No description available.

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.

<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

No description available.

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.

<p>This thesis is based on 9 papers dealing with the synthesis, physicochemical and biochemical properties of two types of chemically modified oligonucleotides which have the potential to down-regulate gene expression: (i) The first set is comprised of antisense oligonucleotides (AONs) conjugated with different chromophores of varying size, charge and π-electron density. Conjugation of the chromophores at the 3'- or 5'-end enhanced the target RNA binding affinity and RNase H recruitment capabilities compared to the native counterpart without changing the global helical conformation of their AON/RNA hybrid duplexes. The 3'-dipyridophenazine (DPPZ) has emerged as the most promising non-toxic chromophore in this series. (ii) The second set encompasses a new class of AONs containing <i>North</i>-<i>East</i> conformationally constrained 1',2'-oxetane-nucleosides. The introduction of oxetane-<b>T</b> and -<b>C</b> units imparts lowering of the T_m by ~ 6º and ~ 3 ºC/modification, respectively, of the AON/RNA hybrids, whereas the incorporation of the corresponding oxetane-<b>A</b> and-<b>G</b> units into AONs did not alter the thermostability in comparison with that of the native hybrid duplex. The oxetane-modified AONs have been found to possess enhanced serum stability compared to that of the native, whereas oxetane-<b>T</b> and -<b>C</b> containing AONs were more endonuclease-resistant than oxetane-<b>A</b> and-<b>G</b> modified AONs. All oxetane-modified mixmer AON/ RNA hybrid duplexes were, however, found to be excellent substrates for RNase H cleavage, which has been analyzed by Michaelis-Menten kinetics. The oxetane-modified mixmer AONs have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells, which was analyzed by QRT-PCR and Western Blot. Based on the amount of AON uptake after delivery, determined by slot blot, it was apparent that the oxetane-modified AONs are 5-6 times more effective antisense agents than the corresponding isosequential phosphorothioate analogues. The electrochemical assay based on sensitive nucleic acid mediated charge transport (CT) has revealed that the presence of oxetane-<b>T</b> unit causes more stacking perturbations in a DNA/DNA duplex than in a DNA/RNA duplex. </p>

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability. The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.

This thesis is based on 9 papers dealing with the synthesis, physicochemical and biochemical properties of two types of chemically modified oligonucleotides which have the potential to down-regulate gene expression: (i) The first set is comprised of antisense oligonucleotides (AONs) conjugated with different chromophores of varying size, charge and π-electron density. Conjugation of the chromophores at the 3'- or 5'-end enhanced the target RNA binding affinity and RNase H recruitment capabilities compared to the native counterpart without changing the global helical conformation of their AON/RNA hybrid duplexes. The 3'-dipyridophenazine (DPPZ) has emerged as the most promising non-toxic chromophore in this series. (ii) The second set encompasses a new class of AONs containing North-East conformationally constrained 1',2'-oxetane-nucleosides. The introduction of oxetane-<b>T</b> and -<b>C</b> units imparts lowering of the Tm by ~ 6º and ~ 3 ºC/modification, respectively, of the AON/RNA hybrids, whereas the incorporation of the corresponding oxetane-<b>A</b> and-<b>G</b> units into AONs did not alter the thermostability in comparison with that of the native hybrid duplex. The oxetane-modified AONs have been found to possess enhanced serum stability compared to that of the native, whereas oxetane-<b>T</b> and -<b>C</b> containing AONs were more endonuclease-resistant than oxetane-<b>A</b> and-<b>G</b> modified AONs. All oxetane-modified mixmer AON/ RNA hybrid duplexes were, however, found to be excellent substrates for RNase H cleavage, which has been analyzed by Michaelis-Menten kinetics. The oxetane-modified mixmer AONs have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells, which was analyzed by QRT-PCR and Western Blot. Based on the amount of AON uptake after delivery, determined by slot blot, it was apparent that the oxetane-modified AONs are 5-6 times more effective antisense agents than the corresponding isosequential phosphorothioate analogues. The electrochemical assay based on sensitive nucleic acid mediated charge transport (CT) has revealed that the presence of oxetane-<b>T</b> unit causes more stacking perturbations in a DNA/DNA duplex than in a DNA/RNA duplex.

La myopathie myotubulaire est une maladie musculaire congénitale très sévère. Le laboratoire d’accueil a démontré que les échantillons de muscle de patients atteints de cette maladie ainsi que le modèle murin présentent une surexpression de DNM2, alors que sa réduction par croisement génétique améliore les signes cliniques et histologiques de la maladie. Le but de ce travail consistait à développer, tester et valider des composés injectables qui ciblent DNM2 et diminuent son niveau. Deux approches thérapeutiques ont été développées l’une basée sur l’utilisation de virus adéno-associés (AAV) exprimant des shRNA, l’autre sur les oligonucleotides antisens (ASO). L’injection des vecteurs AAV-shDnm2 ou bien les ASO-Dnm2 pouvait corriger les défauts histologiques et fonctionnels des muscles des souris myopathes.Les résultats obtenus montrent le potentiel thérapeutique de la réduction de DNM2, et présente une nouvelle approche pour le traitement de la myopathie myotubulaire. / Myotubular myopathy is a severe muscle disease. We previously have shown that muscle specimens of both patients and the mouse model presented an overexpression of DNM2, while its genetic reduction prevents the development of the muscle phenotypes. The aim of this work was to develop, test and validate deliverable compounds. Two therapeutic approaches were used. Injection of antisense oligonucleotide or adeno-associated virus expressing shRNA restores a normal lifespan with improved muscle structure and function of the myopathic mice. These results demonstrate that therapeutic potential of reduction of DNM2 level and provides an attractive therapeutic strategy that could be applied to treat myotubular myopathy.