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Ribonuclease H1 is a conserved enzyme that is localized in the nuclear and mitochondrial compartments of eukaryotic cells, and functions in DNA replication, repair, recombination and transcription. (Arunachandran et al., 2000; Cerritelli et al., 2003) Oligonucleotide binding to a complementary RNA sequence can provide a substrate for RNase H1, and provides the mechanistic basis for antisense oligonucleotide (AON)-directed gene silencing in cells (Opalinska et al., 2002). Effective evaluation of the therapeutic efficacy of next-generation AONs with novel structures requires an in vitro system involving, purified, highly active RNase H1 of human cells, and a full understanding of the catalytic mechanism of the enzyme. The goal of project 1 described in chapter 3, was to determine the involvement of a conserved Histidine (H264) in the catalytic mechanism of human RNase H1. Based on this analysis I was able to conclude that H264 has a dual role in phosphodiester hydrolysis and in product release. The goal of project 2 (Chapter 4) was to examine the reactivities towards human RNase H1 of model hybrid substrates containing specific types of 2'-FANA substitutions (abbreviated as `F', with 2'-deoxyribose abbreviated as `D'), either at the "wings" of the molecule ("7-gapmer"; each wing=7 nt: FFFFFFF-DDDDDDD-FFFFFFF), or with 3 nt alternations ("3-altimer": FFF-DDD-FFF-DDD-FFF-DDD-FFF). The results of this study strongly support the continued examination of the potential therapeutic utility of the 2'-FANA modification in AONs. The highly efficient and selective inhibition of protein expression is a primary basis of action of most antisense therapeutic strategies. These data suggest that the 2'-FANA modification supports sustained silencing after a single administration, either by mRNA cleavage or by a translational block, and at substantially lower concentrations compared to the unmodified AON. The results of this project underscore the proposal that 2'-FANA-modified AONs will be important additions to the repertoire of rational antisense strategies for the effective treatment of disease. / Chemistry

Eicosapentaenoic acid (EPA, C20:5, n-3) is a long chain crucial unsaturated fatty acid, essential for the regulation of critical biological functions in humans. Its benefits include the therapeutic treatment of cardiovascular disease, schizophrenia and Alzheimer's disease. The fungus Pythium irregulare (ATCC 10951) has great potential as a natural EPA producer. In this study, the electroporation conditions for P. irregulare were determined. The auxotrophic selectable genes ura, trp and his were respectively cloned into the plasmid pESC to construct shuttle vectors. Electroporation with 2.0kV and a 0.2cm cuvette was applied as the most effective condition for heterogeneous genes transformation. The yield and content of EPA and other components of total fatty acids (TFA) were further determined by the FAME approach with GC, as well as the analysis of biomass. The EPA content in P. irregulare with heterologous pESC-TRP vector reached 16.68 mg/g if cultured in auxotrophic medium, which showed a 52.33% increase compared to the wild-type P. irregulare. The maximum of EPA yield was 98.52 mg/L from P. irregulare containing the pESC-URA plasmid, a 32.28% increase over the wild-type. However, the maximum cell dried weight of these two organisms were respectively 6.13g/L and 5.3g/L, significantly less than the 6.80g/L of the wild-type. Not only was a feasible approach detected to electro-transform and increase the EPA yield of P. irregulare, this study also inferred that Ï -6 route was mainly involved in the EPA biosynthesis in this organism. An antisense RNA (asRNA) thermodynamic model was developed to design new asRNA constructs capable of fine-tuning gene expression knockdown. The asRNA technology is now identified as an effective and specific method for regulating microbial gene expression at the posttranscriptional