Dissertations / Theses on the topic 'SiRNA'

Supramolecular drug carriers based on physical assembly of “smart” polymers have emerged in last decades to obtain novel carriers for the tumor therapy. These systems are designed to target cancer tissue, preserve the therapeutic activity of loaded drug and reduce their systemic side-effects. Amphiphilic copolymers can be assembled in vesicles that can be loaded with hydrophilic and/or hydrophobic drugs. The research project was aimed at synthesizing a triblock pH-responsive polymer to be assembled in vesicles for the delivery of specific siRNA to cancer cells to silence specific functions involved in the tumor progression. According to its nanometric size, the colloidal drug delivery system is intended to undergo passive accumulation in the cancer tissue by EPR effect and access selectively the cytosol by cancer cell biorecognition. Once the vesicles are localized in the endosomes, their pH responsiveness will guarantee for the disassembling of the carrier and the release of the siRNA payload. The vesicles forming polymer was synthesized in order to respond to the acid environment in the endosomal and lysosomal compartments with prompt disassembly of the nanocarrier and consequent siRNA release. The triblock copolymers include two hydrophilic blocks at the terminal ends, namely 1.9 kDa or 3.5kDa PEG and poly-glycerolmethacrylate (GMA), and a central pH sensitive block, imidazole hexyl methacrylate (ImHeMA) that control the assembly and disassembly of the vesiscles. The polymer obtained with the 3.5kDa PEG is intended to be conjugated with the targeting agent folic acid to confer biorecongnition properties to the polimersomes. The vesicles were prepared by mixing the polymers obtained with 1.9 kDa and 3.5 kDa at adequate ratios. The copolymers with ratio 90:10 w/w polymers 1.9 kDa and 3.5 kDa self-assemble in vesicles at pH 7.4 with a mean size of 100 nm as detected by light scattering analysis and are very stable at room temperature. At 37°C the stability of vesicles was modulated by increasing the ratio of the copolymer 3.5 kDa PEG. Vesicles obtained with ratio 90:10 w/w polymers 1.9 kDa and 3.5 kDa were imaged by TEM microscopy showing a spherical shape and high size homogeneity. The polymeric vesicles were found to loads very efficiently double stranded DNA (dsDNA) sequences with a 14% molar loading yield as shown by UV-Vis spectrometry and release them in 8 hours when incubated at pH 5, while vesicles showed very limited DNA release at pH 7.4. The physical assembly of the dsDNA with the pH responsive triblock copolymer was controlled by the environmental pH: gel retardation electrophoresis showed that the polymer and dsDNA completely associate at a N/P ratio of 2/1 at pH 5, while no association was observed up to a N/P ratio of 20/1 at pH 7.4 where the ImHeMA block is neutral. Cell viability assay performed on B16F10 mouse melanoma cells showed a remarkable biocompatibility of the polymeric vesicles at concentration of 1, 2, 3 mg/mL. The high hemolytic activity of the polymer at acidic pH (pH 5) support for the capacity of the material to induce endosomal membrane disruption. The results displayed a 70% hemolytic activity at pH 5, while in physiological condition (pH 7.4) no red blood cell lysis was detected. Polymer formulations, with and without the folate-tipped terminal ends, were incubated with KB human cervical carcinoma cell line and MCF7 human breast adenocarcinoma cell line, that overexpress and do not express the folate receptor respectively, to investigate active targeting properties of the folate tipped vesicles. The uptake of vesicles loaded with cyanine 3 labeled dsDNA, that was evaluated by fluorescence spectroscopy on cell lisate and by cytometry, was 3 times higher in KB cells compared to MCF-7 cells. Vesicles loaded with double strand siRNA for the silencing of luciferase were investigated on luciferase transfected B16F10 cells that express the folate receptor. The decrease of bioluminescence in cell sample treated with siRNA loaded folate targeted vesicles was 30% with respect to control empty vesicles. The above results were confirmed by confocal microscopy carried out with the same cancer cell lines. Confocal microscopy showed a significantly higher accumulation of the fluorescently labeled dsDNA in KB cells and a major localization of the macromolecule at the nuclear compartment
