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- Obtenção de vacina de DNA plasmídico HPV-16 E6/E7 e avaliação da sua imunogenicidade in vitro e in vivoPublication . Almeida, Ana Margarida Cardoso Valério de; Sousa, Ângela Maria Almeida de; Sousa, Fani Pereira deA constante evolução da ciência tem permitido uma melhor partilha de conhecimentos na área da tecnologia do DNA recombinante, fornecendo um melhor conhecimento da informação contida nos genes e o impacto que alterações nesses genes poderão ter no organismo. A descodificação do genoma humano aliada ao progresso obtido no desenvolvimento de variados vetores de transporte de informação genética permitiu a evolução de terapias baseadas na entrega de genes terapêuticos, como a terapia génica e as vacinas de DNA. O desenvolvimento destas terapias trouxe uma nova esperança para o tratamento de certas patologias que, até então, permaneciam como intratáveis. Vetores biológicos e não biológicos têm evoluído largamente nos últimos anos, no entanto, a toxicidade demonstrada pela maioria dos vetores biológicos tem levado a um aumento de utilização de vetores não biológicos. O DNA plasmídico destaca-se entre os diversos vetores genéticos devido à simplicidade da sua produção, obtenção, baixo custo e ausência de toxicidade. As vantagens deste vetor têm levado a que a sua utilização como vacina de DNA tenha aumentado nos últimos anos, tornando-o o vetor de escolha na maioria dos estudos de investigação. As vacinas de DNA têm como modo de atuação a expressão de proteínas antigénicas com o objetivo de induzir uma resposta imunitária direcionada para essas mesmas proteínas, permitindo a prevenção e/ou tratamento de infeções virais e bacterianas. Torna-se imperativo o desenvolvimento de tecnologias que permitam a produção e purificação destes vetores, obtendo a maior percentagem de recuperação e pureza possíveis do plasmídeo na sua forma biologicamente ativa, a isoforma superenrolada (sc). A área da cromatografia tem progredido bastante no desenvolvimento de estratégias eficazes de purificação de plasmídeo, permitindo o aumento de produtividade e obtenção deste vetor e diminuindo eventuais custos associados à sua produção. O Vírus do Papiloma Humano (HPV) é um vírus sexualmente transmissível que se encontra atualmente associado ao desenvolvimento de massas tumorais devido à produção de duas proteínas oncogénicas, oncoproteínas E6 e E7, capazes de alterar o ciclo de proliferação celular e de provocar o crescimento anormal de células do organismo infetado. A tecnologia de vacinas de DNA apresenta-se assim como uma terapia promissora para infeções provocadas pelo HPV, através da indução de uma resposta imunitária contra as proteínas referidas. Recentemente, o nosso grupo de investigação conseguiu desenvolver de forma eficaz a produção e purificação da vacina de DNA sc HPV-16 E6/E7 através da utilização de um monolito modificado com ligandos de arginina, tirando partido dos princípios básicos da cromatografia de afinidade. Contudo, a recuperação do plasmídeo não foi a esperada, tendo sido apenas recuperado 39% da molécula alvo. O Desenho experimental é uma ferramenta estatística que, através da escolha correta dos fatores a serem avaliados, bem como os seus intervalos em estudo, permite a otimização de respostas de um sistema experimental. Deste modo, através do design experimental foi feita uma otimização ao sistema de purificação da vacina de DNA sc HPV-16 E6/E7 de modo a garantir um aumento de recuperação da molécula, mantendo o elevado nível de pureza. Com esse intuito, após uma avaliação inicial dos fatores e dos intervalos a serem usados, o design ‘Central Composite Face’ (CCF) foi utilizado para delinear um conjunto de experiências cromatográficas de modo a encontrar o ponto ótimo para a percentagem de recuperação do plasmídeo ser maximizada. A otimização foi bem-sucedida, permitindo a obtenção de uma percentagem de recuperação de cerca de 83%, mantendo-se a percentagem de 100% para a pureza. Após a otimização da estratégia de purificação, estudos de transfeção in vitro foram realizados de modo a avaliar a capacidade de transfeção celular e consequente expressão da proteína codificada pelo gene-alvo contido na vacina de DNA. Células CHO-1, isoladas a partir de tecido ovárico de rato chinês, foram cultivadas e transfetadas com a isoforma sc purificada através da estratégia otimizada com o monolito de arginina, bem como com a isoforma circular aberta (oc) e DNA plasmídico obtido através de um kit comercial, de modo a avaliar qual a melhor estratégia para transfeção. Através das técnicas de western blot e imunocitoquímica foi possível verificar que a entrada do pDNA nas células eucarióticas ocorreu com sucesso (processo de transfeção), observando-se um aumento significativo de expressão génica das proteínas E6 e E7 em comparação ao grupo de controlo (células não transfetadas). A avaliação da expressão génica da proteína E6 dos diferentes tipos de plasmídeos utilizados permitiu verificar que o aumento de expressão desta proteína foi mais significativo com a amostra de plasmídeo sc purificado pelo monolito de arginina, concluindo-se que de facto a isoforma sc induz uma maior eficiência de transfeção.