level. This is done by targeting mRNA molecules. Although the study of regulation by small RNAs is advanced in eukaryotes, the regulation of expression through artificially introducing antisense oligodeoxynucleotides into host is still being developed in prokaryotes. To study the thermodynamics of asRNA and mRNA binding, (i) the fluorescence protein genes GFP and mCherry were separately cloned into the common pUC19 vector and (ii) antisense GFP and antisense mCherry DNA fragments were randomly amplified and inserted into the constructed plasmid under the control of an additional plac promoter and terminator. The expression level of fluorescence reporter proteins was determined by plate reader in this combinatorial study. A thermodynamic model to describe the relationship between asRNA binding and observed expression level was created. The study indicates two factors that minimum binding energy of the asRNA-mRNA complex and the percentage of asRNA binding mRNA were crucial for regulating the expression level. The correlation relationship between gene expression level and binding percentage multiplied by the minimum binding energy was found to show a good correlation between the thermodynamic parameters and the observed level of gene expression. The model has the potential to predict the sequence of asRNA and the approach will ultimately be applied to cyanobacteria to increase lipids production. Here, the long-term approach is to build metabolic switches from asRNA that can turn "on/off" various cellular programs and metabolic pathways at will in a fine-tuned manner. This will allow engineers to control metabolic activity in response to reactor conditions. / Master of Science

Em 1978, o trabalho realizado por Stephenson e Zamecnik demonstrou a capacidade de um oligonucleotídeo de impedir a expressão de uma proteína específica. Atualmente, duas tecnologias são mais utilizadas para este propósito: os oligodeoxiribonucleotídeos antisense e o RNA de interferência (siRNA), que se aproveitam da capacidade de anelação entre as fitas complementares. A maior diferença entre as duas técnicas é a maquinaria proteica recrutada, isso é, o complexo RISC atua no funcionamento do siRNA, e a protease RNase H atua na clivagem da fita de RNA quando hibridizada com DNA. Apesar da grande aplicabilidade destas tecnologias, tanto para doenças metabólicas quanto para canceres, o veículo de entrega e proteção dessas sequências é de fundamental importância, visto que a aplicação desses oligonucleotídeos livres está sujeita à rápida degradação e ineficiência. A modificação das bases é uma das estratégias para conferir maior estabilidade às sequências, porém estas tem sido relacionadas a um aumento da toxicidade. Nessa dissertação, a quitosana, um polissacarídeo catiônico é utilizado para síntese de nanopartículas e encapsulamento dos oligodeoxiribonucleotídeos antisense (ASO). Para isso, foram realizadas modificações na quitosana comercial como despolimerização, trimetilação ou conjugação com PEG, seguida da síntese das nanopartículas com a adição de tripolifosfato de sódio (TPP) pelo método de gelatinização ionotrópica. A estabilidade das nanopartículas foi medida em função do tempo, da variação de temperatura e da diferença de pH. Além disso, a toxicidade dessas nanopartículas foi analisada através da viabilidade celular em diferentes linhagens, NB-4, HepaRG, HTC e BHK-570. A expressão da proteína verde fluorescente (GFP) na célula NB-4 foi utilizada para avaliar a entrega do ASO desenhado, sendo sua fluorescência monitorada por microscopia confocal. Os resultados demonstram que as nanopartículas se mantiveram estáveis durante o período de tempo analisado, assim como com a temperatura variando de 22 a 45°C e em pH ácido. Cada linhagem celular respondeu de forma diferente ao tratamento com as nanopartículas sem ASO, sendo a linhagem saudável BHK-570 com a maior resistência. Ademais, todas as células apresentaram viabilidade reduzida quando tratadas com concentrações na