Purificação de uma vacina de DNA plasmídico para prevenção ou tratamento do cancro do colo do útero

• Autor(a) principal: Carapito, Duarte Gonçalves
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/5818
• topic: Cromatografia de Afinidade; Hpv; Monolito de Cadaverina; Monolito de Lisina; Vacinas de Dna Plasmídico.; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

Human papillomavirus (HPV) is a common sexually transmitted virus responsible for malignant progression of cervical cancer. High-risk HPVs have been reported in 99% of cervical cancer cases worldwide, the second most common cancer in women. The cervical cancer development is associated with HPV E6 and E7 protein production, which can interfere with apoptosis and deregulate cell cycle proliferation. Nowadays, prophylactic vaccines are available and commercialized towards HPV eradication. However, these vaccines only stimulate the immune system against future infections, being ineffective when the woman is already infected. Furthermore, not all types of HPV virus are covered by this type of vaccines. DNA vaccines are advantageous compared to conventional vaccines by inducing both cellular and humoral immune responses, leading to prevention and treatment of a target infection. Thus, the development of a DNA vaccine able to produce E6 and E7 antigens arises as a promising therapeutic pathway to control HPV infection. Plasmid DNA (pDNA) has been explored as a versatile non-viral vector to deliver therapeutic genes due to its biosafety, low cost and easy manufacture, which make it a desirable vector to deliver therapeutic genes. In order to guarantee the purity of the pDNA vaccine, it is necessary to explore several chromatographic methods. Our research group has been focused on the purification of mutated pDNA HPV-16 E6/E7, to prevent oncogenecity of E6 and E7 viral proteins, in order to achieve the required purity levels imposed by regulatory agencies for therapeutic applications. In addition, interest around innovative monolithic supports has recently increased due to their advantageous features, like high binding capacities of large molecules and excellent mass transfer properties. Thus, the aim of the present work was to explore two monolithic columns modified with lysine and cadaverine ligands, in order to develop suitable purification strategies to isolate supercoiled (sc) pDNA isoform, which is the conformation with most biological interest for therapeutic applications. Through the manipulation of NaCl concentrations, buffer nature and pHs, the chromatographic conditions were optimized to isolate sc pDNA in cadaverine and lysine monolithic columns. The results obtained with cadaverine and lysine monoliths from purity degree (97.89% and 97.53%, respectively) and recovery yield (98.70% and 96.10%, respectively) of sc pDNA, revealed that the best elution strategy consists on an increasing stepwise gradient of NaCl in 10 mM tris-EDTA (pH 6.0). It was also observed that lower pH values led to stronger interaction between ligands and biomolecules, favoring the sc pDNA purification. The results of genomic DNA, endotoxins and proteins quantification, revealed that all impurities had levels below to those imposed by regulatory agencies. These data suggest the applicability of two supports to purify sc pDNA in order to be applied on a DNA vaccine against HPV.