Rational genetic engineering of insect cells to improve rAAVs and influenza VLPs production yield utilizing the baculovirus expression vector system

• Autor(a) principal: Graça, Miguel Ângelo Matos
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/145992
• topic: IC-BEVS; Cell Line Development; CRISPR/Cas9; Ribonucleoprotein complex; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The insect cells-baculovirus expression vector system (IC-BEVS) is one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, allowing for high expression levels in short time frames. Despite its benefits, IC-BEVS is a transient system due to the baculovirus lytic nature, which causes the productivity and/or quality of products generated to be impacted. Improvements to the expression platform, and in particular to the cell line, is thus essential in order to bring IC-BEVS products faster to market. However, this has been limited by the lack of well-established tools for genetic engineering in insect cells. This thesis aimed at overcoming these bottlenecks by establishing a gene editing pipeline based on the CRISPR-Cas9 system for simple and fast gene(s) knockout in Sf9 cells towards improved phenotypes. To establish the pipeline, different transfection methods were explored as to their potential to deliver the ribonucleoprotein complex comprising the Cas9 enzyme and a guide RNA that targeted the fused lobes (fdl) gene expressing the β-N-acetylglucosaminidase enzyme. Lipofection was identified as the best method with up to 85 % of gene editing efficiency achieved in a single transfection step. To validate the system, Sf-caspase-1, a gene associated to apoptosis (pathway demonstrated by in-house data to be up-regulated upon infection and linked to increased product titers), was used as a model knockout target. Editing efficiency of Sf-caspase-1 was optimized, obtaining a highly edited population. Single-cell cloning by limited dilution was implemented and Sf-caspase-1 knockout clones identified. Isolated clones were evaluated for cell growth, showing similar kinetics to non-edited Sf9 cells. In addition, clones showed improved resistance to apoptosis and generated slightly improved titers of recombinant adeno-associated viruses and influenza virus-like particles when compared to non-edited Sf9 cells. Overall, an efficient CRISPR-Cas9 gene editing pipeline in insect cells was successfully established, which can assist rational engineering of insect cell lines towards improved production titers.