ordem de 1011 nanopartículas/mL a base de quitosana trimetilada. A fluorescência das células NB-4 quando tratada com as nanopartículas com ASO diminuiu consideravelmente nas 18 primeiras horas, seguida de um aumento após 42 horas. Dessa forma, pode-se concluir que as nanopartículas de quitosana propostas nessa dissertação apresentaram uma excelente alternativa para a entrega de material genético, principalmente para o trato gastro-intestinal, devido à sua estabilidade em pH ácido. / The property of an oligonucleotide to interfere in the expression of a protein was observed in 1978 by Stephenson and Zamecnik. To perform such interference, there are today, two main techniques being explored: antisense oligodeoxyribonucleotides and interference RNA. In both cases, the particularity of their chemical structure is taken into account as soon as they can bind in a complementary manner to the messenger RNA and inhibit its translation. The great difference between these techniques is related to the proteases involved in the process, while for interference RNA the RISC machinery acts, for antisense oligodeoxyribonucleotides RNase H cleaves the RNA in the duplex DNA-RNA. Although these tools to edit the translation process are relevant to the treatment and even cure of metabolic disorders and cancers, it is still not effective when employed without a coating to protect the sequences before it reaches the destiny in vivo. Efforts have been made in developing modified bases to be more stable, but they show some toxicity. In this dissertation, chitosan, a natural cationic polyssacharide, is used to produce nanoparticles to protect the antisense oligodeoxyribonucleotide (ASO). For this reason, the commercial chitosan was modified, depolymerized, trimetilated or PEGlated and the nanoparticles were synthesized with sodium tripolyphosphate (TPP) by ionotropic gelation method. The stability along time, in different pHs and temperatures was assessed. The toxicity of nanoparticles without ASO was quantified by MTT tests in NB-4, HepaRG, HTC and BHK-570 cell lines. A green fluorescent protein (GFP) expressed by NB-4 cells was the target to evaluate the delivery efficiency of the ASO, and its fluorescence was measured by confocal microscopy. Results showed that nanoparticles were stable over time as well as in temperatures ranging from 22 to 45°C and in acidic pH. Each cell line responded in a different manner to the treatment, with the health cell BHK-570 showing higher resistance. Furthermore, all of them presented lower viability when treated with trimetilated chitosan nanoparticles in the highest concentrations (ca 1011 nanoparticles/mL). NB-4 cells presented a decrease in fluorescence in 18 hours of treatment followed by an increase after 42 hours. We conclude that chitosan nanoparticles are a good alternative to the delivery of genetic material even more in the gastro intestinal tract due to its great stability in acid pH values.

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.

by Judy, Yuet-wa Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 107-111). / Acknowledgements --- p.i / Contents --- p.ii-iv / Abstract --- p.v-vii / Abbreviations --- p.ix / List of figures and tables --- p.x-xii / Chapter Chapter One: --- Introduction --- p.1-20 / Chapter 1.1 --- Facilitative Glucose Transporter Family (GLUT) / Chapter 1.2 --- Sequence and characterization of GLUT / Chapter 1.3 --- Overexpression of GLUT 1 in human cancer cells / Chapter 1.4 --- Inhibition of gene expression by antisense nucleic acid / Chapter 1.5 --- Types of antisense nucleic acids / Chapter 1.5.1 --- Nuclear expression of RNA by engineered antisense genes / Chapter 1.5.2 --- Antisense oligonucleotides / Chapter 1.6 --- Use of antisense oligomers in cell culture system / Chapter 1.7 --- Modification of antisense oligonucleotides / Chapter 1.8 --- Length and sequence selection of antisense oligomers / Chapter 1.9 --- Controls for measuring antisense effect / Chapter 1.10 --- Internalization