Development of an insect cell factory for the production of complex biopharmaceuticals using a synthetic biology approach

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011Insect cells, in particular the Spodoptera frugiperda Sf9 cell line, are a popular system for the production of biologically active recombinant proteins. However, the current technology uses baculovirus infection which has two main disadvantages: firstly, the recombinant gene is only expressed transiently during the infection cycle, after which cells die; secondly, due to the lytic nature of this system, the cellular protein processing machinery is severely compromised at the end of the infection cycle, affecting the correct formation of recombinant proteins whose expression is usually controlled by very-late baculovirus promoters. Stably transformed insect cell lines represent an alternative system for continuous protein production. However, their establishment is laborious, requiring the identification of cell clones that display the right expression properties due to random integration of the gene-of-interest. Furthermore, independently of the expression system, multimeric biopharmaceuticals continue to have very low yields, mainly because the stoichiometry between components is not properly reached in the producer cells. To overcome these issues, this report shows work towards the development of a novel Sf9 cell factory combining (i) recombinase mediated cassette exchange (RMCE) for targeted gene integration and (ii) a tetracycline (Tet) inducible system for the modulation of gene expression levels. We started by evaluating the strength of different promoters to drive GFP expression in Sf9 cells as well as different transfection protocols. The baculovirus promoter OpIE2 allowed the strongest expression when compared to others, and the efficiency of transfection was significantly higher using the lipotransfection method. From the different transfection protocols, 4 cell populations with distinct fluorescence distributions were obtained after 3 weeks under hygromycin selection. These populations were independently subject to limiting dilution and isolated cell clones were analyzed by southern blot to screen for single-copy integration of the tagging GFP construct. While the majority of clones were single copy, cassette exchange was performed in a few clones by co-transfection with flippase and the target plasmid containing dsRed. Cells were selected in the presence of neomycin and cassette exchange was confirmed by PCR of genomic DNA, revealing the absence of tagging cassette and the presence of the target cassette in the some locus. In parallel, the functionality of a tetracycline inducible system in Sf9 cells was evaluated by substituting mammalian promoters with insect cell specific promoters. However, transactivation was suboptiomal, requiring additional changes to the original system. In summary, this work reports for the first time the implementation of a RMCE system in Sf9 cells and a preliminary assessment of Tet transactivation in these cells. This new cell line will be a major breakthrough since it will combine the advantages inherent to Sf9 cell growth, continuous protein production, the ability of re-using a well characterized locus for targeted DNA integration and inducible gene expression.A cultura de células de insecto conjugada com a infecção por baculovirus representa um dos sistemas biológicos de eleição para a produção de proteínas recombinantes biologicamente activas. Há vários biofármacos em ensaios clínicos e alguns já no mercado, como é o caso da vacina contra o vírus do papiloma humano (Cervarix, GSK), que utilizam este tipo tipo de sistema de expressão. No entanto, o uso de baculovirus representa um sistema de expressão transiente / lítico inerente ao processo de infecção. Em contraste, quando estavelmente transformadas, as células de insecto podem ser usadas como um sistema de expressão contínuo e não-lítico. Contudo, o desenvolvimento de linhas celulares estáveis é significativamente demorado e laborioso, sendo necessário identificar e isolar clones que exibam elevadas taxas de expressão. A grande variabilidade existente nos níveis de expressão entre clones deve-se ao chamado efeito de posição, ou seja a aleatoriedade de integração no genoma da célula. Além disso, e independentemente do sistema de expressão, quando se pretende expressar biofármacos multiméricos, como as particulas semelhantes a vírus, estes continuam a ter rendimentos muito baixos. A estequiometria entre os componentes é um factor chave e, quando não é alcançada adequadamente nas células produtoras, resulta na formação de uma grande percentagem de partículas incorrectamente formadas. De forma a superar estas limitações, neste trabalho é iniciado o desenvolvimento de uma linha celular derivada de Spodoptera frugiperda, usando uma combinação de duas tecnologias: (i) sistema de troca de cassete mediada por recombinase (RMCE) e (ii) circuito transcricional indutível. Os sistemas RMCE utilizam um eficiente e avançado processo de troca génica localizada por intermédio de uma reacção de recombinação. Anteriormente implementada em células de mamífero, este tipo de tecnologia foi agora aplicada num cenário de expressão estável em células de insecto com o objectivo de permitir o uso repetido de locais genómicos pré-caracterizados com elevada taxa de expressão. Já os circuitos indutíveis sintéticos, como o sistema de indução por tetraciclina, permitem ajustar a expressão de diferentes genes de interesse por intermédio de um indutor, facilitando a produção de produtos multiméricos correctamente formados. O trabalho desenvolvido nesta dissertação de mestrado começou por centrar-se na análise de diferentes promotores no controlo da expressão de genes repórter na linha celular Sf9. Foram estudados promotores com diferentes origens mas com uma base de funcionamento comum em células de insecto, como os promotores de baculovirus OpIE2 e OpIE1, o promotor da metalotioneína e o das proteínas de choque térmico (hsp70) de Drosophila. Em resultado, verificou-se que o promotor OpIE2 superou consideravelmente os restantes na expressão de eGFP. Por outro lado, o promotor da metalotioneína induzido por cobre revelou ser um sistema desadequado para expressão de proteínas de interesse em células Sf9, uma vez que apenas para doses citotóxicas se verificou uma expressão de eGFP mensurável. Para o funcionamento do sistema RMCE são necessários dois vectores distintos e complementares (Tagging / Target). Em ambos os vectores foi utilizado o promotor OpIE2 para a expressão das proteínas repórter (diferentes nos dois vectores: Tagging – dsRed; Target - eGFP), e o promotor OpIE1foi usado para a expressão do agente de selecção, necessário para garantir a expressão estável dos dois vectores no genoma da célula (Tagging – higromicina; Target -neomicina). Enquanto a integração do plasmídeo Tagging no genoma se dá através de um processo aleatório, a integração do Target é o resultado da troca bem-sucedida por recombinação enzimática. A selecção pelo agente neomicina assegura o sucesso da troca génica uma vez que o gene de resistência é activado pela inserção de um códão de iniciação (ATG) presente apenas no Target. Antes do processo de troca, é importante que apenas uma cópia da cassete Tagging seja integrada no genoma da célula para que o sistema RMCE seja funcional. Utilizaram-se dois métodos em paralelo para transfectar as células Sf9 com o plasmídeo Tagging: lipotransfecção usando cellfectin e eletroporação, donde resultaram quatro populações celulares com distribuições de fluorescência distintas. Para cada população de células foi posteriormente realizada uma diluição progressiva para a obtenção de clones produtores, os quais foram analisados por citometria de fluxo para comparar o nível de expressão de eGFP. Posteriomente, foi implementado um protocolo de Southern blot para analisar o número de cópias integradas da cassete Tagging em cada clone., Dos nove clones analisados, apenas um revelou ter mais do que uma cópia. Em alguns dos clones com apenas uma cópia procedeu-se à co-transfecção da construção Target com a enzima recombinase (flippase). Foi possível seleccionar células resistentes à neomicina sugerindo o sucesso da troca de cassetes, tendo sido mais tarde confirmado por meio de PCR do DNA genómico e de RT-PCR do RNA dos genes repórteres.No que toca o circuito indutível de transcrição, o trabalho conduzido teve como objectivo fazer uma avaliação preliminar da sua funcionalidade em células Sf9 antes da implementação directa na cassete de recombinação. No entanto, os resultados revelaram uma fraca indução por parte do agente tetraciclina, justificando a necessidade de uma optimização do sistema original. Em conclusão, com este trabalho reportamos pela primeira vez a implementação bem sucedida de um sistema de troca de cassete por intermédio de recombinação enzimática em células Sf9. Esta tecnologia representa um grande avanço na produção de novas linhas celulares de insecto, combinando as vantagens inerentes à cultura deste tipo de células com a possibilidade de atingir elevados níveis de expressão contínua de diferentes proteínas recombinantes através da fácil integração de genes de interesse no genoma celular

