils als Standardtherapie zur Behandlung von Brust- und kolorektalem Karzinom gewählt. Die ip. Applikation von 20mg/kg KG 4625 mit oder ohne Paclitaxel/5-FU führte zu einem verlangsamten Wachstum humaner Tumor Xenotransplantaten in Nacktmäusen, im Vergleich mit denen die mit dem Kontrolloligonukleotid 4626 mit oder ohne Chemotherapie behandelt wurden. Bcl-2 und Bcl-xL spielen unterschiedliche Rollen in der Tumorentwicklung und sind häufig heterogen in soliden Tumorgeweben exprimiert. Diese Daten zeigen, daß die moderne Antisense Technologie eine wirksame Methode zur Herunterregulierung zweier Hauptinhibitoren der Apoptose mit einem einzigen Oligonukleotid darstellt, wovon möglicherweise mehr Patienten mit malignen Erkrankungen in Zukunft profitieren könnten. Die Expression bestimmter Zelloberflächenmoleküle ist ein häufiger Prozess in vielen soliden Tumoren, was sie für eine zielgerichtete Antikörpertherapie angreifbar macht. Das epitheliale Glykoprotein-2 (Ep-CAM) wird reichlich von epithelialen Tumoren und Tumorzellinien exprimiert. Die antineoplastische Aktivität des Ep-CAM spezifischen 4D5MOC-B-ETA Immunotoxin wird im zweiten Teil dieser Arbeit beschrieben. In vitro hemmt 4D5MOC-B-ETA spezifisch die Proteinsynthese in Ep-CAM positiven Krebszellen unterschiedlichen histologischen Ursprungs ermittelt durch [H3]leucin Aufnahme und reduzierte die Überlebensrate dieser Zellen in Konzentrationen von 0.01 bis 1 pM. Ep-CAM negative Zellen wurden als negative Kontrolle genutzt und blieben durch das Immunotoxin in Konzentration bis zu 10.000 pM unversehrt, was dessen hochgradige Ep-CAM Spezifität beweist. Die tägliche Applikation von 0.01 mg 4D5MOC-B-ETA im Nacktmausmodell führte zu einem Schrumpfen der Tumor Xenotransplantate während der Behandlungszeit. Diese hohe Wirksamkeit des scFv Immunotoxin bedarf weiterer Beachtung in der zukünftigen Krebstherapie. / Impaired apoptosis contributes to cancer development and resistance towards chemotherapy, since apoptosis normally eliminates cells with damaged DNA or increased malignant potential. The increased resistance towards cell death often limits therapeutic options in the clinic and is one major problemin current tumor therapy. Different approaches, which have been described so far intend to lower the apoptotic threshold in order to eliminate chemoresistant cancer cells. In the first part of this thesis the anti-tumor potential of the bispecific 4625 oligonucleotide was investigated in combination with chemotherapeutic drugs in vitro and in vivo. The second part describes the anti tumor activity of the recombinant Ep-CAM specific scFv immunotoxin 4D5MOC-B-ETA in vitro and in nude mice. Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in malignant tumor cells. Downregulation of either Bcl-2 or Bcl-xL lowers the apoptotic threshold and tumor cells undergo apoptosis. The 4625 antisense oligonucleotide targets a region of high homology shared by the bcl-2/bcl-xL mRNAs and simultaneously downregulates Bcl-2 and Bcl-xL. The 4625 bispecific Antisense Oligonucleotide downregulates Bcl-2 and Bcl-xL expression in cancer cell lines of diverse histological origins assessed by immuno blotting. It further leads to proliferation inhibition of cancer cells at concentrations ranging from 75-600 nM in MTT assay in a dose-dependent manner. For combination experiments Paclitaxel and 5-FU were chosen as standard therapy for the treatment of breast and colorectal cancer, respectively. The ip. application of 20 mg/kg 4625 with