Native and artificial miRNAs as versatile tools for glycoengineering in CHO production cells

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Date

2024-12-19

Authors

Schlossbauer, Patrick

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Dissertation

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Abstract

Market analysis clearly shows that the biopharmaceutical industry is growing rapidly, and with the increasing demand for biosimilars, and the introduction of tailor-made therapies the need for new technologies to modulate complex molecules emerges simultaneously. Monoclonal antibodies (mAbs) produced in Chinese hamster ovary (CHO) cells still reflect approximately 50 % of newly approved biologicals in the last decade. As the effector function of mAbs is highly sensitive to changes in the glycosylation pattern attached to the protein, this modification has been subject to numerous engineering approaches. Standard methods like radical gene knockout or energy consuming overexpression, however, often fail to meet moderate effector functions. Therefore, the present work aimed to elucidate the possibilities to use microRNAs (miRNAs) as fine-tuning regulators of mAb glycosylation in CHO cells. MiRNAs were identified regulating mAb fucosylation and galactosylation in transient experiments and in stable miRNA overexpressing CHO production cell lines. Here, strategies for the overexpression of miRNAs were tested with subsequent analysis of regulation on transcript and glycosylation level and potentially bioprocessing relevant parameters. Furthermore, artificial miRNAs (amiRNAs) which reflect improved synthetic variants of their native counterparts, were designed and applied in stable cell line development. Thereby, basic principles of efficient amiRNA regulation and potential pitfalls were identified. The experiments were accompanied by miRNA target prediction, which served as a valuable tool to hint at potential regulations and was evaluated by the generated data. Summarized, numerous cell lines with varying fucosylation and galactosylation properties could be generated which clearly demonstrated the applicability of miRNAs or tailor-made amiRNAs as versatile engineering tools in cell line development to generate moderate phenotypic changes required in biosimilar production.

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Universität Ulm
Institut für Pharmazeutische Biotechnologie

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DFG Project uulm

EU Project THU

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Keywords

CHO, Chinese hamster ovary, miRNA, miRNS, Glykosidierung, Monoklonaler Antikörper, CHO-Zelle, Biopharmaceutics, Cricetulus; Genetics, MicroRNAs, Antibodies, Monoclonal, Glycosylation, DDC 570 / Life sciences