Tesi sul tema "Antisense nucleic acids"

Antisense oligonucleotides are short strands of DNA, which bind to their complementary mRNA target to prevent protein translation. Although conceptually appealing, for their practical use as drugs, these oligonucleotides must have better cellular uptake, resistance to enzymatic degradation, and target selectivity. In this work, new synthetic chemistry is established to prepare a novel group of chemically modified oligonucleotides. The anionic phosphodiester backbone is partially replaced with a neutral triazole and, at the same time, the 2'-position of the ribose sugar is functionalised with pyrene, anthraquinone, or guanidine moieties. Being unnatural, the triazole backbone is inherently resistant to enzymatic degradation, while the reduced negative charge potentially improves cell penetration. The limitation of introduction of a triazole backbone into the antisense strand is its destabilising effect on the duplex formation with their complementary target. In this study, the 2'-modifications are used to restore the lost duplex stability and they have been found to be very efficient stabilising moieties. To evaluate the viability of this strategy, reporter gene assays based on splice-switching model are used. Promisingly, these modified oligonucleotides have successfully shown antisense splice-switching activity, suggesting there is further scope for their improvement.

Antisense oligodeoxynucleotides can selectively inhibit individual gene expression provided they remain stable at the target site for a sufficient period of time. Thus, the efficacy of antisense oligodeoxynucleotides may be improved by employing a sustained release delivery system which would protect from degradation by nucleases whilst delivering the nucleic acid in a controlled manner to the site of action. Biodegradable polymer films and micro spheres were evaluated as delivery devices for the oligodeoxynucleotides and ribozymes. Polymers such as polylactide, polyglycolide, polyhydroxybutyrate and polyhydroxyvalerate were used due to their biocompatability and non toxic degradation products. Release profiles of antisense nucleic acids from films over 28 days was biphasic, characterised by an initial burst release during the first 48 hours followed by a more sustained release. Release from films of longer antisense nucleic acids was slower compared to shorter nucleic acids. Backbone type also affected release, although to a lesser extent than length. Total release of the nucleic acids is dependent upon polymer degradation, no degradation of the polymer films was evident over the 28 day period, due to the high molecular weight and crystallinity of the polymers required to make solvent cast films. Backbone length and type did not affect release from microspheres, release was generally faster than from films, due to the increased surface area, and low molecular weight polymers which showed signs of degradation over the release period, resulting in a triphasic release profile. An increase in release was observed when sphere size and polymer molecular weight were decreased. The polymer entrapped phosphodiester oligodeoxynucleotides and ribozymes had enhanced stability compared to free oligodeoxynucleotides and ribozymes when incubated in serum. The released nucleic acids were still capable of hybridising to their target sequence, indicating that the fabrication processes did not adversely effect the properties of the antisense nucleic acids. Oligodeoxynucleotides loaded in 2μm spheres had a 10 fold increase in macrophage association compared to free oligodeoxynucleotides. Fluorescent microscopy indicates that the polymer entrapped oligodeoxynucleotide is concentrated inside the cell, whereas free oligodeoxynucleotides are concentrated at the cell membrane. Biodegradable polymers can reduce the limitations of antisense therapy and thus offer a potential therapeutic advantage.

Peptide nucleic acids (PNA) are analogues of DNA that bind to DNA and RNA via Watson-Crick base-pairing rules. Due to the lack of a negatively-charged backbone, hybridisation of PNA to DNA or RNA occurs without electrostatic repulsion thus binding is typically stronger and more rapid than when traditional DNA probes are used. This is reflected in the increased melting temperature (Tm) of the conjugates. These properties, as well as the chemical and biological stability of PNA, make these molecules attractive for use in diagnostic and therapeutic applications. Amino acids are routinely conjugated to PNA probes to enhance the synthesis and solubility of the probes or assist with their cellular delivery, however little thought is given to the impact these modifications have on their hybridisation properties. In this work, a series of PNA-peptide chimeric assemblies based around a single PNA sequence were used to investigate the effect different amino acids have on the stability and specificity of PNA/DNA hybridisation. These experiments demonstrated that the positively charged amino acid lysine, which is routinely conjugated to PNA probes, increases the stability of the resultant PNA/DNA duplexes such that at many experimental temperatures, single base mismatched target DNA will also stably hybridise with PNA. In contrast, the negatively charged amino acid glutamic acid decreases the thermal stability of the mismatch duplexes sufficiently so that they are not stable at most experimental temperatures, whilst the fully complementary duplex is. This indicates that glutamic acid should replace lysine as the routine solubility enhancing group used for PNA probes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Thesis (Ph. D.)--West Virginia University, 2002.
Title from document title page. Document formatted into pages; contains xii, 161 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 140-158).

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Theses (Ph. D.)--Marshall University, 2004.
Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.