Negli ultimi decenni sono stati sviluppati, in maniera sempre più consistente, drug carriers supramolecolari per la terapia tumorale capaci di aggregare in nanostrutture grazie all'utilizzo di polimeri "intelligenti". Questi sistemi sono stati progettati in modo tale da essere direzionati selettivamente al tessuto tumorale, mantenere l'efficacia terapeutica del farmaco caricato e ridurre gli effetti collaterali a livello sistemico. Polimeri con proprietà anfifiliche sono in grado di formare vescicole, chiamate anche polimerosomi, che possono essere caricate con farmaci idrofilici/idrofobici. Il progetto di ricerca qui riportato ha avuto come fine ultimo la sintesi di un copolimero pH-sensibile a tre blocchi in grado di aggregare in vescicole utilizzate per il delivery di specifici silencing RNA (siRNA) alle cellule cancerose con lo scopo di rendere silenti specifici meccanismi coinvolti nel processo di progressione tumorale. Grazie alle sue nano-dimensioni, il sistema di drug-delivery colloidale è previsto andare incontro ad accumulazione passiva nel tessuto tumorale per Enhanced Permeability and Retention Effect (EPR) ed avere accesso selettivamente al comparto cellulare citosolico grazie al bioriconoscimento da parte della cellula tumorale. Una volta all'interno degli endosomi, la capacità delle vescicole polimeriche di rispondere in maniera differente ai diversi pH renderà possibile la disaggregazione del carrier e il rilascio del siRNA caricato. I polimeri utilizzati sono stati sintetizzati in modo tale da rispondere con una rapida disaggregazione del nano-sistema una volta in contatto con l'ambiente acido caratteristico dei compartimenti endosomiali e lisosomiali, ottenendo quindi il rilascio del siRNA. I polimeri a tre blocchi utilizzati presentano due monomeri idrofilici alle estremità, chiamati poly-ethilenglycole (PEG) 1.9 kDa - 3.5 kDa e poly-glycerolmethacrylate (GMA), e inoltre un blocco centrale pH sensibile, poly-imidazole hexyl methacrylate (ImHEMA) che guida la formazione e la disaggregazione delle vescicole. Il polimero ottenuto con PEG 3.5 kDa verrà coniugato all'agente direzionante acido folico per conferire proprietà di bioriconoscimento cellulare ai polimerosomi. I polimerosomi sono stati preparati miscelando i polimeri ottenuti con 1.9 e 3.5 kDa in rapporti adeguati. I copolimeri in rapporto 90:10 w/w formano vescicole stabili a pH 7.4 e temperatura ambiente con un diametro medio di 100 nm. La stabilità dei polimerosomi a 37°C è stata modulata aumentando il rapporto del copolimero PEG 3.5 kDa. Vescicole ottenute con rapporto di polimeri 90/10 w/w 1.9 kDa e 3.5 kDa sono state caratterizzate morfologicamente al microscopio elettronico a trasmissione TEM mettendo in evidenza forma sferica e alta omogeneità dimensionale. Le vescicole polimeriche caricano efficacemente sequenze di DNA doppia elica (dsDNA) con una resa molare del 14% come dimostrato da analisi spettrofotometriche. Il dsDNA viene rilasciato in 8 ore quando i polimerosomi vengono incubati a pH 5; a pH 7.4 invece il rilascio è risultato essere quasi nullo. La capacità del polimero di complessare il dsDNA è controllata dal pH esterno: studi di ritardo elettroforetico hanno evidenziato che il polimero e il dsDNA sono completamente associati per rapporti N (gruppi amminici del polimero) /P (gruppi fosfato del DNA) di 2/1 a pH 5. Nessuna formazione di complessi è stata osservata per N/P ratio fino a 20/1 a pH 7.4, condizioni cui l’unità imidazolica risulta pressochè neutra. Studi di citotossicità eseguiti su cellule B16F10 da melanoma di topo hanno mostrato una buona biocompatibilità delle vescicole polimeriche a concentrazioni di 1, 2, 3 mg/mL. La alta attività emolitica del polimero a pH acido (pH 5) conferma la capacità del materiale nell’indurre la lisi della membrana endosomiale. In dettaglio, i risultati hanno mostrato un’attività emolitica pari al 70% a pH 5, mentre in condizioni fisiologiche (pH 7.4) non è stata rilevata alcuna lisi dei globuli rossi. Le formulazioni polimeriche, con e senza agente di targeting, sono state incubate con cellule KB da cancro alla cervice uterina e cellule MCF7 da adenocarcinoma mammario, le quali rispettivamente sovreaesprimono e non sovraesprimono il recettore folato, in modo tale da studiare l’efficacia di direzionamento di polimerosomi aventi il folato sulla loro superficie. L’internalizzazione di vescicole caricate con dsDNA marcato per mezzo del fluoroforo cyanine-3, valutato mediante analisi fluorimetrica su lisato cellulare e per mezzo di citofluorimetria, ha dimostrato essere di circa 3 volte maggiore per cellule KB comparate a MCF7. Quindi, polimerosomi caricati con ds-siRNA per il silenziamento dell’enzima luciferasi sono state testate su cellule B16F10 trasfettate con il promotore per l’enzima e sovraesprimenti il recettore per il folato. L’esperimento ha mostrato una diminuzione della bioluminescenza imputata all'attività luciferasica del 30% rispetto alle vescicole vuote. I risultati riportati sono stati confermati grazie a studi di microscopia confocale eseguiti sulle stesse linee cellulari sopra descritte. Le immagini hanno evidenziato un accumulo di dsDNA marcato in modo significativamente più elevato in cellule KB e con una maggiore localizzazione della macromolecola nel compartimento nucleare

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.