or without Paclitaxel/5-FU led to a growth inhibition of established human carcinomas xenografts in nude mice, relative to those treated with the 4626 control oligonucleotide with or without chemotherapy. Bcl-2 and Bcl-xL play nonredundant roles in tumor growth and are often heterogeneously expressed in solid tumor tissues. This data suggests that state-of-the-art antisense technology offers a potent approach to inhibit the expression of the two major anti-apoptotic proteins Bcl-2 and Bcl-xL with one single oligonucleotide, which could make additional patients benefit from a treatment with this antisense compound. Expression of certain cell surface antigens is a common process in many solid tumors making them suitable for targeted antibody therapy. The epithelial glycoprotein-2 (Ep-CAM) is abundantly expressed on carcinomas and cancer cell lines. The anti tumor activity of the Ep-CAM specific 4D5MOC-B-ETA immunotoxin is described in the second part. In vitro 4D5MOC-B-ETA specifically inhibited protein synthesis in Ep-CAM positive cancer cells of diverse histological origin assessed by [H3]leucin incorporation and reduced cell viability with IC50 ranging from 0.01 to 1 pM. Ep-CAM negative cells were taken as control and were not harmed by the immunotoxin at concentrations up to 10.000 pM, which proves the 4D5MOC-B-ETA Ep-CAM specific potential. In athymic mice, the systemic application of 4D5MOC-B-ETA at a dose of 0.01 mg per day resulted in the regression of established tumor xenografts during the time of treatment. This highly potent anti-tumor activity of a recombinant scFv immunotxin deserves further attention for use in cancer therapy.

Une nouvelle approche thérapeutique est de moduler l'expression d'un gène en ciblant l'ARN messager par des oligonucléotides antisens et des siRNA. Nous avons étudié des formulations à base de lipides cationiques, formant des complexes particulaires avec les acides nucléiques (lipoplexes) et aux propriétés transfectantes. Nous avons l'unique possibilité dans notre laboratoire de formuler les oligonucléotides dans des lipoplexes soit chargés positivement, soit chargés négativement. Nous avons démontré qu'ils protégent et vectorisent efficacement les acides nucléiques dans les cellules in vitro ainsi in vivo. Ces vecteurs synthétiques possèdent de nombreux avantages : particules homogènes et de faible taille, reproductibilité, faible toxicité, stabilité au cours du temps et efficacité en présence de sérum. Nous avons appliqué ces vecteurs à la délivrance d'oligonucléotides antisens et de siRNA in vitro, respectivement dans un modèle de correction de l'épissage alternatif contenant l'intro aberrant de la B-thalassémie et dans un modèle de cancer du sein. Nos résultats démontrent une grande efficacité de délivrance des oligonucléotides via nos vecteurs lipidiques dans les 2 modèles, à faible concentration et en présence de sérum. Enfin, nous avons étudié le mécanisme d'entrée dans les cellules in vitro et évalué la proportion de transport actif (principalement endocytose), de transport passif (principalement fusion) et de fixation à la membrane des lipoplexes : celles-ci semblent dépendre du temps et du milieu de transfection, du type de formulation, de la charge ainsi que de la taille des complexes. Des études in vivo doivent être poursuivies pour évaluer l'efficacité et les propriétés respectives des différentes formulations développées, dans les modèles de correction de l'épissage alternatif et de cancer du sein.