- HPV-16 targeted DNA vaccine expression: The role of purificationPublication . Almeida, Ana Margarida; Tomás, Joana; Pereira, Patrícia; Queiroz, João; Sousa, Fani; Sousa, ÂngelaDNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546-551, 2018.
- Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligandsPublication . Almeida, Ana Margarida; Queiroz, João; Sousa, Fani; Sousa, ÂngelaMinicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.
- Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteinsPublication . Almeida, Ana Margarida; Queiroz, João; Sousa, Fani; Sousa, ÂngelaCervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology. Nowadays, the main treatment often requires the use of invasive techniques. From well-established strategies, like DNA vaccines and gene therapy, to innovative gene silencing technologies; different methodologies are currently under scrutiny that target the E6 and E7 oncoproteins and/or their modes of action.
- Preparation of well-defined brush-like block copolymers for gene delivery applications under biorelevant reaction conditionsPublication . Góis, Joana R.; Reis, Fábio; Almeida, Ana Margarida; Pereira, Patrícia; Sousa, Fani; Serra, Armenio; Coelho, JorgeWell-defined oligo(ethylene glycol) methyl ether methacrylate (OEOMA) based block copolymers with cationic segments composed by N,N-(dimethylamino) ethyl methacrylate (DMAEMA) and/or 2-(diisopropylamino) ethyl methacrylate (DPA) were developed under biorelevant reaction conditions. These brush-type copolymers were synthesized through supplemental activator and reducing agent (SARA) atom transfer radical polymerization (ATRP) using sodium dithionite as SARA agent. The synthesis was carried out using an eco-friendly solvent mixture, very low copper catalyst concentration, and mild reaction conditions. The structure of the block copolymers was characterized by size exclusion chromatography (SEC) analysis and 1H nuclear magnetic resonance (NMR) spectroscopy. The pH-dependent protonation of these copolymers enables the efficient complexation with plasmid DNA (pDNA), yielding polyplexes with sizes ranging from 200 up to 700 nm, depending on the molecular weight of the copolymers, composition and concentration used. Agarose gel electrophoresis confirmed the successful pDNA encapsulation. No cytotoxicity effect was observed, even for N/P ratios higher than 50, for human fibroblasts and cervical cancer cell lines cells. The in vitro cellular uptake experiments demonstrated that the pDNA-loaded block copolymers were efficiently delivered into nucleus of cervical cancer cells. The polymerization approach, the unique structure of the block copolymers and the efficient DNA encapsulation presented can open new avenues for development of efficient tailor made gene delivery systems under biorelevant conditions.