L’utilisation de siRNA est une nouvelle approche thérapeutique très prometteuse. Néanmoins leur transfection à visée thérapeutique est un réel défi. Les obstacles à franchir pour élaborer des agents de transfection sûrs et fiables sont nombreux. Afin de les contourner nous nous sommes attachés à la construction d’un système dynamique qui, à l’image des virus, est constitué de briques moléculaires, s’emboitant et interagissant avec des acides nucléiques selon des interactions supramoléculaires. Ainsi, nous avons élaboré des polymères supramoléculaires polycationiques à base de monomères de cyclodextrines pontées, fonctionnalisées par un groupement adamantyle. Ce type de conjugué pallie un problème manifeste dans la littérature concernant les assemblages de β-CD souvent insolubles ou bien auto-inclus. L’ajout éventuel d’une autre fonction cationique pour améliorer l’interaction avec les siRNA a aussi été réalisé. Ainsi, la capacité à s’auto-assembler de quatre composés a été étudiée par RMN-1H, RMN-ROESY, ITC, RMN-DOSY, et SANS. Par ailleurs, ces composés ont montré une certaine capacité à complexer et à protéger les siRNA. L’un de ces composés a de plus montré une bonne aptitude à transfecter des siRNA in vitro, sans induire de toxicité. Les assemblages CD-siRNA ont finalement été observés par cryo-TEM et ont montré la formation de fibres, organisées de manière hiérarchique et hautement coopérative. Nous avons ainsi créé des assemblages supramoléculaires uniques à base d’acides nucléiques, rappelant la structure, la taille et la fonction d’un virus
SiRNA based therapeutics are very promising. A key challenge for their development is the design of sophisticated, safe and effective delivery methods. To address all the biological obstacles for the conception of such therapeutics, we focused on the construction of a virus-like dynamic system, built with molecular bricks, able to self assemble and to interact with nucleic acid through supramolecular interactions. Bridged cyclodextrin based supramolecular polymers were developed to form host-guest interactions. To do so, cyclodextrins were conjugated with cationic and hydrophobic moiety in a spatially controlled way. These conjugates solved problems well known in the literature about the self-inclusion and the solubility in water of such molecules. The ability to self-assemble of 4 compounds were studied by RMN-1H, RMN-ROESY, ITC, RMN-DOSY and SANS. All these compounds showed a good capability to complex and protect siRNA. Moreover, one of these compounds is able to transfect siRNA in vitro without any toxicity, and therefore, to induce gene silencing. Assembly of CD and siRNA were finally observed by cryo-microscopy, which showed long fibres organised in a hierarchical and cooperative manner. This unique system is therefore strongly reminiscent of the structure, size and function of a virus

To direct post-transcriptionally gene silencing, RNAi machinery exploits the formation of base pairs between the loaded guide strand and the complementary mRNA. The Ago2 protein is the “slicer” effector of the RISC and drives the endonucleolytic cleavage only when the siRNA guide strand is full paired with its RNA counterpart. Ago2 is able to incorporate a duplex siRNA molecule, unwinds the double helix and holds one strand while discarding the other. Ago2 bearing only the guide strand is defined “active” and can guide multiple cleavage reactions against the complementary mRNAs. Structural insights into Ago2 assembly process have speculated that early interactions between the siRNA and the Ago2 relies on specific recognition by the PAZ domain. Thus, proper PAZ domain recognition contributes to the specific and productive incorporation of siRNAs into the Ago2. Interactions between PAZ domain and siRNA molecule are essentially asymmetric. The guide strand with its 2-nt 3’overhang is involved in the majority of the contacts between the PAZ pocket and the siRNA, whereas the passenger strand interacts only with its 5’ end residue. In principle, overhang modifications (i.e. 2’-deoxy units) were just introduced to protect the RNA duplex integrity. Only after the understanding of the Ago2 architecture, overhang modifications were also harnessed to improve the siRNA potency and specificity. The comprehension of the PAZ lodging/dislodging motion during the formation of