The application of ICP-MS to the fields of proteomics and genomics has arisen in part due to its ability to detect and quantify trace levels of S and P, which are major constituents in proteins and nucleic acids respectively. The development of collision/reaction cell technology and high resolution instruments has enabled these biologically important elements to be measured and quantified at the pg - ng ml-1 level. Despite these advances, the detection limits of P and S are still inferior compared to other elements. Oligonucleotides containing biotin functionality were labelled with Au nano-particles attached to a streptavidin protein to achieve site specific labelling, with 100% labelling efficiency. Each nano-particle contained ~86 Au atoms, resulting in an 882 fold signal enhancement for 24 base length oligonucleotides. However, this enhancement factor was only observed when one oligonucleotide bound to one nano-particle in a 1:1 ratio. Much lower Au labelling efficiencies and signal enhancements were observed when thiolated oligonucleotides were labelled with maleimide functionalised gold nano-particles. This was attributed to the extensive and difficult sample preparation steps that were required prior to labelling. The detection and quantification of adducts formed between DNA and the Pt anti-cancer drugs cisplatin and oxaliplatin were also investigated with ICP-MS. Acid digestion of the carbon based DNA matrix enabled Pt adducts to be quantified at low dose rates of 1 Pt atom per 1 500 000 nucleotides in ~12 μg DNA. Such sensitive mass spectrometric determinations could be employed in clinical tests to detect and quantify low level adducts formed in patients in-vivo. To complement ICP-MS analysis, electrospray ionisation linear ion trap mass spectrometry was employed to study the interaction of oxaliplatin with the four DNA nucleobases. Multiple stage mass spectrometry enabled detailed Pt-nucleobase adduct fragmentation pathways to be established. The method of DNA detection using P in conjunction with the collision cell, or cool plasma to form PO+ was also demonstrated and the limitations of the method, namely, polyatomic interferences and severe matrix effects were highlighted.

L'épissage des ARN pré-messagers est une étape essentielle pour l'expression des gènes chez les eucaryotes supérieurs. La reconnaissance des exons par la machinerie d'épissage est réalisée grâce à différents éléments cis-régulateurs incluant les séquences consensus d'épissage et les séquences auxiliaires activatrices ou inhibitrices d'épissage. Le pré-ARNm représente une nouvelle cible thérapeutique pour le traitement des maladies génétiques. L'approche du saut d'exon thérapeutique, destinée à restaurer l'expression d'une protéine totalement ou partiellement fonctionnelle en interférant avec le processus d'épissage, suscite un grand intérêt notamment pour la dystrophie musculaire de Duchenne où la modification du transcrit permettrait d'obtenir une forme modérée de la maladie, la Dystrophie musculaire de Becker. Des oligonucléotides antisens sont utilisés pour masquer les signaux d'épissage de reconnaissance d'un exon par le spliceosome, et induire son excision (ou saut) du transcrit mature. La détermination de la meilleure séquence cible des AONs est une difficulté majeure de cette approche. Pour le gène DMD, nous avons pu établir grâce à des analyses bioinformatiques et statistiques combinées avec des tests fonctionnels utilisant des minigènes rapporteurs d'épissage, que le ciblage de motifs exoniques qui fixent le facteur d'épissage SF2/ASF permettait d'obtenir la meilleure efficacité des AONs. Par ailleurs, nous avons exploré la régulation de l'épissage des pseudoexons dans le gène DMD, et notamment les mécanismes conduisant à l'inclusion de ces séquences introniques dans le transcrit mature en condition pathologique. L'étude de deux cas exceptionnels d'activation de pseudoexons associée à des remaniements introniques rares (double délétion, inversion) élargit le spectre des mutations à l'origine de ces défauts d'épissage, et illustre le rôle encore mal connu des remaniements introniques en pathologie humaine. / Splicing of pre-messenger RNAs to mature transcripts is a crucial step in eukaryotic gene expression. The recognition of exon by the splicing machinery involves different cis-regulatory elements, including the splice site motifs and auxiliary sequences, which can act by stimulating or repressing splicing. The pre-mRNA represents a new therapeutic target for the treatment of genetic diseases. Notably, the exon skipping strategy is currently one of the most promising therapeutic approaches for the Duchenne muscular dystrophy. It intends to restore the expression of a partially functional protein by interfering with the splicing process, and converts the severe DMD phenotype into the moderate form of the disease, Becker muscular Dystrophy (BMD). Antisense oligonucleotides are used to mask the splicing signals involved in exon recognition by the spliceosome to induce its skipping from the mature transcript and restore an open reading frame. The determination of the best target sequence of the AONs is one of the major hurdles to overcome. For the DMD gene, a bioinformatic and statistical analysis combined with minigenes studies allowed us to establish that targeting binding sites for the splicing factor SF2/ASF maximizes the AONs efficiency. In a second part of this work, we investigated the splicing regulation of pseudoexons in the DMD gene, in particular the mechanisms leading to the inclusion of these intronic sequences in the mature transcript in pathological conditions. The study of two exceptional cases of pseudoexons activation associated with rare intronic rearrangements (double-deletions, inversion) expands the spectrum of missplicing mutations, and demonstrates the potential role of pure intronic rearrangements in human pathology.

