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The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and VLPs. More recently IC-BEVS has been also used as an alternative to produce adeno-associated virus (AAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of recombinant AAV using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-seq (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated to cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, this being linked to the expression of baculovirus genes as well as to differences in AAV transgenes ( rep, cap and gfp) expression. Noteworthy, at 24 hours post-infection (hpi) only 29 % of cells showed to enclose all three necessary AAV transgenes to produce packed AAV particles, indicating limitations of the dual baculovirus system. In addition, the trajectory analysis herein performed highlighted biological processes such as protein folding, metabolic processes, translation and stress response has been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight for rational cell and process engineering towards improved AAV production in IC-BEVS.

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.

Additional file 1: Supplementary information (Supplementary Figures S1–S9, Supplementary Table S1 and Supplementary Methods).

Additional file 1: Supplementary information (Supplementary Figures S1–S9, Supplementary Table S1 and Supplementary Methods).

"The insect cell-baculovirus expression vector system (IC-BEVS) has been established as an important platform for the manufacturing of therapeutical products, including vectors for gene therapy such as recombinant adeno-associated virus (rAAV). Nevertheless, further characterisation of the underlying biological mechanisms of the IC-BEVS as well as improved engineering strategies are needed to further leverage this system towards optimal product titers and quality. In this thesis, transcriptome analysis approaches (bulk and single-cell) were applied to understand the impact on Sf9 cells of a dual baculovirus co-infection process to produce rAAV. In addition, an improved CRISPR-Cas9 delivery and gene knockout strategy was established to enhance the efficiency of target gene knockout in IC-BEVS." Associate Laboratory LS4FUTURE A/P/0087/2020

Ovarian cancer is the first cause of death among gynaecological malignancies in women. Most patients are diagnosed at late stages and present ascites, the accumulation of ascitic fluid in the perito- neal cavity, associated to metastasis and poor prognosis. Ascitic fluids are enriched in pro-tumorigenic components, such as extracellular vesicles (EVs) involved in the tumour microenvironment intercellular crosstalk. Despite metabolic alterations being a hallmark of cancer and highly correlated with sensitivity to treatment, the metabolite cargo is still the least explored component of EVs in ovarian cancer. The aim of this thesis was to characterize the metabolic signature of EVs isolated from ascitic fluids in ovarian cancer patients. Methods for the characterization of EVs were first implemented using EVs isolated from an ovarian cancer cell line (ES-2), namely for the presence of EV and non-EV com- ponents by Western blot, as well as the analytical platform for metabolomics and lipidomics by LC- MS. Ultimately, EVs were isolated using differential centrifugation from ovarian cancer biofluids, in- cluding ascitic fluids and peritoneal washes as controls. Isolated and discarded fractions were charac- terized by Western blot; by transmission electron microscopy (TEM) for morphology, and by nanopar- ticle tracking analysis (NTA) for particle concentration and size, following the International Society for Extracellular Vesicles (ISEV) guidelines. We demonstrated the recovery of EVs and increasing purity of isolates and confirmed the pres- ence of EVs on biofluids. The metabolic characterization of ES-2 EVs suggested an enrichment in metabolic pathways involved in cancer processes. This methodology for characterization of the meta- bolic content of EVs can be further applied in EVs from ascitic fluids and peritoneal washes, which may elucidate the role of EVs as mediators of metabolic crosstalk in ovarian cancer tumour microenvi- ronment and eventually uncover potential biomarkers for ovarian cancer prognosis. O cancro do ovário é a principal causa de morte por doenças ginecológicas em mulheres. Geral- mente o diagnóstico é tardio e acompanhado por ascites, a acumulação de fluído ascítico na cavidade peritoneal associada a processos metastáticos e mau prognóstico. Os fluídos ascíticos são constituídos por componentes pró-tumorigénicos, como vesículas extracelulares (EVs) envolvidas no crosstalk in- tercelular estabelecido em microambientes tumorais. Alterações metabólicas são um marcador de can- cro e estão relacionadas com a sensibilidade ao tratamento. Contudo, o conteúdo metabólico dos EVs é ainda o menos estudado no contexto de cancro do ovário. O objetivo desta tese foi caraterizar o perfil metabólico de EVs isolados de fluídos ascíticos em pacientes de cancro do ovário. Métodos para caraterização de EVs foram primeiro implementados usando EVs de uma linha celular (ES-2), nomeadamente para a deteção de componentes e contaminan- tes de EVs por Western blot e para a análise de metabolómica/lipidómica por LC-MS. Por fim, EVs de fluídos biológicos, incluindo fluídos ascíticos e lavados peritoneais (controlos), foram isolados por cen- trifugação diferencial. Frações isoladas e descartadas foram caraterizadas por Western blot, por micros- copia eletrónica de transmissão (TEM) para análise morfológica das partículas e por nanoparticle tracking analysis (NTA) para determinação da concentração e tamanho, seguindo as diretrizes da Inter- national Society for Extracellular Vesicles. Foi demonstrada a recuperação de EVs no final do isolamento, o aumento de pureza das frações isoladas e a presença de EVs em fluídos biológicos. A caraterização dos EVs de ES-2 sugeriu o enri- quecimento em vias metabólicas envolvidas em processos tumorais. Esta metodologia para carateriza- ção do conteúdo metabólico pode futuramente ser aplicada em EVs de fluídos ascíticos e lavados peri- toneais para elucidar o papel dos EVs como mediadores do crosstalk metabólico no microambiente tumoral e eventualmente revelar potenciais biomarcadores para o prognóstico de cancro do ovário.

"Since the first publication introducing the concept in 1972, gene therapy has had a series of success stories and setbacks. However, the recent rise of awareness, public interest, promising results in clinical trials and recent market approvals indicate that gene therapy has come to stay. Currently there is a growing interest from the biopharmaceutical industry in gene and cell therapy, mostly using viral vectors. (...)"

"The application of stem cell-derived products for human health is in its infancy. Different applications of stem cell bioprocesses – cell therapy, discovery of new targets for regenerative medicine, disease modeling, drug and toxicity testing – have common hurdles. The most relevant hurdle is the lack of quality of cell products due to inefficient and ineffective stem cell differentiation procedures. Differentiated cells in vitro do not behave similarly to mature cells in vivo despite presenting similar surface and intracellular protein biomarkers. Our current weak understanding of cell “behavior” contributes to lack of cell functionality of produced cells and make these inadequate for cell therapy, disease modeling, drug screening and toxicity. Cells communicate with the environment and with neighboring cells, usually through secreted factors and through metabolites. This communication is guaranteed by an adjustable and controlled internal system in which metabolism is very important. (...)"