PROPOSTA DE MODELO PARA IMPLEMENTAÇÃO E DESENVOLVIMENTO DA CONTABILIDADE ANALÍTICA NA UNIVERSIDADE DE ÉVORA (PORTUGAL).

Desde a publicação, em 2000, do Plano Oficial de Contabilidade para o Setor da Educação (POC-Educação) que as universidades portuguesas estão obrigadas a implementar um sistema de contabilidade analítica que permita, entre outros, o cálculo do custo das atividades intermédias e finais, a determinação o custo referente aos serviços internos, o custo por curso, por disciplina e por aluno, o custo de cada projeto de investigação, o custo de outras atividades internas e da prestação de serviços à comunidade, a análise da eficiência no uso dos recursos financeiros públicos e a determinar e analisar desvios entre os custos/proveitos previsionais e os custos/proveitos, constituindo, em simultâneo, um sistema de apoio à gestão. O POC-Educação estabelece um modelo orientador para a contabilidade analítica nas universidades, mas são as especificidades e a organização de cada instituição que determinam a forma que a contabilidade analítica vai tomar. O presente trabalho apresenta o modelo de contabilidade analítica desenvolvido para a Universidade de Évora, tendo em linha de conta os objetivos que presidem à sua implementação e que foram anteriormente enunciados. Far-se-á referência aos principais problemas identificados e às propostas identificadas para a sua resoluçã