binary (Ago2 + guide) and ternary complex (Ago2 + guide + mRNA) pointed out the importance of adequate affinity between the guide overhang and the PAZ cleft during the Ago2 multi-turnover cleavage process. Affinity analysis on PAZ/siRNA overhang complex has proved the influence of the overhang presence for efficient binding. SiRNA duplexes with shorter overhang (1-nt) or blunt end have respectively highlighted 85-fold and >5000-fold reduced affinity. Hence, taking advantage of more efficient interactions between the PAZ pocket and the strand bearing the unpaired di-nucleotides structure, structural asymmetric siRNA molecules bearing only the antisense overhang were successful employed to bias the RISC strand selection. Moreover, competition between siRNAs, resulting in preferential incorporation of one siRNA type into the RISC machinery, is influenced by the distinct loading kinetics of siRNA molecules. Thus, the knockdown ability of siRNA mixtures is often compromised due to competition between siRNAs. It also has been reported that the simultaneous transfection of two or more siRNAs causes reduced silencing activity of one siRNA species whereas the potency of the other siRNAs were not affected. Even if siRNA competition effects are essentially produced by the interactions with the Ago2 protein, up to now, no available data about a specific Ago2 domain involvement into the siRNA competition have been described. We have been hypothesized that the PAZ domain, playing an important role in the first steps of the strand loading could be specifically involved in the siRNA competition. Given this background we are questioning how the di-nucleotide unpaired structure can influence the siRNA silencing efficiency and specificity. To explore the structural hallmarks critical for the PAZ pocket interaction, we modified the siRNA overhangs with several modifications. In detail, 2 units of β-L-nucleosides (mirror image L-Thymidine), 2’-deoxyribitol, GNA (glycerol nucleic acids)-Thymine and acyclic L-threoninol were introduced at overhang level and the silencing potency (IC50) was measured. Such modifications may provide fundamental clues on structural prerequisite needed for the PAZ recognition and strand loading into the Ago2.
Para dirigir el silenciamiento génico post-transcripcional, la maquinaria de RNAi explota la formación de pares de bases entre la hebra guía cargado y el ARNm complementario. La proteína Ago2 (Argonauta 2) es la "máquina de cortar" del complejo RISC y dirige la rotura endonucleolítica sólo cuando la hebra guía del siRNA está completamente apareada con su homóloga de ARN. Ago2 es capaz de incorporar una molécula de dúplex de siRNA, desenrolla la doble hélice y mantiene una hebra mientras se descarta la otra cadena. Ago2 cargada con la hebra guía se define "activa" y puede guiar múltiples reacciones de escisión contra los ARNm complementarios. El análisis estructural del proceso de ensamblaje de Ago2 ha llevado a la conclusión de que las primeras interacciones entre el siRNA y la proteina Ago2 se basa en el reconocimiento específico por el dominio PAZ. Por lo tanto, el correcto reconocimiento de dominio PAZ contribuye a la incorporación específica y productiva de los siRNAs en el Ago2. La hebra guía con su extremo 3' protuberante que tiene 2-nt está implicada en la mayoría de los contactos entre la cavidad presente en el dominio PAZ. En principio, las modificaciones en los extremos protuberantes se introdujeron para proteger la integridad del dúplex de ARN. Sólo después de la comprensión de la arquitectura de Ago2, se pensó en la utilización de las modificaciones en los extremos protuberantes para mejorar la potencia y especificidad de los siRNAs. Para explorar las características estructurales críticas para la interacción entre la cavidad PAZ, modificamos los extremos protuberantes de los siRNA con varias modificaciones. Específicamente, 2 unidades de un beta-L-nucleósido como la L-timidina (imagen especular de la timidina), de 2'-desoxiribitol, de GNA (glycerol nucleic acids)- timina y del derivado acíclico L-treoninol se introdujeron a los extremos protuberantes y se midió la potencia de silenciamiento (IC50). Tales modificaciones pueden proporcionar pistas fundamentales sobre el requisito estructural necesario para el reconocimiento y carga de la cadena del dominio PAZ de Ago2.

