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Otimização do bioprocesso de obtenção da vacina de DNA minicircular contra o cancro do colo do útero
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Resumo(s)
O cancro do colo do útero é um dos tumores mais prevalentes entre as mulheres a nível
mundial, estando maioritariamente associado à infeção persistente pelo Vírus do
Papiloma Humano (HPV), sobretudo pelos genótipos de alto risco HPV-16 e HPV-18.
Nestes casos, as oncoproteínas virais E6 e E7 desempenham um papel central na
transformação maligna, ao promoverem a degradação das proteínas supressoras de
tumor p53 e pRB, respetivamente.
As vacinas de DNA têm emergido como uma estratégia promissora para a prevenção e
tratamento de diversas doenças infeciosas e oncológicas. Esta abordagem baseia-se na
introdução de DNA exógeno em células hospedeiras, com o objetivo de induzir a
expressão de antigénios específicos capazes de desencadear uma resposta imunitária
preventiva e terapêutica. Entre os vetores não virais utilizados, o DNA plasmídico
(pDNA) tem sido o mais explorado, devido o seu baixo custo e facilidade de produção.
No entanto, a presença de elementos bacterianos, como genes de resistência a
antibióticos, pode desencadear respostas imunitárias indesejadas e reduzir a segurança
da sua utilização.
Como alternativa, o DNA minicircular (mcDNA) constitui uma geração mais avançada
de vetores, resultante de uma recombinação intramolecular do plasmídeo parental (PP)
numa cultura bacteriana, que se divide em duas moléculas filhas, o miniplasmídeo (mP)
(constituído somente pelos genes procariotas necessários durante o processo de
produção) e o mcDNA (sendo formado exclusivamente pela cassete de expressão
eucariótica). Esta característica confere-lhe vantagens significativas face ao pDNA
convencional, incluindo maior segurança, menor dimensão, eficiência acrescida de
transfeção, maior estabilidade intracelular e expressão génica mais duradoura,
traduzindo-se num elevado potencial terapêutico. Contudo, a sua aplicação clínica
requer o desenvolvimento de métodos de produção e purificação robustos, capazes de
cumprir os requisitos das entidades reguladoras.
Neste contexto, o presente trabalho teve como objetivo explorar a cromatografia de
interação hidrofóbica (HIC) como estratégia para a purificação de pDNA e mcDNA.
Numa primeira fase, foram estudadas as condições de separação entre pDNA e RNA,
seguindo-se a avaliação da seletividade entre mcDNA e RNA. Observou-se que o mcDNA
tende a eluir em fases mais precoces, o que pode estar associado à sua menor
hidrofobicidade relativamente ao RNA. Os ensaios de quantificação permitiram avaliar a pureza e recuperação dos vetores de
DNA em função das estratégias exploradas, verificando a sua conformidade com os
parâmetros definidos pelas agências reguladoras para as condições otimizadas. Contudo,
não foi possível obter uma separação clara entre isoformas de DNA nem entre mcDNA e
PP. Em suma, os resultados demonstram que a HIC constitui uma abordagem
promissora para a purificação de mcDNA, fornecendo uma base sólida para otimizações
futuras e para a consolidação deste vetor como ferramenta terapêutica de nova geração.
