Minicircular DNA as a potential gene therapy vector in cervical cancer

• Autor(a) principal: Alves, Joel Marques
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6389
• topic: Cancro Cervical; Cromatografia de Afinidade; Dna Minicircular; Proteína Supressora de Tumor P53; Terapia Génica.; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

The Human Papillomavirus (HPV) infects a large number of people worldwide and is associated with more than 99% of cervical cancer, being a second major cause of cancer death in women. HPV E6 oncoprotein is responsible for the progression of this tumor due to the inactivation of the p53 tumor suppressor. Thus, novel therapeutic approaches for the treatment of this cancer have been explored, for example gene therapy, which is based on the insertion of genetic material of interest into the cells, resulting in the expression of proteins encoded by the respective genes. Plasmid DNA has been the non-viral vector mainly used in DNA therapeutics due to its simple manufacture, low toxicity and immunogenicity. Despite some successful cases, the possibility of using a DNA vector of reduced size, decreased toxicity by elimination of the bacterial cassette and able to favor the target gene expression by increased transfection efficiency, makes the minicircular DNA (mcDNA) a very promising gene therapy vector. Therefore, the present work aims to construct a mcDNA vector encoding p53 to reestablish the levels of this tumor suppressor protein in cervical cancer cells and to induce apoptosis. The production of mcDNA started by cloning the p53-encoding sequence into a parental plasmid (PP). Then, Escherichia coli ZYCY10P3S2T strain was transformed by heat shock with the p53-PP vector. The use of this E. coli strain is of major importance since it allows the plasmid recombination to form mcDNA and miniplasmid species by induction with L-arabinose during cell growth. Moreover, after the recombination process, this strain is also able to digest the contaminant miniplasmid through endonucleases action. A highest mcDNAp53/ PP-p53 ratio was achieved by optimizing the recombination step to use 0.01% of Larabinose and 2h of induction. After the production, the mcDNA must be purified for further evaluation of transfection efficiency and cellular effect through in vitro studies. For that, a new chromatographic column was studied in order to explore the simultaneous affinity character of two ligands (arginine and lysine) immobilized in the same functional group (triazine), in which hydrophobic binding/elution conditions were explored and optimized in order to purify the sc mcDNA-p53. When it was used lower salt concentration in the equilibrium step, it seemed that the selectivity of the column was favored, obtaining a sample of sc mcDNA-p53 with lower content of impurities, although the total recovery of the injected sample has been sacrificed. On the other hand, using the strategy of effective binding of the sample to the column and manipulating the pH was seemed to favor the recovery of sc mcDNA-p53 despite containing some impurities. In vitro transfection studies were performed to evaluate the transfection efficiency of PP and mcDNA vectors and the p53 expression, by using HeLA cells. Through immunocytochemistry it was observed that PP and mcDNA vectors were able to transfect HeLa cells, being more efficient with mcDNA. By RT-PCR it was confirmed the presence of p53 transcripts and by western blot it was verified the correct expression of p53 tumor suppressor in transfected cells Overall, this work allowed the construction, biosynthesis and recovery of a minicircular DNA vector encoding the p53 tumor supressor protein, showing great potential to proceed to in vitro and in vivo studies in order to develop a strategy for cervical cancer treatment in the future.