The discovery of RNA interference and the increasing understanding of disease genetics have created a new class of potential therapeutics based on oligonucleotides. This therapeutic class includes antisense molecules, small interfering RNA (siRNA), and microRNA modulators such as antagomirs (antisense directed against microRNA) and microRNA mimics, all of which function by altering gene expression at the translational level. While these molecules have the promise of treating a host of diseases from neurological disorders to cancer, a major hurdle is their inability to enter cells on their own, where they may render therapeutic effect. Nanotechnology is the engineering of materials at the nanometer scale and has gained significant interest for nucleic acid delivery due to its biologically relevant length-scale and amenability to multifunctionality. While a number of nanoparticle vehicles have shown promise for oligonucleotide delivery, there remains a lack of understanding of how nanoparticle coating and size affect these delivery processes. This dissertation seeks to elucidate some of these factors by evaluating oligonucleotide delivery efficiencies of a panel of iron oxide nanoparticles with varying cationic coatings and sizes. A panel of uniformly-sized nanoparticles was prepared with surface coatings comprised of various amine groups representing high and low pKas. A separate panel of nanoparticles with sizes of 40, 80, 150, and 200 nm but with the same cationic coating was also prepared. Results indicated that both nanoparticle surface coating and nanoparticle hydrodynamic size affect transfection efficiency. Specific particle coatings and sizes were identified that gave superior performance. The intracellular fate of iron oxide nanoparticles was also tracked by electron microscopy and suggests that they function via the proton sponge effect. The research presented in this dissertation may aid in the rational design of improved nanoparticle delivery vectors for nucleic acid-based therapy.

Thesis (Ph. D.)--Ohio State University, 2005.
Document formatted into pages; contains 375 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 9.

Les PNAs ciblant la région codante d’un ARN messager permettent par stratégie antisens de bloquer l’élongation de la traduction. Avant ce travail, seuls les PNAs riches en thymines et capables de former des triplexes avaient été démontrés capables d’une telle activité. Afin d’élargir la variété de séquences ciblées, nous avons montré que des PNAs polypyrimidiques, formant des duplexes ou des triplexes, arrêtent aussi efficacement l’élongation de la traduction. Ces PNAs AS ciblent la séquence polypurine tract du messager de la protéine intégrase du virus VIH-1. Nous avons montré que cette séquence, qui est composée de guanines contiguës, est engagée dans des structures très stables que les PNAs sont capables d’envahir. Ces mêmes PNAs présentent également une activité antisens spécifique dans des cellules humaines perméabilisées. En collaboration avec l’équipe de Gérard Chassaing1, nous avons évalué la capacité de différents peptides pénétrants à véhiculer ces PNAs dans les cellules. Nous avons sélectionné un transporteur peptidique RW9(RRWWRRWRR) qui permet au PNA transporté d’inhiber spécifiquement l’expression du messager cible et l’efficacité est presque identique à celle obtenue dans les cellules perméabilisées. De plus, des concentrations submicromolaires du conjugué PNA-RW9 sont suffisantes pour induire une inhibition et un transport efficace. Aussi, cette internalisation est rapide et aboutit à une forte localisation nucléaire du PNA, qui échappe à un emprisonnement vésiculaire. Ainsi, ce travail ouvre des perspectives importantes pour l’utilisation des conjugués PNA afin de générer des protéines tronquées ayant un rôle de dominants négatifs d’intérêt biologique
PNAs targeting the coding region of a messenger RNA allow, by antisense strtategy, to block translation elongation. Before this work, only thymine-rich PNAs able to form triplex, have been shown with such an activity. To enlarge sequence variety that can be targeted, we have shown that duplex and triplex-forming polypyrimidic PNAs are both able to block efficiently the elongation step. These PNAs target the polypurine tract sequence inside the coding region of the integrase protein mRNA of the HIV-1 virus, that is made of contiguous guanines, and is implicated in very stable secondary structures that PNAs can invade. These same PNAs have also an antisense activity in permeabilized human cells. In collaboration with Gérard chassaing’s group1, we assessed the ability of different Cell Penetrating Peptides (CPPs) to deliver PNAs inside human cells. We thus selected a very efficient peptidic carrier, named RW9 (RRWWRRWRR) that allow the carried PNA to inhibit messenger expression with an antisense activity efficiency that is quite similar to the one obtained in artificially permeabilized cells. Moreover, submicromolar concentrations of PNA-RW9 conjugate are sufficient to observe an efficient delivery and inhibition. This uptake is a rapid phenomenon that entails a strong nuclear PNA localization outside endosomal or lysosomal vesicles. Consequently, this work opens important outlooks for using PNA-CPPs conjugates to induce truncated proteins that can play a dominant-negative role with a therapeutic interest

Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of antisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecules with 18-mer units which show significant improvement in binding properties, this gives birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.

