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Abstract(s)
O cancro do colo do útero é um dos cancros mais prevalentes em mulheres em todo o
mundo. Este tipo de cancro é causado pelo Vírus do Papiloma Humano (HPV),
nomeadamente pelos seus genótipos de alto risco mais virulentos, o HPV-16 e o HPV-18.
Após uma infeção persistente, as oncoproteínas E6 e E7 expressas por esses vírus são
responsáveis pela inibição e degradação das proteínas supressoras de tumor p53 e pRB,
respetivamente.
A terapia génica tem sido explorada nos últimos anos devido ao seu potencial no
tratamento de doenças adquiridas e incuráveis. Esta abordagem terapêutica baseia-se na
entrega de uma sequência específica de DNA exógeno a uma célula hospedeira, para
alterar a função de uma determinada molécula que pode desempenhar um papel
importante no desenvolvimento de uma doença. O DNA minicircular (mcDNA) tornouse um vetor de DNA não viral promissor, porque carece de sequências procarióticas
consideradas prejudiciais (como genes de resistência a antibióticos, origem de replicação
e motivos CpG, geralmente associados a questões de segurança) e, porque é um vetor de
menor dimensão quando comparado com o DNA plasmídico convencional (pDNA),
apresentando assim melhor eficiência de transfeção, permanência celular e expressão
génica. Adicionalmente, os micro RNAs foram identificados como silenciadores de genes
que degradam ou inativam um RNA mensageiro específico. Particularmente, foi
comprovado que o micro-RNA-375 (miR-375) silencia a expressão das oncoproteínas E6
e E7 em células infetadas por HPV de alto risco.
Assim, o presente trabalho visa a produção e purificação de um vetor de mcDNA que
codifica o gene pri-miR-375, que futuramente poderá ser utilizado para terapia génica
contra o cancro do colo do útero. No entanto, o processo biotecnológico para obtenção
de mcDNA envolve várias etapas (nomeadamente centrifugações; concentração com
solventes orgânicos; clarificação com altas concentrações de sal; métodos de purificação
que consistem em cromatografia de exclusão molecular ou cromatografia de afinidade
que são demorados ou não estão disponíveis comercialmente, respetivamente) que
tornam o processo downstream bastante caro do ponto de vista da indústria
farmacêutica, mostrando assim a necessidade do desenvolvimento de estratégias
alternativas.
Neste sentido, a presente dissertação sugere uma nova abordagem de recuperação do
mcDNA, baseada no tratamento de lisado bruto com terra diatomácea para filtração
direta e remoção de todos os detritos celulares e impurezas precipitadas, combinada com uma segunda etapa usando uma membrana cromatográfica Sartobindâ para purificar o
DNA eliminando o RNA. Esta abordagem permite a substituição de várias etapas préestabelecidas para a preparação do lisado, nomeadamente a centrifugação dos restos
celulares no final da lise alcalina, a etapa de concentração com isopropanol (e respetiva
centrifugação) e a etapa de clarificação com sulfato de amónio (e respetiva centrifugação)
poupando deste modo recursos, muito tempo ao processo global e, diminuindo o impacto
ambiental. Na etapa de purificação, foi explorada a cromatografia de troca aniónica
usando duas membranas, a Sartobind® Q75 e a Sartobind® D75. Após alguns estudos
preliminares das condições de equilíbrio e eluição, foi otimizada a eliminação de grande
quantidade de RNA diretamente no flowthrough, depois do carregamento da amostra de
lisado simplificado, e o restante RNA foi eliminado durante um gradiente crescente de
NaCl. A eficiência de ambas as etapas deste processo combinado foi comprovada através
da quantificação das amostras recuperadas no final de cada etapa, usando uma coluna
analítica CIMac™ pDNA e por fim a densidade de bandas do gel de agarose.
No geral, os estudos realizados demonstraram a eficiência da estratégia combinada. Esta
nova abordagem de preparação do lisado simplificada torna o processo mais rápido,
passando a ser realizado em cerca de 40 min, enquanto que a abordagem convencional
demora aproximadamente 180 min. Quanto à purificação do lisado, com a membrana
Sartobind® Q75 foi possível obter uma recuperação quase total do DNA (» 100%), com
uma pureza superior a 99%. No entanto, ainda é necessário realizar mais alguns estudos
para melhorar o isolamento e o rendimento máximo do vetor mcDNA recuperado.
Assim, espera-se que esta estratégia combinada possa ser ampliada e aplicada com
sucesso na indústria farmacêutica para purificar vetores de DNA de forma rápida e
económica.
