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Projeto de investigação
Bolsa de Doutoramento FCT: Isolamento, purificação e entrega de DNA plasmídico (pDNA) codificador de p53 para aplicação em terapia genética do cancro [SFRH/BD/96809/2013]
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The biological performance of purified supercoiled p53 plasmid DNA in different cancer cell lines
Publication . Valente, J. F. A.; Sousa, Ângela; Gaspar, V. M.; Queiroz, João; Fani, Sousa
Tumor suppressor p53 remains one of the most interesting therapeutic targets in cancer gene therapy due to itsconsistent mutation in numerous cancers. Thus, the reinstatement of the p53 expression and function can be seenas an effective alternative for cancer treatment, motivating research in thisfield. In this study,L-methioninematrix was used to purify the supercoiled topoisoform of a plasmid DNA encoding the p53 protein. This purebiopharmaceutical was conjugated with liposomes to comprehensively analyze itsin vitroperformance andtherapeutic potential in different cancer cell lines, including the lung and cervix models. A different profile ofcellular responses was attained after the transfection of these cancer cell lines with the p53-pDNA. Actually, thein vitrotransfection with pure sc p53-pDNA resulted in a higher expression of the tumor suppressor protein incancer cells when compared with the native pDNA samples (oc + sc topoisoforms). Also, wild-type p53 ex-pression following transfection was significantly higher in HeLa cervix cancer cells compared to that obtained inA549 lung cancer cells. Overall, ourfindings emphasize the potential of sc pDNA gene-based therapy, alsoraising awareness of the need to adjust the therapeutics, considering the feature of high heterogeneity of cancer cells.
DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography
Publication . Valente, Joana; Sousa, A.; Queiroz, João; Sousa, Fani
P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.
Affinity purification and delivery of a p53-encoding plasmid DNA for gene mediated cancer therapy
Publication . Valente, Joana Filipa Abreu Pereira; Sousa, Fani Pereira de
Cancer is still one of the leading causes of death worldwide. In order to treat this scourge, gene therapy and DNA vaccination have been proposed as an alternative to the common treatments. Among the several gene abnormalities that could be responsible for the oncogenic process, the ones presented in p53 stands up. p53 is one of the most important tumour suppressor gene being considered the “genome guardian” since when occurs exposure to stressful stimuli it is activated through post-transcriptional modifications increasing its stability and activity. This gene is directly and indirectly implicated in different cellular functions, including DNA damage repair, cell cycle arrest in G1/S and apoptosis. Several studies shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into apoptosis and/or growth arrest, suggesting that a gene therapy approach for cancer treatment can be related to the re-establishment of the normal p53 function. Recently, the supercoiled (sc) conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than open circular conformation. Aiming to successfully isolate this bioactive isoform, several chromatographic techniques have been used, namely amino acids-based affinity chromatography. Concerning this chromatographic approach, in this doctoral work different amino acids like, Lmethionine, L-tyrosine, and arginine, were used to isolate the sc p53 encoding plasmid. From this work, it was achieved a better recovery yield and purity levels for O-Phospho-L-tyrosine when compared with L-methionine agarose matrix. Regarding the macroporous arginine resin, it was possible to recover the sc p53 encoding pDNA with high purity, and an increase of more than 50% in the dynamic binding capacity was achieved, when comparing with their homologous commercial agarose matrix. To understand the activity and the therapeutic effect induced by this sc isoform, different cell lines (HeLa, A549 and human dermal fibroblasts) were transfected with the pDNA purified either by the affinity purification strategy or with a commercial kit, for further in vitro evaluation. In particular, the cytotoxicity, the expression of the p53 transgene and the resulting apoptotic effect were evaluated in these in vitro cancer models. The results brought relevant information concerning the potential application of a sc p53 encoding plasmid in cancer gene therapy. To eliminate different cancer types, treatments must be applied systematically and therefore, must be targeted to cancer cells. Concerning this and, to enable an easy and safe systemic therapy, stable and non-viral gene vectors have been developed to encapsulate and deliver foreign genetic materials in specific cell types, such as cancerous cells. The use of non-viral vectors usually promotes low immune response, they are easily prepared, have a low production cost and also, they can be easily produced at large scale. Other important characteristic about these vectors is the ability to transfer different and large transgenes being also able to be stored for long periods due to their stability. Regarding this, in this doctoral work, chitosan (CH) and polyethyleneimine (PEI) were complexed with different plasmids in order to search for the suitable non-viral nanocomplex combination to be applied for gene therapy. Through the obtained results it was found that p53-encoding pDNA/PEI polyplexes demonstrate some toxicity in normal cells which could be a handicap for future therapeutic application of this nanocarriers. Also, from the track of the nanocarriers inside the cells, it was achieved a better transfection efficiency for the carriers delivering the smaller pDNA. The ability of the polyplexes to promote P53 protein expression was also evaluated using HeLa cancer cells and an increase of 54.2% and 32% of the P53 levels was achieved when CH and PEI nanocarriers were respectively applied. Overall, the scientific work performed in this thesis hopes to lead the scientific community to give more and more relevance to the use of the supercoiled pDNA isoform for gene therapy involving the reestablishment and restoration of the levels of p53. Moreover, it has been shown that the use of chromatography using specific amino acid ligands is a crucial factor in the final quality of the recovered sc pDNA sample, being this a predominant parameter for the accomplishment of the desired therapeutic results. Finally, the effort in the search and development of suitable non-viral vectors should stand up since it can guarantee the stability and activity of the sc p53 encoding plasmid during the delivery process.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/96809/2013