Les insuffisances des traitements actuels des infections à Virus de l’Hépatite B (VHB), orientent la recherche vers le développement de nouvelles stratégies antivirales. Ainsi, nous avons étudié à l’aide du modèle du VHB du canard, la capacité des PNAs (Peptide Nucleic Acids), une nouvelle classe de molécules antisens, à inhiber la réplication virale. Nous avons amélioré la pénétration intracellulaire des PNAs par leur couplage à des peptides perméabilisants (CPPs) et optimisé leur voie d’administration. Nous montrons que la voie intraveineuse est plus efficace que la voie intrapéritonéale. Aussi, nous démontrons pour la première fois que le PNA anti-epsilon couplé aux CPPs est capable d’inhiber la réplication virale in vitro et in vivo. Cependant, la spécificité de cette inhibition peut être affectée par l’activité antivirale de certains CPPs. Ces molécules pourraient représenter ainsi de nouveaux agents potentiellement intéressants pour les traitements des hépatites B chroniques
Giving the partial efficacy of nucleoside analogues, novel approaches against chronic hepatitis B virus (HBV) infection need to be developed. Thus, Peptide Nucleic Acid (PNAs), a novel generation of antisense agents, appears of particular value for HBV therapy. We have evaluated the capacity of the PNA to inhibit viral replication in vitro and in vivo in DHBV-infected duck model. Because the major problem of their therapeutic application is their poor intracellular penetration, we have used the PNA coupled to cell penetrating peptide (CPP). First, we have optimized the administration route and show that intravenous route led to a better liver delivery of PNA that intraperitoneal route. We provided here the first evidence that CPP-PNA conjugate and CPPs themselves inhibit viral replication suggesting their usefulness for HBV therapy. Our results also demonstrate that the choice of CPPs used as a vehicle for delivery plays an important role in the specificity and inhibition of viral replication

L'infection par le virus de l'hépatite C (VHC) demeure une cause majeure de cirrhose et de carcinome hépatocellulaire. Compte tenu d'une efficacité encore insuffisante des thérapies actuelles (55% de succès en moyenne), le développement de nouvelles stratégies antivirales reste une priorité. Dans ce contexte, nos travaux ont porté sur l'évaluation de nouveaux variants de l'interféron alpha dans un modèle de réplicon VHC. Nous avons pu identifier un variant, GEA007.1 qui présente une activité anti-VHC supérieure à celle de l'IFN alpha-2b utilisé en clinique associé à une plus grande efficacité de transduction du signal. Nous avons également évalué une approche antisens avec des molécules de type Peptide Nucleic Acids (PNA) et montré qu'elle permettait d'inhiber la traduction in vitro du VHC par blocage de la région interne d'entrée du ribosome (IRES), de manière plus efficace et spécifique qu'une approche antisens avec des molécules de premières générations. Une telle stratégie se justifie par la conservation de la cible qui pourrait se révéler utile avec l'utilisation prochaine des anti-protéases et anti-polymérases du VHC et le risque inhérent d'apparition de résistances au traitement. L'ensemble de ces résultats montre que l'amélioration de la bithérapie actuelle est possible et que de nouvelles approches thérapeutiques fonctionnent.

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

Bibliography: leaves 175-186.
186 leaves : ill. (some col.) ; 30 cm.
Aims to examine the AP in A. thaliana and to produce an inducible antisense plant to assist future studies of the role of AOX.
Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1999?