Cervical cancer (CC) is one of the most prevalent cancers in women worldwide. This type of cancer is caused by the human papillomavirus (HPV), namely by its most virulent high-risk genotypes, HPV-16 and HPV-18. After a persistent infection, E6 and E7 oncoproteins expressed by this virus are responsible for the inhibition and degradation of p53 and pRB tumor suppressor proteins. Gene therapy has been explored in last years due to its potential in the treatment of acquired and incurable diseases. This therapeutic approach is based on delivering a specific exogenous DNA to a host cell, to change the function of a particular molecule that may play an important role in the development of a disease. Minicircular DNA (mcDNA) has become a promising non-viral DNA vector, because it lacks prokaryotic sequences, which are considered harmful (such as antibiotic resistance genes, replication origin, and CpG motifs that are generally associated with safety issues) and, because it is a small dimension vector when compared with the conventional plasmid DNA (pDNA), thus presenting better transfection efficiency, cell permanence and gene expression. Additionally, the micro RNAs have been identified as gene silencers that degrade or inactivate a specific target messenger RNA. Particularly, micro-RNA-375 (miR-375) has been proven to silence the expression of E6 and E7 oncoproteins in high-risk HPVinfected cells. Thus, the present work aims the production and purification of a mcDNA vector that encodes the pri-miR-375 gene, which can be used for gene therapy against CC in the future. However, the biotechnological process to obtain mcDNA involves several steps (namely centrifugations; concentration with organic solvents; clarification with high salt concentrations; purification methods consisting of molecular exclusion or affinity chromatography that are time-consuming or are not available commercially, respectively) that make the downtream process quite expensive from the pharmaceutical industry's point of view, leading that the development of alternative strategies become essential. Therefore, this Master thesis a new mcDNA recovery approach, based on the crude lysate treatment with diatomaceous earth to promote direct filtration and removal of all cell debris and precipitated impurities. Moreover, this step will be combined with a second step using a Sartobindâ chromatographic membrane to purify DNA and eliminate the RNA. This approach allows the substitution of several steps pre-established for the lysate preparation, namely the cell debris centrifugation at the end of alkaline lysis, the isopropanol concentration step and respective centrifugation, and the ammonium sulfate clarification step and respective centrifugation, saving a lot of time in the global process, resources and environmental impact. In the purification step, anion exchange chromatography was explored using two Sartobind® membranes, Sartobind® Q75 and Sartobind® D75. After some preliminary studies of equilibrium and elution conditions, it was optimized the elimination of large amount of RNA directly into the flowthrough, after loading the simplified lysate sample, and the remaining RNA was eliminated during an increasing NaCl gradient. The efficiency of both steps of this combined process was proven through the quantification of recovered samples at the end of each step, using a well-studied CIMac™ pDNA analytical column and band density of the agarose electrophoresis gel. Overall, both studies prove the combined strategy’s efficiency. This new simpliflied lysate preparation approach makes the process faster, starting in about 40 min, while the convencional approach takes approximately 180 min. As for the purification of lysate, with the membrane Sartobind® Q75 was possible obted an almost total recovery of DNA (» 100%), with a purity greater than 99%. Although further studies are needed to improve the isolation and maximize the yield of the recovered mcDNA vector. Thus, it is expected that this combined strategy can be scaled-up and successfully applied in the pharmaceutical industry to purify the DNA vectors in a faster and more economically way.
Cervical cancer (CC) is one of the most prevalent cancers in women worldwide. This type of cancer is caused by the human papillomavirus (HPV), namely by its most virulent high-risk genotypes, HPV-16 and HPV-18. After a persistent infection, E6 and E7 oncoproteins expressed by this virus are responsible for the inhibition and degradation of p53 and pRB tumor suppressor proteins. Gene therapy has been explored in last years due to its potential in the treatment of acquired and incurable diseases. This therapeutic approach is based on delivering a specific exogenous DNA to a host cell, to change the function of a particular molecule that may play an important role in the development of a disease. Minicircular DNA (mcDNA) has become a promising non-viral DNA vector, because it lacks prokaryotic sequences, which are considered harmful (such as antibiotic resistance genes, replication origin, and CpG motifs that are generally associated with safety issues) and, because it is a small dimension vector when compared with the conventional plasmid DNA (pDNA), thus presenting better transfection efficiency, cell permanence and gene expression. Additionally, the micro RNAs have been identified as gene silencers that degrade or inactivate a specific target messenger RNA. Particularly, micro-RNA-375 (miR-375) has been proven to silence the expression of E6 and E7 oncoproteins in high-risk HPVinfected cells. Thus, the present work aims the production and purification of a mcDNA vector that encodes the pri-miR-375 gene, which can be used for gene therapy against CC in the future. However, the biotechnological process to obtain mcDNA involves several steps (namely centrifugations; concentration with organic solvents; clarification with high salt concentrations; purification methods consisting of molecular exclusion or affinity chromatography that are time-consuming or are not available commercially, respectively) that make the downtream process quite expensive from the pharmaceutical industry's point of view, leading that the development of alternative strategies become essential. Therefore, this Master thesis a new mcDNA recovery approach, based on the crude lysate treatment with diatomaceous earth to promote direct filtration and removal of all cell debris and precipitated impurities. Moreover, this step will be combined with a second step using a Sartobindâ chromatographic membrane to purify DNA and eliminate the RNA. This approach allows the substitution of several steps pre-established for the lysate preparation, namely the cell debris centrifugation at the end of alkaline lysis, the isopropanol concentration step and respective centrifugation, and the ammonium sulfate clarification step and respective centrifugation, saving a lot of time in the global process, resources and environmental impact. In the purification step, anion exchange chromatography was explored using two Sartobind® membranes, Sartobind® Q75 and Sartobind® D75. After some preliminary studies of equilibrium and elution conditions, it was optimized the elimination of large amount of RNA directly into the flowthrough, after loading the simplified lysate sample, and the remaining RNA was eliminated during an increasing NaCl gradient. The efficiency of both steps of this combined process was proven through the quantification of recovered samples at the end of each step, using a well-studied CIMac™ pDNA analytical column and band density of the agarose electrophoresis gel. Overall, both studies prove the combined strategy’s efficiency. This new simpliflied lysate preparation approach makes the process faster, starting in about 40 min, while the convencional approach takes approximately 180 min. As for the purification of lysate, with the membrane Sartobind® Q75 was possible obted an almost total recovery of DNA (» 100%), with a purity greater than 99%. Although further studies are needed to improve the isolation and maximize the yield of the recovered mcDNA vector. Thus, it is expected that this combined strategy can be scaled-up and successfully applied in the pharmaceutical industry to purify the DNA vectors in a faster and more economically way.
Description
Keywords
Cancro do Colo do Útero Cromatografia de Troca Aniónica Dna Minicircular Membranas Cromatográficas Mirna-375 Terapia Génica Terra Diatomácea