RNA silencing is a plant antiviral defence mechanism. A hallmark of RNA silencing is the production of small interfering (si) RNAs of 21-25 nucleotides that are generated from the viral genome. Moreover, profiles of viral siRNAs suggest that certain regions, namely hotspots, of the viral genome are more prone to RNA silencing-mediated degradation. African cassava mosaic virus (ACMV) causes cassava mosaic disease which is considered one of the greatest threats to the production of cassava in Africa and throughout the world. Geminiviruses such as ACMV are believed to be both inducers and targets of RNA silencing. The hypothesis of this research is that by targeting siRNA hotspots in the viral genome a novel strategy can be developed to control viral infection. This project has three main objectives. First, it aims to profile siRNAs associated with cassava-infecting DNA viruses via hybridisation and deep sequencing technology. Second, it aims to test the interaction between hotspot vsiRNAs and ACMV. Third, it aims to induce resistance to ACMV, and assess the level of resistance developed, by direct delivery of hotspot vsiRNA in plants. As a preliminarily study, it was demonstrated that synthetic exogenous siRNA targeting one of the siRNA hotspots in the genome of Tomato mosaic virus (ToMV) had a negative impact on virus infection in terms of RNA accumulation and symptom severity in Nicotiana benthmiana plants. In further experiments, different sized siRNAs associated with ACMV infection was detected by both northern blotting and high throughput 454 sequencing of samples from infected plants. Based on the 454 sequencing data obtained sense and anti-sense strand siRNAs have been produced corresponding to siRNA hotspot and coldspot regions. The effects of these exogenous siRNAs on ACMV infection in N. benthamiana plants were investigated. The results revealed that the co-inoculation of the hotspot siRNAs with ACMV reduced viral DNA replication and disease development. ACMV/siRNA co-inoculated plants remained symptomless throughout the 3-month course of the experiments. On the other hand, when ACMV was coinoculated with coldspot targeted siRNA, the viral DNA accumulation was comparable to infection with ACMV alone, and plants displayed typical ACMV infection symptoms. The effect of hotspot siRNA in inducing viral resistance was dose-dependent. The results also revealed that the effectiveness of hotspot siRNA to elicit durable resistance to ACMV infection requires a functional host RNAdependent RNA polymerase 6 (RDR6). In conclusion, these data demonstrate that hotspot vsiRNA could offer a novel strategy for controlling major destructive viral diseases in plants.

The effectiveness of radiotherapy treatment could be significantly improved if tumour cells could be rendered more sensitive to ionising radiation without altering the sensitivity of normal tissues. However, many of the key mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. This thesis is concerned with the identification of novel determinants of tumour radiosensitivity. A siRNA screen of 200 genes involved in DNA damage repair was conducted using γH2AX foci post-irradiation as a marker of cell damage. This screen identified POLQ as a potential tumour-specific contributor to radioresistance. Subsequent investigations demonstrated that POLQ knockdown resulted in radiosensitisation of a panel of tumour cell lines, whilst having little or no effect on normal tissue cell lines. It was subsequently shown that POLQ depletion rendered tumour cells significantly more sensitive to several classes of cytotoxic agents. Following exposure to etoposide, it was found that tumour cells depleted of POLQ had reduced RAD51 foci formation, suggesting that POLQ is involved in homologous recombination. A homologous recombination assay was used to confirm that POLQ depletion does indeed result in reduced homologous recombination efficiency. These findings led to the investigation of the clinical significance of tumour overexpression of POLQ. The clinical outcomes of patients with early breast cancer were correlated with tumour expression levels of POLQ. It was found that POLQ overexpression was correlated with ER negative disease and high tumour grade, both of which are associated with poor clinical outcomes. POLQ overexpression was associated with extremely poor relapse free survival rates, independently of any other clinical or pathological feature. The mechanism that causes this adverse outcome may in part arise from resistance to adjuvant chemotherapy and radiotherapy treatment. These findings, combined with the limited normal tissue expression of POLQ, make it an appealing target for possible clinical exploitation.