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

Chung Ka Wing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 151-162).
Abstracts in English and Chinese.
Contents --- p.i
Acknowledgements --- p.v
Abstract --- p.vi
論文摘要 --- p.ix
List of Abbreviations --- p.xi
List of Figures --- p.xiii
List of Tables --- p.xv
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Breast Cancer --- p.2
Chapter 1.1.1 --- Incidence Rate of Breast Cancer --- p.2
Chapter 1.1.2 --- Risk Factors Lead to Breast Cancer --- p.5
Chapter 1.1.3 --- Conventional Treatments --- p.5
Chapter 1.2 --- Relationship between Breast Cancer and Glucose Transporters --- p.7
Chapter 1.2.1 --- Importance of Glucose and Fructose --- p.7
Chapter 1.2.2 --- Facilitative Glucose Transporters (Gluts) and The Relationship with Breast Cancer --- p.7
Chapter 1.3 --- Antisense Oligonucleotides --- p.13
Chapter 1.3.1 --- Characteristics of Antisense Oligonucleotides --- p.13
Chapter 1.3.2 --- Action Mechanism of Antisense Oligonucleotides --- p.15
Chapter 1.3.3 --- Sequence Selection --- p.19
Chapter 1.3.4 --- Chemical Modifications of Antisense Oligonucleotides --- p.20
Chapter 1.3.5 --- Uptake and Delivery Means of Antisense Oligonucleotides --- p.24
Chapter 1.4 --- Objectives of Present Study --- p.26
Chapter Chapter 2 --- Materials and Methods --- p.31
Chapter 2.1 --- Materials --- p.32
Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.32
Chapter 2.1.2 --- Buffers and Reagents --- p.33
Chapter 2.1.3 --- Reagents for Transfection --- p.34
Chapter 2.1.4 --- Reagents for D-[U14C]-Fructose and 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.35
Chapter 2.1.5 --- Reagents for ATP Assay --- p.35
Chapter 2.1.6 --- Reagents for RT-PCR --- p.36
Chapter 2.1.6.1 --- Reagents for RNA Extraction --- p.36
Chapter 2.1.6.2 --- Reagents for Reverse Transcription --- p.36
Chapter 2.1.6.3 --- Reagents for Gel Electrophoresis --- p.37
Chapter 2.1.7 --- Reagents for Real Time-PCR --- p.38
Chapter 2.1.8 --- Reagents and Chemicals for Western Blotting --- p.39
Chapter 2.1.8.1 --- Reagents for Protein Extraction --- p.39
Chapter 2.1.8.2 --- Reagents for SDS-PAGE --- p.39
Chapter 2.1.9 --- Reagents for Flow Cytometry --- p.42
Chapter 2.1.10 --- In Vivo Study --- p.43
Chapter 2.2 --- Methods --- p.44
Chapter 2.2.1 --- Oligonucleotide Design --- p.44
Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.47
Chapter 2.2.3 --- Transfection --- p.47
Chapter 2.2.4 --- MTT Assay --- p.47
Chapter 2.2.5 --- D-[U14C]-fructose and 2-deoxy-D-[l-3H] Glucose Uptake Assay --- p.48
Chapter 2.2.6 --- Detection of Intracellular ATP Concentration --- p.49
Chapter 2.2.7 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.51
Chapter 2.2.7.1 --- RNA Extraction by TRIzol Reagent --- p.51
Chapter 2.2.7.2 --- Determination of RNA Concentration --- p.51
Chapter 2.2.7.3 --- Reverse Transcription --- p.52
Chapter 2.2.7.4 --- Polymerase Chain Reaction (PCR) --- p.52
Chapter 2.2.8 --- Real-Time PCR --- p.55
Chapter 2.2.8.1 --- Analysis of the Real-Time PCR Data --- p.57
Chapter 2.2.9 --- Western Blot Analysis --- p.58
Chapter 2.2.9.1 --- Protein Extraction --- p.58
Chapter 2.2.9.2 --- Protein Concentration Determination --- p.58
Chapter 2.2.9.3 --- Western Blotting --- p.60
Chapter 2.2.10 --- Flow Cytometry --- p.62
Chapter 2.2.10.1 --- Detection of Cell Cycle Pattern with PI --- p.62
Chapter 2.2.10.2 --- Detection of Apoptosis with Annexin V/PI --- p.62
Chapter 2.2.11 --- In Vivo Study --- p.63
Chapter 2.2.11.1 --- Establishment of Tumor-Bearing Animal Model --- p.63
Chapter 2.2.11.2 --- Treatment Schedule --- p.63
Chapter 2.2.11.3 --- Toxicity of Antisense Oligonucleotides --- p.64
Chapter Chapter 3 --- Results --- p.66
Chapter 3.1 --- In Vitro Study --- p.67
Chapter 3.1.1 --- Effect of Tamoxifen on MCF-7 cells and MDA-MB-231 cells --- p.67
Chapter 3.1.2 --- Cytotoxicity of Antisense Oligonucleotides against Glut 5 on MCF-7 cells and MDA-MB-231 cells by MTT Assay --- p.69
Chapter 3.1.3 --- Effect of Antisense Oligonucleotides against Glut 5 on Fructose and Glucose Uptake of MCF-7 cells and MDA-MB-231 cells by D-[U14C]-Fructose & 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.77
Chapter 3.1.4 --- Effect of Antisense Oligonucleotides against Glut 5 on Intracellular ATP Content of MCF-7 cells and MDA-MB-231 cells by ATP Assay --- p.81
Chapter 3.1.5 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 RNA Expression of MCF-7 cells and MDA-MB-231 cells by RT-PCR and Real-Time PCR --- p.83
Chapter 3.1.5.1 --- RT-PCR --- p.83
Chapter 3.1.5.2 --- Real-Time PCR --- p.87
Chapter 3.1.6 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 Protein Expression of MCF-7 cells and MDA-MB-231 cells by Western Blot Analysis --- p.89
Chapter 3.1.7 --- "Effect of Antisense Oligonucleotides against Glut 5 on Change in Cell Cycle Pattern of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using PI Stainning" --- p.93
Chapter 3.1.8 --- "Effect of Antisense Oligonucleotides against Glut 5 on Induction of Apoptosis of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using Annexin V-FITC Stainning" --- p.98
Chapter 3.2 --- In Vivo Study --- p.101
Chapter 3.2.1 --- Animal Model: Nude Mice --- p.101
Chapter 3.2.2 --- Effect of Antisense Oligonucleotides against Glut 5 on the MCF-7 cells-Bearing Nude Mice --- p.101
Chapter 3.2.2.1 --- Change of Weight of the Tumor-Bearing Nude Mice --- p.101
Chapter 3.2.2.2 --- Tumor Growth Rate --- p.105
Chapter 3.2.2.3 --- Glut 5 RNA Expression by Real-Time PCR --- p.109
Chapter 3.2.2.4 --- Glut 5 RNA Expression by Western Blotting --- p.111
Chapter 3.2.3 --- "Assessment of Side Effects of Antisense Oligonucleotides against Glut 5, by Measuring the Plasma Enzyme Level" --- p.113
Chapter Chapter 4 --- Discussion --- p.118
Chapter 4.1 --- Antisense Oligonucleotides against Glut 5 on Human Breast Cancer --- p.119
Chapter 4.1.1 --- Antisense Oligonucleotides Strategy --- p.119
Chapter 4.1.2 --- Role of Glut 5 in Breast Cancer --- p.123
Chapter 4.1.3 --- Effects of Tamoxifen on MCF-7 and MDA-MB-231 --- p.126
Chapter 4.2 --- In Vitro Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.127
Chapter 4.3 --- In Vivo Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.135
Chapter 4.3.1 --- Effects of Antisense Oligonucleotides against Glut 5 on Body Weight and Tumor Size --- p.137
Chapter 4.3.2 --- Expression Level of Glut 5 of the Tumor --- p.138
Chapter 4.3.3 --- Assessment of Side Effects of Antisense Oligonucleotides against Glut 5，by Measuring the Plasma Enzymes Level --- p.140
Chapter 4.4 --- Possible Mechanism of Antisense Oligonucleotides against Glut 5 on Breast Cancer --- p.141
Chapter Chapter 5 --- Future Prospectus and Conclusions --- p.143
Chapter 5.1 --- Future Prospectus of Antisense Oligonucleotides --- p.144
Chapter 5.1.1 --- Antisense Oligonucleotides and Treatment of Breast Cancer --- p.144
Chapter 5.1.2 --- Role of Glut 5 in Breast Cancer --- p.147
Chapter 5.2 --- Conclusions and Remarks --- p.148
References --- p.151