- Biosynthesis and purification of DNA vectors as therapeutic approaches against Human Papillomavirus infectionPublication . Almeida, Ana Margarida Cardoso Valério de; Sousa, Ângela Maria Almeida de; Sousa, Fani Pereira deCervical cancer is one of the leading causes of death amongst women, especially in countries lacking suitable access to health and hygiene care. Contrarily to most cancers, which usually take origin in mutations related with the action of mutagenic or carcinogenic agents, the development of cervical cancer is strongly associated with infection by Human Papillomavirus (HPV). This virus is responsible for the expression of E6 and E7 oncoproteins, which interfere with the cell cycle regulation, affecting the cellular mechanisms of repair and proliferation of infected cells. In the last decades, DNA-based therapies, such as DNA vaccines or gene therapy, have been highly explored by researchers in the search of an efficient non-viral vector for the cervical cancer treatment. Plasmid DNA (pDNA) is a popular non-viral vector, which has been widely studied in the past decades for the development of a variety of DNA vaccines and gene therapy strategies. However, the presence of CpG motifs necessary to its amplification in prokaryotic hosts may lead to the activation of the immune system and to the degradation of this molecule before it can reach the target cells, in a gene therapy perspective. However, these same motifs may contribute to the recognition of this bioproduct by antigen presenting cells, facilitating the processing of pDNA as a DNA vaccine. Minicircle DNA (mcDNA) consists in a non-viral vector whose popularity has been largely increasing in the last years. This vector results from the recombination of a molecule, which is similar to conventional pDNA, named parental plasmid (PP). However, PP presents two recombination sites which, upon induction, give origin to the recombination of this molecule into two different molecules: one containing all the prokaryotic information necessary for PP amplification in prokaryotic hosts (miniplasmid – mP) and another containing all the information necessary for the therapeutic gene expression in eukaryotic cells (mcDNA). Considering its innovative character, it is crucial to develop suitable strategies for mcDNA preparation, with a purity that fits the requirements established by regulatory agencies such as European Medicines Agency (EMA) or Food and Drug Administration (FDA). Thus, this work was performed with two different perspectives regarding cervical cancer treatment, namely DNA vaccines and gene therapy. Firstly, concerning DNA vaccines, the application of an adequate purification strategy allowed to understand that the purity degree of the pDNA sample can significantly contribute for the increased expression of the target antigen. The application of a monolith modified with arginine ligands, comparatively to the use of commercially available techniques, allowed to retrieve a final DNA vaccine sample with higher supercoiled (sc) content, leading to a higher expression of the target proteins. Concomitantly, the development of nanocarriers composed by calcium carbonate and gelatin allowed the delivery of pDNA vaccine to dendritic cells, also known as antigen presenting cells, without detecting cytotoxicity. On the other hand, two different strategies for mcDNA purification were explored. Firstly, the preparation of a monolith modified with cadaverine ligands and its use in the purification of sc mcDNA allowed a recovery yield of 78.6% coupled with 98.4% purity. These results turned out to be very interesting considering the current mcDNA purification scenario in the literature. On the other side, size exclusion chromatography was also explored through the use of Sephacryl SF-1000 matrix. This strategy allowed to obtain a sc mcDNA yield of 66% alongside a purity of 98.35%. Despite 12 times more mass of pure sc mcDNA was obtained in one assay, the assay turned out to be approximately 8 times longer than the assays performed with the cadaverine modified monolith. In the end, two strategies were developed for sc mcDNA purification, each presenting its advantages and disadvantages. Nonetheless, both allowed the purification of mcDNA without resorting to PP specific genetic modifications, which implies that these strategies can be universally used for mcDNA purification. However, the need to implement an analytical methodology that allows to quantify sc mcDNA content in a fast, low cost and effective way led to the study of cadaverine-modified monolith with this purpose. Thus, a chromatographic method was implemented and validated to quantify sc mcDNA concentration present in a complex or pure sample, within a range of 1-25 μg/mL. Lastly, to study the effectiveness of miR-375 in the treatment of cervical cancer, in vitro studies were performed to evaluate the effect displayed by mcDNA-pri-miR-375 in the expression of E6 an E7 oncoproteins. Therefore, CaSki cell line model was used, which consists in metastatic cervical cancer cells infected by HPV-16. Also, a fibroblast cell line was used as a non-carcinogenic model for control. It was possible to identify the correct expression of miR-375, coupled with a decrease in E6 and E7 transcript and protein levels. Furthermore, it was observed that fibroblast viability was not affected, instead observing a decrease in transfected CaSki proliferation and viability. Thus, the application of mcDNA-pri-miR-375 as a possible therapeutic strategy should be more amply studied in the future. In the end, this thesis presents a scientific work that intends to contribute towards the scientific community with useful information for the potential development of new approaches for cervical cancer treatment, through the implementation of two biotechnological platforms. One of the strategies is based on the obtainment of a DNA vaccine, encoding E6 and E7 HPV antigens with the aim of activating two pathways of the immune system, the preventive/humoral and the therapeutic/cellular pathways, allowing the consequent elimination of the antigen-expressing and HPV-infected cervical cancer cells. The second strategy is focused on the obtainment of innovative gene therapy mcDNA vector, encoding pri-miR-375, to silence E6 and E7 HPV oncoproteins, allowing the direct targeting of infected cervical cancer cells, through the re-establishment of tumor suppressor proteins expression or function.