Insect cell platforms for production of pseudo-typed VLPs for drug and vaccine development

Conformational-complex membrane proteins (MPs) are vaccine/drug targets in many diseases, but drug and vaccine development has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of ordered nanoscale architectures such as virus-like particles (VLPs), preserving their native lipidic environment. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect cell platforms for fast production of enveloped VLPs pseudo-typed with target MPs. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept (PoC) demonstrated using two membrane proteins, the influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes were designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve HIV Gag-VLPs production in the developed stable insect cells. Under hypothermic culture conditions, adapted cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the later alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. PoC was successfully demonstrated in 0.5 L stirred-tank bioreactors. Profiting from the platforms developed above, a modular system comprising stable and baculovirus-mediated expression in insect cells was established for the production of a multi-HA influenza VLP as vaccine candidate that otherwise could not be obtained due to baculovirus vector instability. By combining stable with transient expression systems, we could rationally distribute the number of genes to be expressed per platform and thus generate the target VLP for subsequent animal studies. In addition, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth resulting in a 4-fold increase in HA titers per mL. PoC was successfully demonstrated in 2 L stirred-tank bioreactors. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate drug and vaccine development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

DEEP BRAIN STIMULATION COMO ALTERNATIVA PARA O TRATAMENTO DE DISTÚRBIOS NEUROPSIQUIÁTRICOS

A Deep brain stimulation é uma técnica de neuroestimulação que vem se destacando no tratamento de alguns distúrbios psiquiátricos, como a doença de Parkinson, a depressão resistente ao tratamento convencional e a epilepsia. O objetivo com este trabalho foi analisar as principais aplicações e identificar as potencialidades e limitações da técnica. A coleta de dados realizou-se por meio de pesquisa bibliográfica de periódicos, revistas e jornais nas bases de dados Scopus, MEDLINE/PubMed, Science Citation Index, Expanded Web of Science e ScienceDirect, publicados depois de 2000. A literatura demonstra que a DBS é capaz de fornecer notáveis benefícios terapêuticos para pacientes com uma variedade de distúrbios neuropsiquiátricos não responsivos às terapias convencionais. A técnica também fornece uma importante ferramenta de pesquisa das conexões cerebrais. Sua estimulação pode ser ajustada como diversas atividades cerebrais são medidas, na tentativa de entender as bases da fisiopatologia dos distúrbios psiquiátricos e seus mecanismos de ação terapêuticos. Os principais riscos da técnica incluem a ocorrência de infecções e hemorragias, e as principais limitações são o desconhecimento do mecanismo de ação exato da DBS, a vida útil dos hardwares e seu elevado preço de instalação e manutenção e os elevados custos do acompanhamento médico pós-operatório dos pacientes. A superação dessas limitações por meio de pesquisa e investimentos pode, no futuro, tornar a DBS viável como alternativa para o estudo da fisiopatogenia e tratamento das doenças neuropsiquiátricas.Palavras-chave: Estimulação cerebral profunda. Parkinson. Neuromodulação. Depressão. Epilepsia

Bioprocess engineering of insect cells for accelerating vaccines development

Insect cells emerged as a powerful and versatile platform for vaccines production, mostly using the lytic baculovirus expression vector system (BEVS). Stable expression in such hosts has been increasingly explored to circumvent BEVS-related drawbacks, but protein titers achieved to date are still seemingly low. The design of new or improved cell factories and bioprocess intensification strategies are therefore necessary to increase productivities and thus accelerate implementation of stable insect cell lines as a fast, cost-effective platform for vaccines manufacturing. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect (Sf-9 and High Five) cell platforms for fast production of enveloped virus-like particles (VLPs). Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins: the influenza HA protein (for vaccines) and the human beta-2 adrenergic receptor (for drug screening or antibody discovery). Aiming to improve production yields in developed stable cell lines, two bioprocess engineering schemes were evaluated (either individually or in combination): (i) adaptive laboratory evolution of insect cells to hypothermic culture conditions, and (ii) supplementation of insect cell cultures with productivity enhancers. The stable cell line expressing HIV Gag-VLPs was used as model. Under hypothermic culture conditions, adapted Sf-9 cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the latter alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. No synergistic effect was observed when combining adapted cells and supplementation with productivity enhancers. Production of HIV Gag-VLPs was successfully scaled-up to stirred-tank bioreactors. The adapted cell line was then pseudo-typed with influenza HA protein for production of Gag-HA VLPs, and their performance benchmarked against (i) parental Sf-9 cells stably expressing Gag-HA VLPs and (ii) insect cells-BEVS, both cultured under standard temperature conditions (27C). Adapted cells showed increased production of Gag-HA VLPs when compared to parental/stable cells, corroborating previously obtained data, but still lower when compared to insect cells-BEVS. Bioprocess intensification strategies are currently under in-house testing to further improve yields of adapted cells and thus shorten the gap between stable insect cells and IC-BEVS. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist and accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Differential lipid accumulation on HepG2 cells triggered by palmitic and linoleic fatty acids exposure