Cette thèse décrit deux thèmes principaux: 1) la conception, la synthèse, et l'évaluation biophysique des nucléosides tricycliques, et 2) la synthèse de nagilactone B, un produit naturel norditerpenoïde dilactone de la famille de produits naturels “podolactone”. Le premier chapitre décrit la stratégie de design rationnel des nucléosides nommé “restriction conformationnelle double” basée sur les études de modélisation structurales des duplex ADN–ARN modifiés. Cette stratégie implique un blocage du cycle furanose dans une configuration de type N- ou S, et une restriction de la rotation torsionelle autour de l’angle γ. La première contrainte a été incorporée avec un pont méthylène entre l’oxygène en position 2′ et le carbone 4′ du nucléoside. Cette stratégie a été inspirée par les acides nucléiques bloqués (ou “locked nucleic acid”, LNA). La deuxième contrainte a été réalisée en ajoutant un carbocycle supplémentaire dans l'échafaud de l’acide nucléique bloqué. Les défis synthétiques de la formation des nucléotides modifiés à partir des carbohydrates sont décrits ainsi que les améliorations aux stabilités thermiques qu’ils apportent aux duplex oligonucléïques dont ils font partie. Chapitres deux et trois décrivent le développement de deux voies synthétiques complémentaires pour la formation du noyau de nagilactone B. Ce produit naturel a des implications pour le syndrome de Hutchinson–Gilford, à cause de son habilité de jouer le rôle de modulateur de l’épissage d’ARN pré-messager de lamine A. Ce produit naturel contient sept stereocentres différents, dont deux quaternaires et deux comprenant un syn-1,2-diol, ainsi que des lactones à cinq ou six membres, où le cycle à six ressemble à un groupement α-pyrone. La synthèse a débuté avec la cétone de Wieland-Miescher qui a permis d’adresser les défis structurels ainsi qu’explorer les fonctionnalisations des cycles A, B et D du noyau de nagilactone B.
The present thesis comprises two major themes: 1) the design, synthesis, and biophysical evaluation of conformationally restricted tricyclic nucleosides for antisense applications, and 2) strategic approaches for synthesizing the core of nagilactone B, a norditerpenoid dilactone from the podolactone family of natural products. Guided by structural studies of modified DNA–RNA duplexes, Chapter One focuses on a proposed dual-conformational-restriction strategy, in which two modes of conformational restriction are incorporated into a single nucleotide modification: 1) locking the furanose ring in an N- or S-type configuration and 2) restricting rotation around backbone torsion angle γ. The first constraint was incorporated by way of a 2′,4′-anhydro bridge that is found in the scaffold of locked nucleic acid (LNA), while the second was realized by annealing an additional carbocyclic ring to the modified nucleoside. The synthetic challenges associated with preparing these highly constrained molecules from carbohydrate-derived starting materials are described, in addition to the corresponding improvements in duplex thermal stability they provide to oligonucleotide sequences containing them. Chapters Two and Three describe complementary approaches for the synthesis of the core of nagilactone B, a natural product with implications for Hutchinson–Gilford progeria syndrome, as a consequence of its ability to act as a modulator of splicing events leading to lamin A. This natural product contains seven stereogenic centers overall, including a syn-1,2-diol moiety, a γ-lactone, and a pair of quaternary stereocenters, which are complemented by the presence of an α-pyrone moiety. To address the synthesis of these structural features, the utility of the Wieland–Miescher ketone was explored with an emphasis on synthesizing rings A, B, and D of the core of nagilactone B.

