Generazione mediata dal trasposone piggyBac di linee cellulari HEK293 stabili per la produzione di proteine terapeutiche ricombinanti

The development of stable high-producing mammalian cell lines represents a major bottleneck in the production of recombinant therapeutic proteins. Conventional transfection methods entirely rely on random transgene integration, a rare event which generally produces very heterogeneous cell pools requiring long screening procedures before the identification of clones with the desired characteristics in terms of productivity and stability. In this work, a transposon-based gene delivery method was used for the generation of stable HEK293 pools expressing the anti- human Rhesus-D Immunoglobulin G1. This system leverages the ability of the piggyBac(PB) transposase to catalyze the excision of a transgene flanked by specific PB termini from a donor plasmid and its integration into highly transcribed regions of the host genome. A preliminary evaluation of the PB transposition efficiency revealed that the PB system allows the generation of stable HEK293 cell lines with a frequency more than 35-fold higher than conventional transfection. Moreover, several parameters potentially influencing the cell line generation process (e.g. type and amount of transposase used for transfection, type and concentration of antibiotic used for selection) were analyzed and optimized. Interestingly, a relatively short selection (14 days) with 200 ?g/ml of zeocin was found out to enable the generation of cell pools with high productivity and outstanding stability. The work performed in this thesis opens a new attractive possibility for the fast and efficient development of stable high-producing HEK293 cell lines.