Nanotechnology builds devices from the bottom up with atomic accuracy. Among the basic nano-components to fabricate such devices, semiconductor nanoparticle quantum dots (QDs), metal nanocrystals, proteins, and nucleic acids have attracted most interests due to their potential in optical, biomedical, and electronic areas. The major objective of this research was to prepare nano-components in order to fabricate functional nano-scale devices. This research consisted of three projects. In the first two projects, we incorporated two desirable characteristics of QDs, which are their abilities to serve as donors in fluorescence energy transfer (FRET) and surface energy transfer (SET) as well as to do multiplexing, to engineer QD-based nanoconjugates for optical and biomedical applications. Immobilizing luminescent semiconductor CdSe/ZnS QDs to a solid platform for QD-based biosensors offers advantages over traditional solution-based assays. In the first project, we designed highly sensitive CdSe/ZnS QD SET-based probes using gold nanoparticles (AuNPs) as FRET acceptors on polystyrene (PS) microsphere surfaces. The emission of PS-QD was significantly quenched and restored when the AuNPs were attached to and then removed from the surface. The probes were sensitive enough to analyze signals from a single bead and for use in optical applications. The new PS-QD-AuNP SET platform opens possibilities to carry out both SET and FRET assays in microparticle-based platforms and in microarrays. In the second project, we applied the QD-encoded microspheres in FRET-based analysis for bio-applications. QDs and Alexa Fluor 660 (A660) fluorophores are used as donors and acceptors respectively via a hairpin single stranded DNA. FRET between QD and A660 on the surface of polystyrene microspheres resulted in quenching of QD luminescence and increased A660 emission. QD emission on polystyrene x microspheres was restored when the targeted complementary DNA hybridized the hairpin strand and displaced A660 away from QDs. The third project involved fabrication of different nanoconjugates via self-assembly of template-based metal nanowires and metal nanoparticles using oligonucleotides as linkers. These nanoconjugates can serve as building blocks in nano-electronic circuits. The template method restricted the oligonucleotides attachment to the tip of the nanowires. Nanowires tagged with hybridizable DNA could connect to complementary DNA-modified metal crystals in a position-specific manner.