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015
Epigenética é a área da genética que se foca no estudo das alterações biológicas da célula que não envolvem alterações na sequência de nucleótidos do DNA. Um dos componentes da epigenética que tem vindo a ganhar interesse na comunidade científica são os RNAs longos não codificantes (do inglês long noncoding RNAs - lncRNAs) que são transcritos que contém mais de 200 nucleótidos. Estes não possuem quadros de leitura abertos (do inglês open reading frames – ORFs) e desempenham papéis biológicos importantes em diferenciação celular, pluripotência, regulação da transcrição, processamento e tradução de moléculas de RNAs. Têm sido também muito associados ao desenvolvimento de cancro, nomeadamente, na progressão tumoral e desenvolvimento de metáteses. Várias classes de lncRNAs têm sido descritas tendo em conta, maioritariamente, a localização destes transcritos no genoma em relação a transcritos com potencial codificante, os RNAs mensageiros (mRNAs). Uma classe de lncRNAs com interesse neste projecto é a dos transcritos anti-direccionais naturais (do inglês natural antisense transcripts – NAT). Estes transcritos têm a particularidade de serem codificados na cadeia anti-direccional de genes que codificam mRNAs, podendo haver sobreposição parcial com a região promotora ou com intrões. Pensa-se que poderão estar implicados na regulação da transcrição dos genes codificantes de mRNA ou na regulação da remoção dos intrões (splicing). O presente trabalho é parte de um projecto que tem como objectivo principal investigar a biologia dos lncRNAs no contexto do desenvolvimento de leucemia. Embora já exista evidências recentes que destacam a importância de lncRNAs na regulação da expressão genética, pouco se sabe sobre o seu papel na diferenciação das células T e na transformação leucémica. O objectivo final do projecto em si, é encontrar possíveis alvos para terapias direccionadas a moléculas de RNA em células cancerígenas de Leucemia Linfoblástica Aguda de células T (do inglês T-cell Acute Lymphoblastic Leukaemia - T-ALL). A metodologia proposta neste projecto combina técnicas de alta resolução de epigenómica, transcriptómica e biologia molecular com abordagens para monitorizar a síntese, tempos de semi-vida e localização sub-celular de lncRNAs. A análise está focada em precursores de células T primárias purificadas a partir de tumores do timo de ratinho e em modelos celulares de T-ALL de ratinho. O factor que diferencia e dá o carácter leucémico a estas células é a sobreexpressão do oncogene TLX3 que é considerando um dos genes mais mutados neste tipo de leucemias. No entanto, estudos anteriores mostraram que, por si só, esta sobreexpressão do oncogene não é suficiente para induzir a T-ALL, deste modo, poderão existir outros factores, tais como lncRNA, que estejam envolvidos no desenvolvimento da T-ALL. No âmbito deste estudo, foi seleccionado a partir da literatura uma lista de lncRNAs que são expressos em células T e podem ser relevantes no contexto da leucemia. Para monitorizar o tempo de semi-vida de lncRNAs realizou-se marcação do RNA nascente nas células com um pulso de incorporação (do inglês, pulse labelling) do análogo do uracilo, 4-thioridine (4sU), que é incorporado em todo o RNA sintetizado na célula durante esse pulso de marcação. Segue-se a extracção de todo o RNA da célula e purificação dos RNAs nascente marcados com 4sU, e dos pré-existentes e dos não marcados. A quantificação por RT-qPCR dos lncRNAs de interesse nas diferentes populações de RNA permite o cálculo da semi-vida desses lncRNA. Os resultados obtidos neste trabalho corroboram os dados já conhecidos de outras investigações dando validade e eficácia da técnica experimental executada. Para determinar a localização sub-celular de lncRNAs foi desenvolvido um ensaio de hibridação in situ de fluorescência com base em sondas de LNA e amplificação do sinal de hibridação das sondas com base em sistemas que utilizam métodos enzimáticos associados a fluorescência. Neste caso, os resultados não foram conclusivos, precisando esta técnica experimental ser melhorada e optimizada. Os lncRNAs que serão analisados por estes ensaios, no futuro, serão fornecidos por meio de análise bioinformática do transcritoma de células T em diferentes fases de transformação leucémica. No final deste estudo iniciou-se esta análise bioinformática com dados de sequenciação de RNA (RNA-seq) obtidos de células T não transformadas e de células T em diferentes fases de transformação leucémica Nesta análise pretendeu-se ter uma ideia geral dos lncRNAs e mRNAs que se encontram diferencialmente expressos entre fases diferentes de transformação leucémica. Os resultados preliminares desta análise sugerem que existe uma percentagem maior de mRNAs diferencialmente expressos do que lncRNAs, quando se comparam células não transformadas com células em diferentes fases de transformação leucémica. O objectivo é identificar entre os lncRNAs diferencialmente expressos aqueles que poderão ser relevantes na transformação leucémica. Estes serão alvo de estudos funcionais utilizando as técnicas optimizadas neste estudo, de modo a que se perceba o seu mecanismo de acção e se possa avaliar se têm potencial para ser alvos para terapias direccionadas.
Epigenetics is the field of genetics that studies the alterations in the transcriptional potential of a cell without interfering with the DNA sequence. One of its component is the long noncoding RNAs (lncRNAs) which are transcripts with more than 200 nucleotides and no evident open reading frames (ORFs) that play important biological roles like transcription and splicing regulation and have been associated with carcinogenesis. Several classes of lncRNAs have been described according to their genomic location in relation to protein‐coding genes. The present work is part of a project aiming at gaining novel insights into the biology of lncRNAs in the context of leukemogenesis. Although recent evidence highlights the importance of lncRNAs in regulation of gene expression, little is known about their role in T-cell differentiation and leukaemic transformation. The ultimate goal of the project is finding possible targets for RNA therapeutics in T-cell Acute Lymphoblastic Leukaemia (T-ALL). The proposed methodology combines high-throughput epigenomics, transcriptomics and systems biology approaches with techniques to monitor synthesis, lifetime and sub-cellular localization of lncRNAs. The analysis is focused on primary T-cell precursors purified from the mouse thymus and on cellular mouse models of T-ALL. The present study aims to develop functional assays to monitor lifetime and sub-cellular localization of lncRNAs in a cellular mouse model of T-ALL. To monitor the lifetime of lncRNAs we carried out pulse labeling with the uridine analogue 4-thioridine (4sU) followed by purification of labeled nascent, pre-existing unlabeled and total cellular RNAs. RT-qPCR quantification of the RNA subsets allows the estimation of the lncRNA’s half-life. To determine the sub-cellular localization of lncRNAs we developed a fluorescence in situ hybridization assay based on LNA probes and enzyme-based signal amplification. In this study we selected from the literature a list of lncRNAs that are expressed in T‐cells and may be relevant in leukemogenisis. The candidate lncRNAs that will be analysed by these assays in the future will be provided by genome-wide transcriptomic analysis of different stages of T-cell leukemic transformation.