Lipid metabolism pathways such as β-oxidation, lipolysis and, lipogenesis, are mainly associated with normal liver function. However, steatosis is a growing pathology caused by the accumulation of lipids in hepatic cells due to increased lipogenesis, dysregulated lipid metabolism, and/or reduced lipolysis. Accordingly, this investigation hypothesizes a selective in vitro accumulation of palmitic and linoleic fatty acids on hepatocytes. After assessing the metabolic inhibition, apoptotic effect, and reactive oxygen species (ROS) generation by linoleic (LA) and palmitic (PA) fatty acids, HepG2 cells were exposed to different ratios of LA and PA to study the lipid accumulation using the lipophilic dye Oil Red O. Lipidomic studies were also carried out after lipid isolation. Results revealed that LA was highly accumulated and induced ROS production when compared to PA. Lipid profile modifications were observed after LA:PA 1:1 (v/v) exposure, which led to a four-fold increase in triglycerides (TGs) (mainly in linoleic acid-containing species), as well as a increase in cholesterol and polyunsaturated fatty acids (PUFA) content when compared to the control cells. The present work highlights the importance of balancing both PA and LA fatty acids concentrations in HepG2 cells to maintain normal levels of free fatty acids (FFAs), cholesterol, and TGs and to minimize some of the observed in vitro effects (i.e., apoptosis, ROS generation and lipid accumulation) caused by these fatty acids.info:eu-repo/semantics/publishedVersio

Phytosterols and novel triterpenes recovered from industrial fermentation coproducts exert in vitro anti‐inflammatory activity in macrophages

The unstoppable growth of human population that occurs in parallel with all manufacturing activities leads to a relentless increase in the demand for resources, cultivation land, and energy. In response, currently, there is significant interest in developing strategies to optimize any available resources and their biowaste. While solutions initially focused on recovering biomolecules with applications in food, energy, or materials, the feasibility of synthetic biology in this field has been demonstrated in recent years. For instance, it is possible to genetically modify Saccharomyces cerevisiae to produce terpenes for commercial applications (i.e., against malaria or as biodiesel). But the production process, similar to any industrial activity, generates biowastes containing promising biomolecules (from fermentation) that if recovered may have applications in different areas. To test this hypothesis, in the present study, the lipid composition of by‐products from the industrial production of β‐farnesene by genetically modified Saccharomyces cerevisiae are studied to identify potentially bioactive compounds, their recovery, and finally, their stability and in vitro bioactivity. The assayed biowaste showed the presence of triterpenes, phytosterols, and 1‐ octacosanol which were recovered through molecular distillation into a single fraction. During the assayed stability test, compositional modifications were observed, mainly for the phytosterols and 1‐octacosanol, probably due to oxidative reactions. However, such changes did not affect the in vitro bioactivity in macrophages, where it was found that the obtained fraction decreased the production of TNF‐α and IL‐6 in lipopolysaccharide (LPS)‐induced inflammation.info:eu-repo/semantics/publishedVersio

Insect cells platforms for fast production of Pseudo-Typed VLPs for drug and vaccine development

Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays. Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. For the latter, we will present recent results on the feasibility of combining stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs towards the development of an “universal” vaccine. This strategy surpasses standard methods for production of multivalent Influenza VLPs such as coinfections or the use of larger, unstable vectors. Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as receptor display particles for drug screening or antibody discovery. Acknowledgments: Funding from European Commission (Project EDUFLUVAC; Grant nr. 602640) and Fundação para a Ciência e a Tecnologia through the project EXPL/BBB-BIO/1541/2013 and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012