Malgré des progrès importants réalisés ces dernières années, la lutte contre les infections virales (SIDA, hépatites etc.) et les cancers demeurent un problème de santé mondiale. Ce bref bilan met en évidence la nécessité de développer de nouvelles molécules pour contourner les limites des traitements disponibles actuellement. Cette thèse, articulée autour de trois grands thèmes, s’inscrit dans ce contexte. Nous avons d’abord mis au point de manière rationnelle de nouveaux ligands d’ARN capables de se lier sélectivement à certaines structures secondaires de type tige-boucle ou tige-renflement de l’ARN TAR du VIH-1. Ces ligands interagissent avec l’ARN grâce à l’action coopérative de deux motifs de reconnaissance : (i) une nucléobase modifiée qui peut reconnaitre spécifiquement une paire de base de l’ARN et (ii) des acides aminés qui agissent avec les bases non appariées de l’ARN. Ces deux motifs sont reliés grâce à une matrice aliphatique (ligands non nucléosidiques) ou une matrice 2-désoxyribose (ligands nucléosidiques). Des études biophysiques et biologiques ont été menés en collaboration avec l’équipe du Dr. L. Briant (CEAPBS, UMR5236-CNRS) pour connaitre leur activité antivirale et leur site d’interaction sur la cible. Nous avons ensuite développé des molécules de type benzènesulfonamide thiazoles pour cibler le mélanome résistant aux inhibiteurs de B-Raf. Des modulations effectuées sur ce squelette nous ont permis d’établir des relations structure/activité, en collaboration avec l’équipe de Dr. S. Rocchi (C3M, INSERM U895). Enfin, nous avons développé une stratégie de modification post-synthétique d’oligonucléotides en position anomérique par réaction clic. / Despite significant progress made in recent years, the fight against viral infections (AIDS, Hepatitis, etc.) and cancer remains a global health problem. This brief summary underlines the need for new compounds in order to overcome the limitations of currently available drugs. To this end, the main objective of this thesis is to address these issues by the investigation of three major research projects. We first developed new RNA ligands that selectively bind to RNA secondary structures such as the stem-loop or the stem-bulge of HIV-1 TAR RNA. These ligands interact with RNA thanks to the presence of two RNA binding domains acting in a cooperative manner: (i) a modified nucleobase that can specifically recognize an RNA base pair and (ii) basic amino acids that interact with strong affinity with surrounding free RNA nucleobases. These two patterns are connected by an aliphatic matrix (non-nucleoside ligands) or a 2-desoxyribose matrix (nucleoside-based ligands). Biophysical and biological studies were conducted in collaboration with the team of Dr. L. Briant (CEAPBS, UMR5236-CNRS) in order to study their antiviral activity and their mode of action. We next developed new bioactive molecules featuring a thiazole benzenesulfonamide scaffold to target melanoma cells resistant to B-Raf inhibitors. The modular synthesis of a large number of analogs allowed us to establish the structure/activity relationships, in collaboration with the team of Dr. S. Rocchi (C3M, INSERM U895). Finally, we developed a straightforward and convenient strategy for post-synthetic modification of oligonucleotides at the anomeric position using click chemistry.

O objetivo deste trabalho foi estudar a utilização de uma nanoemulsão lipídica (LDE) como vetor de oligonucleotídeos antissenso (OAS). A LDE é uma nanoemulsão constituída por 48% de éster de colesterol, 47,8% de fosfolipídeos, 2,3% de triglicérides e 1,9% de colesterol não-esterificado. É capaz de adquirir apoE de HDL e, desta forma, a emulsão pode interagir com o receptor B/E. O comportamento metabólico da LDE se assemelha ao da LDL. OAS agem como inibidores da função de genes, ligando-se à fita oposta (complementar) do RNA mensageiro (mRNA) ou à dupla fita do DNA. Previnem que o mRNA codifique uma proteína funcional. Os mecanismos celulares de resistência a drogas representam diversas formas de proteção da célula e do organismo e estão presentes na maioria das células normais, exercendo funções fisiológicas. Infelizmente, muitos tumores utilizam esses mecanismos para sua própria proteção. A proteína codificada pelo gene MDR1 (ABCB1), a P-gp, é uma glicoproteína de membrana com peso molecular de 170Kda, que funciona como uma bomba orgânica catiônica. Neste trabalho foi observado que o OAS se ligam à LDE, sendo a constante de ligação de 4,2 X 10-3M-1. O complexo OAS/LDE foi capaz de se ligar especificamente ao receptor de LDL e através desta via ser internalizado, pelas células de sarcoma uterino resistentes a doxorrubicina. Os OAS apresentaram após 24 horas distribuição citoplasmática e nuclear e após 48 horas, somente distribuição citoplasmática. Utilizando-se dois diferentes OAS, verificou-se que ambos foram capaz de inibir (70%) a