Światowa Organizacja Zdrowia (WHO) umieściła zakażenia powodowane przez antybiotykooporne bakterie Escherichia coli na pierwszym miejscu listy chorób zakaźnych, których zwalczaniu oraz zapobieganiu należy nadać najwyższy priorytet. Pojawienie i rozprzestrzenienie się bakteryjnych szczepów antybiotykoopornych jest istotnym problemem, którego rozwiązanie wymaga zastosowania nowatorskich strategii przeciwbakteryjnych. Syntetyczne antysensowne oligonukleotydy, stosowane do zahamowania procesu syntezy białek niezbędnych do życia bakterii, mogą okazać się pomocne w zwalczaniu zakażeń bakteryjnych. Peptydowe kwasy nukleinowe (PNA) są syntetycznymi analogami kwasów nukleinowych, które zawierają neutralny szkielet, są odporne na działanie enzymów degradujących kwasy nukleinowe i białka oraz tworzą stabilne kompleksy zarówno z DNA, jak i RNA. Celem mojej rozprawy doktorskiej było (a) zbadanie potencjału naturalnych bakteryjnych systemów toksyna-antytoksyna (TA) jako nowych celów dla związków indukujących efekt przeciwbakteryjny oraz (b) opracowanie skutecznej i nieinwazyjnej metody wprowadzania krótkich, modyfikowanych oligonukleotydów do komórek bakterii. W pracy opisano trzy strategie związane z wykorzystaniem systemów TA (i powiązanych z nimi białek) jako celów dla cząsteczek antybakteryjnych. Pierwsza z nich zakładała bezpośrednią aktywację toksyn MazF i HipA E. coli poprzez wykorzystanie antysensownych oligomerów PNA hamujących translację odpowiednich antytoksyn (odpowiednio MazE i HipB). Druga strategia prowadziła do pośredniej aktywacji działania toksyny MazF, co osiągnięto poprzez „wyciszenie” genu thyA, kodującego syntazę tymidylanową (stres wywołany niedoborem tyminy powoduje aktywację systemu mazEF i w konsekwencji zahamowanie wzrostu komórki). Trzecia strategia polegała na „naśladowaniu” działania toksyny HipA (inaktywującej syntazę glutamylo-tRNA) poprzez wyciszenie genu gltX, kodującego ww. syntazę. Na podstawie przewidzianych struktur drugorzędowych i trzeciorzędowych docelowych mRNA zaprojektowano cztery antysensowne sekwencje PNA: anty-mazE, anty-hipB, anty-thyA oraz anty-gltX. Badania przeprowadzono na trzech szczepach E. coli: laborotoryjnym K-12, pochodnym szczepu enterokrwotocznego O157:H7 oraz szpitalnym szczepie WR3551/98. Wszystkie zaprojektowane sekwencje PNA prowadziły do zahamowania wzrostu komórek badanych szczepów E. coli. Wartość MIC (ang. Minimal Inhibitory Concentration) dla poszczególnych PNA różnił się w zależności od badanego szczepu E. coli i były w przedziale 1-16 µM. Przy pomocy RT-qPCR zbadano wpływ PNA na poziom ekspresji poszczególnych genów. Stwierdzono znaczne obniżenie poziomu docelowych mRNA po inkubacji z komplementarnymi PNA, potwierdzając specyficzność oddziaływania tych związków z odpowiednimi transkryptami. Zbadano także oddziaływania synergistyczne między PNA a wybranymi chemioterapeutykami (trimetoprym oraz sulfametoksazol). Synergię działania zaobserwowano w połączeniu PNA anty-thyA z trimetoprymem. W końcowym etapie zbadano wpływ sekwencji PNA na powstawanie populacji komórek przetrwałych (ang. persister cells). Modyfikowane oligonukleotydy wykazują wiele interesujących właściwości biologicznych i mogłyby być potencjalnymi lekami, niestety ograniczony transport przez błony biologiczne sprawia, że nie znalazły one szerszego zastosowania. W pracy wykorzystano witaminę B12 jako nośnik antysensownych oligonukleotydów – PNA i 2’OMe RNA – do komórek E. coli. Przetestowano łącznie 17 koniugatów witamina B12-PNA, udzielając odpowiedzi na trzy zasadnicze pytania: (1) które elementy strukturalne witaminy B12 są kluczowe dla rozpoznawania koniugatów z oligonukleotydem, (2) która pozycja w strukturze jest najlepsza do syntezy koniugatów oraz (3) czy rodzaj oraz długość łącznika ma znaczenie w dostarczaniu koniugatów do komórek E. coli. Wykazano, że modyfikacje witaminy B12 w obrębie pierścienia koryny wpływają niekorzystanie na transport PNA, w porównaniu do modyfikacji w miejscu R5’. Ponadto zaobserwowano, że struktura i długość łącznika miały istotny wpływ na dostarczanie PNA. Docelowo, wykazano także antybakteryjny potencjał koniugatów B12-PNA. Podsumowując, wyniki badań przedstawione w mojej rozprawie doktorskiej dowodzą, że systemy TA są wrażliwe na działanie antysensownych PNA, a zaburzenie ich funkcjonowania in vivo prowadzi do zahamowania wzrostu bakterii. Wyniki te stanowią dowód poprawności koncepcji i zachęcają do kontynuacji prac nad wykorzystaniem natywnych systemów TA w opracowywaniu nowych strategii antybakteryjnych. Ważny jest również wybór optymalnych nośników wprowadzających antysensowne oligonukleotydy do komórek bakteryjnych, takich jak badana przeze mnie witamina B12.