Cervical cancer is one of the most prevalent malignancies among women worldwide, being predominantly associated with persistent infection by Human Papillomavirus (HPV), particularly the high-risk genotypes HPV-16 and HPV-18. In these cases, the viral oncoproteins E6 and E7 play a central role in malignant transformation by promoting the degradation of the tumor suppressor proteins p53 and pRB, respectively. DNA vaccines have emerged as a promising strategy for the prevention and treatment of various infectious and oncological diseases. This approach is based on the introduction of exogenous DNA into host cells, with the aim of inducing the expression of specific antigens capable of triggering a preventive and therapeutic immune response. Among non-viral vectors, plasmid DNA (pDNA) has been the most widely explored due to its low cost and ease of production. However, the presence of bacterial elements, such as antibiotic resistance genes, can trigger undesired immune responses and compromise its safety. As an alternative, minicircle DNA (mcDNA) represents a more advanced generation of vectors, generated through intramolecular recombination of the parental plasmid (PP) in bacterial cultures, which divides into two daughter molecules, the miniplasmid (mP) (containing only the prokaryotic genes required during the production process) and the mcDNA (composed exclusively of the eukaryotic expression cassette). This feature provides mcDNA with significant advantages over conventional pDNA, including greater safety, smaller size, enhanced transfection efficiency, higher intracellular stability, and prolonged gene expression, resulting in increased therapeutic potential. Nonetheless, its clinical application requires the development of robust production and purification methods capable of meeting the requirements of regulatory authorities. In this context, the present work aimed to investigate hydrophobic interaction chromatography (HIC) as a strategy for the purification of pDNA and mcDNA. Initially, separation conditions between pDNA and RNA were assessed, followed by the evaluation of the selectivity between mcDNA and RNA. It was observed that mcDNA tends to elute at earlier stages, which may be related to its lower hydrophobicity compared to RNA. The quantification assays made it possible to evaluate the purity and recovery of the DNA vectors according to the strategies explored, verifying their compliance with the parameters defined by regulatory agencies under the optimized conditions. However clear separation of DNA isoforms or between mcDNA and PP was not achieved. In summary, the results demonstrate that HIC is a promising approach for mcDNA purification, providing a solid foundation for future optimizations and for consolidating this vector as a next-generation therapeutic tool.
Cervical cancer is one of the most prevalent malignancies among women worldwide, being predominantly associated with persistent infection by Human Papillomavirus (HPV), particularly the high-risk genotypes HPV-16 and HPV-18. In these cases, the viral oncoproteins E6 and E7 play a central role in malignant transformation by promoting the degradation of the tumor suppressor proteins p53 and pRB, respectively. DNA vaccines have emerged as a promising strategy for the prevention and treatment of various infectious and oncological diseases. This approach is based on the introduction of exogenous DNA into host cells, with the aim of inducing the expression of specific antigens capable of triggering a preventive and therapeutic immune response. Among non-viral vectors, plasmid DNA (pDNA) has been the most widely explored due to its low cost and ease of production. However, the presence of bacterial elements, such as antibiotic resistance genes, can trigger undesired immune responses and compromise its safety. As an alternative, minicircle DNA (mcDNA) represents a more advanced generation of vectors, generated through intramolecular recombination of the parental plasmid (PP) in bacterial cultures, which divides into two daughter molecules, the miniplasmid (mP) (containing only the prokaryotic genes required during the production process) and the mcDNA (composed exclusively of the eukaryotic expression cassette). This feature provides mcDNA with significant advantages over conventional pDNA, including greater safety, smaller size, enhanced transfection efficiency, higher intracellular stability, and prolonged gene expression, resulting in increased therapeutic potential. Nonetheless, its clinical application requires the development of robust production and purification methods capable of meeting the requirements of regulatory authorities. In this context, the present work aimed to investigate hydrophobic interaction chromatography (HIC) as a strategy for the purification of pDNA and mcDNA. Initially, separation conditions between pDNA and RNA were assessed, followed by the evaluation of the selectivity between mcDNA and RNA. It was observed that mcDNA tends to elute at earlier stages, which may be related to its lower hydrophobicity compared to RNA. The quantification assays made it possible to evaluate the purity and recovery of the DNA vectors according to the strategies explored, verifying their compliance with the parameters defined by regulatory agencies under the optimized conditions. However clear separation of DNA isoforms or between mcDNA and PP was not achieved. In summary, the results demonstrate that HIC is a promising approach for mcDNA purification, providing a solid foundation for future optimizations and for consolidating this vector as a next-generation therapeutic tool.
Descrição
Palavras-chave
Dna Plasmídico Cancro do Colo do Útero Cromatografia de Interação Hidrofóbica Dna Minicircular Hpv