expressão do gene de resistência a múltiplas drogas após 48 horas de incubação, tornando as células mais susceptíveis à ação da doxorrubicina. Assim, o complexo OAS/LDE é um sistema potencialmente promissor para ser utilizado em terapia gênica. / The objective of this study was to evaluate the usefulness of a nanolipid emulsion (LDE) as a vector to carry antisense oligonucleotides (OAS). LDE is a nanoemulsion consisting of 48% cholesterol esters, 47,8% phospholipid, 2,3% triglycerides and 1,9% unesterified cholesterol. It is able to obtain apoE from HDL and interact with B/E receptor. The metabolic behavior of LDE is similar to LDL. OAS are able to inhibit specific gene expression since they bind to a complementary sequence in the mRNA or in the DNA. This binding impairs the synthesis of a functional protein. The cell resistance mechanisms are present in most of normal cells, been involved in physiological process. Tumors are able to use these mechanisms to their own protection. The protein P-gp (MDR1 gene) is a glycoprotein with 170Kda that works as an organic cationic pump. We have observed that LDE was able to bind to the OAS; the binding constant was 4,2 X 10-3M-1. The complex was shown to bind to LDL receptors and then been internalized into a human sarcoma cell line resistant to doxirrubicine. After 24 hours the complex have shown citoplasmatic and nuclear distribution, after 48 hours only citoplasmatic distribution was observed. Two OAS were used. Both OAS strongly inhibited (by 70%) the cell MDR-1 gene expression after 48 hours of incubation and cells turned out to be more susceptible to doxorrubicine action. Therefore, OAS/LDE is promising complex to be used in gene therapy studies.

Compostos carcinogênicos estruturalmente diversos ligam-se covalentemente ao DNA formando adutos que, se não reparados, provocam mutações. Inicialmente relacionados apenas a compostos exógenos, atualmente há várias evidências de que compostos gerados endogenamente poderiam modificar o DNA gerando adutos. Dentre os compostos endógenos, os produtos carbonílicos α,β-insaturados destacam-se pois reagem como agentes alquilantes bifuncionais com as bases do DNA, formando adutos exocíclicos. O 2,4-decadienal (DDE) é um aldeído α,β-insaturado que além de estar presente em alimentos e poluentes, é um dos mais importantes produtos da lipoperoxidação. Embora há várias indicações sobre a ação genótoxica do DDE, nenhum aduto deste composto com nucleobases havia sido caracterizado. O presente trabalho mostrou que o DDE é um agente alquilante versátil sendo capaz de gerar cinco adutos diferentes. Este estudo também mostrou que o DDE é capaz de gerar os mesmos adutos que outros dois importantes produtos da lipoperoxidação: o 4-0H-nonenal e o 2-octena1. Todos os adutos foram detectados em DNA tratado in vitro com o DDE. Para possibilitar a detecção dos adutos em sistemas mais complexos, foi desenvolvido um método extremamente sensível baseado em HPLC interfaceado com espectrometria de massa em tandem com ionização por electrospray. Foi também desenvolvida uma estratégia de incorporação de um dos adutos (III) em oligonucleotídeos por via química. A estratégia foi utilizada com sucesso na incorporação do aduto em oligonucleotídeos de sequências diversas. Os oligonucleotídeos foram utilizados em ensaios de reparo por excisão de base e replicação in vitro. / Structurally diverse carcinogenic compounds bind covalently to DNA producing adducts that can, if not repaired, lead to mutations. Firstly restricted to exogenous compounds, nowadays there are evidences that compounds endogenously generated can modify DNA forming adducts. Among these endogenous compounds, α,β-unsaturated carbonyl products are of special interest due their reactivity as bifunctional alkylating agents towards DNA bases leading to exocyclic adducts. 2,4-decadienal (DDE) is an α,β-unsaturatedaldehyde that in addition to be present in food and pollutants, it is one of the most important lipid peroxidation products. Despite of many indications about the DDE genotoxic properties, there was no information about adducts produced between this compounds and nucleobases. This work showed DDE as a versatile DNA alkylating agent being able to generate five different adducts. This study also showed that DDE can generate the same adducts produced from two other important lipid peroxidation products: 4-0H-nonenal and 2-octena1. All adducts were detected in DNA treated in vitro with DDE. To allow adduct detection in more complex systems, we developed a highly sensitive method based on HPLC coupled to electrospray/tandem mass spectrometry. A strategy to incorporate one adduct (III) into oligonucleotides was also developed. The strategy was successfully applied in the adduct incorporation into diverse sequences of oligonucleotides. They were utilized in base excision repair and in vitro replication experiments.