and targeting of oligonucleotides / Chapter 1.11 --- Possible action mechanisms of antisense nucleotides / Chapter 1.12 --- Clinical applications of antisense approach / Chapter 1.13 --- Aim of the project / Chapter Chapter Two: --- Materials and Methods --- p.21-45 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Cell line and culture medium / Chapter 2.1.1a --- Cell line / Chapter 2.1.1b --- Culture medium / Chapter 2.1.2 --- Reagents and Buffers / Chapter 2.1.2a --- Phosphate-Buffered Saline (PBS) / Chapter 2.1.2b --- 50XTAE Buffer / Chapter 2.1.2c --- Tris-EDTA Buffer / Chapter 2.1.2d --- MTT solution / Chapter 2.1.2e --- Lipofectin Reagent / Chapter 2.1.3 --- Reagents for Northern Analysis / Chapter 2.1.3a --- DEPC-treated water (0.1% DEPC) / Chapter 2.1.3b --- 20X SSC / Chapter 2.1.3c --- 20X SSPE / Chapter 2.1.3d --- 10X Formaldehyde gel-running buffer / Chapter 2.1.3e --- Formaldehyde gel-loading buffer / Chapter 2.1.3f --- Prehybridization buffer / Chapter 2.1.3g --- Hybridization buffer / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Synthesis of oligonucleotides and phosphorothioated oligonucleotides / Chapter 2.2.2 --- Cloning of human GLUT 1 cDNA into pRc/CMV expression vector at sense and antisense orientation / Chapter 2.2.2a --- Primer designed for cloning of sense and antisense GLUT 1 cDNA / Chapter 2.2.2b --- Isolation of sense and antisense GLUT 1 clone by PCR / Chapter 2.2.2c --- Restriction Digestion / Chapter 2.2.2d --- Purification of Restriction Digested DNA / Chapter 2.2.2e --- DNA Ligation / Chapter 2.2.2f --- Preparation of competent bacterial cells for transformation / Chapter 2.2.2g --- Plasmid DNA Transformation / Chapter 2.2.3 --- Large scale preparation of plasmid DNA / Chapter 2.2.4 --- Formation of Lipofectin-encapsulated oligonucleotides / Chapter 2.2.5 --- [32P]-labeled oligonucleotides uptake assay / Chapter 2.2.6 --- Methods to monitor antisense effect / Chapter 2.2.6a --- MTT assay / Chapter 2.2.6b --- Northern Analysis / Chapter (i) --- Preparation of radiolabeled probe / Chapter (ii) --- Isolation of total RNA from HL-60 cells / Chapter (iii) --- Separation of total RNA by eletrophoresis and blotting onto a membrane / Chapter (iv) --- Prehybridization of the Northern blot / Chapter (v) --- Hybridization of the Northern blot / Chapter 2.2.6c --- [3H]-deoxyglucose uptake assay / Chapter Chapter Three: --- Results --- p.46-88 / Chapter 3.1 --- Synthesis of Oligonucleotides / Chapter 3.2 --- Multiple alignment of cDNA sequence of Glucose Transporter isoforms / Chapter 3.3 --- [32P]-labeled oligonucleotide uptake assay / Chapter 3.4 --- Antisense oligonucleotides designed against different regions of GLUT 1 cDNA sequence / Chapter 3.4.1 --- Effects on HL-60 cell proliferation / Chapter 3.4.2 --- Effects on GLUT 1 mRNA level / Chapter 3.5 --- The effects of different oligonucleotide concentrations on HL- 60cell proliferation / Chapter 3.6 --- The effects of modified oligonucleotides on HL-60 cell proliferation / Chapter 3.7 --- The effects of different oligonucleotide lengths on HL-60 cell proliferation / Chapter 3.8 --- [3H]-deoxyglucose uptake assay / Chapter 3.9 --- Cloning of sense and antisense GLUT 1 cDNA into pRc/CMV vector / Chapter 3.10 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter Chapter Four: --- Discussion --- p.89-106 / Chapter 4.1 --- Importance of GLUT 1 gene / Chapter 4.2 --- HL-60: the target cancer cell line / Chapter 4.3 --- "Importance of ""Antisense Approach""" / Chapter 4.4 --- Optimization of condition for antisense inhibition by oligonucleotides / Chapter 4.4.1 --- Oligonucleotide length / Chapter 4.4.2 --- Oligonucleotide Modification / Chapter 4.4.3 --- Sequence selection / Chapter 4.4.4 --- Uptake efficiency / Chapter 4.5 --- Intracelluar distribution of oligonucleotides / Chapter 4.6 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter 4.7 --- Mechanisms for antisense inhibition of gene expression / Chapter 4.8 --- Further Directions / References --- p.107-117