The World Health Organization has placed antibiotic-resistant Escherichia coli at the top of the list of most important pathogens at a global level for which there is an urgent need for new treatments. The emergence of antibiotic-resistant bacterial strains is a critical issue which requires innovative antibacterial strategies to solve. One approach is the use of short, synthetic, antisense oligonucleotides (ASO) as inhibitors of bacterial growth. Such specifically designed oligonucleotides suppress proper expression of bacterial genes by complementary binding to bacterial RNA. One of the most extensively used ASOs are Peptide Nucleic Acids (PNA) which can be used to modulate the expression of target genes. PNA are synthetic DNA analogues with high affinity to natural nucleic acids. Due to their resistance to nucleases and proteases, PNAs exhibit high stability in the intracellular environment. The aim of this dissertation was (a) to explore the potential of natural bacterial toxin-antitoxin (TA) systems as new targets for compounds inducing antibacterial effects, and (b) to develop an effective and non-invasive method of introducing short, modified oligonucleotides into bacterial cells. This dissertation describes three innovative strategies that utilize antibacterial ASOs to target the TA systems and related proteins. The first method was the artificial activation of the E. coli MazF and HipA toxins by using antisense PNA oligomers to inhibit the translation of the corresponding antitoxins (MazE and HipB, respectively). The second strategy involved the indirect activation of the MazF toxin by inducing thymine starvation. This was achieved by silencing the thyA gene which encodes for thymidylate synthase, an enzyme involved in folic acid metabolism, that has been shown to interfere with mazEF-mediated growth inhibition. The third strategy was to mimic the action of the HipA toxin by silencing the gltX gene encoding its cellular target glutamyl-tRNA synthase. Based on the predicted secondary and tertiary structures of the targeted mRNAs, four antisense PNA sequences were designed: anti-mazE, anti-hipB, anti-thyA and anti-gltX. To evaluate the potential of antisense PNAs to inhibit the growth of E. coli strains, the minimal inhibitory concentrations (MIC) of the compounds were determined. Experiments were performed with three E. coli strains: K-12 – wild type; O157:H7 – the derivative of an enterohaemorrhagic strain and a clinical, multi-drug resistant strain WR3551/98. All tested PNAs inhibited the growth of the tested E. coli strains, however the MIC values depended on the PNA sequence as well as on the tested strain. The effectiveness of antisense silencing was estimated by quantifying mRNA abundance by RT-qPCR. Upon treatment with sequence-specific PNAs, a significant decrease in the level of corresponding gene transcript levels was observed. Next, this study investigated the synergistic interactions between PNAs and selected chemotherapeutics (trimethoprim and sulfamethoxazole). The synergy was found through a combination of PNA anti-thyA with trimethoprim. In the final stage of the project, the effect of the PNA sequences on the formation of persister cells was also studied. Modified oligonucleotides present many interesting biological properties and are exciting candidates as antibacterial drugs. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall, which precludes their use as antibiotics. To solve this problem, vitamin B12 was used as a carrier of ASOs: PNA and 2'OMe RNA, into E. coli cells. A total of 17 conjugates of vitamin B12-PNA were tested. Three basic questions were answered: (1) which structural elements of vitamin B12 are essential for the recognition of oligonucleotide conjugates by E. coli receptors, (2) which position in the vitamin B12 structure is optimal for the synthesis of conjugates, and (3) whether the type and length of the spacer and linker are relevant for the delivery of conjugates to E. coli cells. It was shown that vitamin B12 modifications within the corrin ring affect the PNA transport as compared to the non-corrin ring modification (R5’-OH). Moreover, it was observed that the structure and length of the linker had a significant influence on the delivery of PNA. Finally, the antibacterial potential of B12-PNA conjugates was also demonstrated. In conclusion, the results of the research presented in this doctoral dissertation demonstrate that TAs are susceptible to sequence-specific antisense agents and provide a proof-of-concept for their further exploitation in antimicrobial strategies. This research also illustrates, through the example of vitamin B12, the importance of choosing the optimal carrier for introducing antisense oligonucleotides into bacterial cells.