La dystrophie facio-scapulo-humérale (FSHD) est une maladie musculaire autosomique dominante rare. Cette pathologie est causée par la perte des marques épigénétiques répressives au macrosatellite D4Z4 en région subtélomérique du chromosome 4, ce qui conduit à la relaxation de la chromatine, l’expression aberrante du facteur de transcription DUX4 et la dérégulation de centaines de gènes. A ce jour, aucun traitement thérapeutique n’existe pour la FSHD. Le but de ce projet était de déterminer si cibler ou non par des oligonucléotides antisens (AOs) les séquences clés impliquées dans la polyadénylation des ARNm peut-être une stratégie thérapeutique pour inhiber l’expression de DUX4 chez les patients FSHD. En effet, le clivage et la polyadénylation en 3’ des ARNm sont des mécanismes fondamentaux de la maturation des ARNm nécessaires à leur export nucléaire, leur stabilité ou leur traduction efficace. Ces mécanismes représentent donc des cibles intéressantes pour une suppression de l’expression d’un gène dans des maladies à gain de fonction. Pour la première fois, nous avons pu montrer in vitro que l’utilisation d’AOs ciblant les séquences clés impliquées dans l’ajout d’une queue poly(A), notamment le signal de polyadénylation ou le site de clivage, conduit à une sous-expression de l’ARNm gène ciblé, et en particulier DUX4. Les AOs présentant in vivo une faible capacité à pénétrer les cellules et une forte clairance, les séquences des AOs les plus prometteurs ont été insérées dans un vecteur AAV sous promoteur U7. Les premiers résultats obtenus avec ces vecteurs sur un modèle murin sont prometteurs. Cette stratégie innovante apparait comme une option thérapeutique pour la FSHD. / FacioScapuloHumeral Dystrophy (FSHD) is a rare autosomal dominant neuromuscular disorder. This disease is caused by a loss of epigenetic marks within the D4Z4 macrosatellite located in the subtelomeric region of chromosome 4 leading to chromatin relaxation, aberrant expression of the DUX4 transcription factor and a cascade of gene deregulations. So far, there is no curative treatment for FSHD. The aim of this project was to determine whether or not targeting 3’-end key sequences involved in the polyadenylation of mRNA by antisens oligonucleotides (AOs) can be used as an efficient strategy for DUX4 gene silencing in FSHD. Indeed, cleavage and polyadenylation of the 3’-end of mRNAs are fundamental mechanisms of mRNAs maturation required for nuclear export, stability of the mRNAs and efficient translation and consequently could represent interesting targets for suppression of gene expression for gain of function diseases. For the first time, we demonstrated in vitro that targeting 3’-end key sequences involved in the addition of the poly(A) tail, such as the polyadenylation signal and the cleavage site, leads to an efficient extinction of the mRNA targeted and in particular DUX4. Because AOs have a weak cellular uptake and a rapid clearance in vivo, the sequences of the most promising AOs have been vectorised into an AAV vector under the control of the U7 promoter. The first results that we obtained with a FSHD mouse model treated with these vectors are promising. This innovating strategy appears as a therapeutic option for FSHD.