Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20

• Published in: Metabolic engineering 2017-03, Vol.40, p.69-79
• Main Authors: Pieper, Lisa A., Strotbek, Michaela, Wenger, Till, Gamer, Martin, Olayioye, Monilola A., Hausser, Angelika
• Format: Article
• Language: English
• Subjects: Animals; Cell line engineering; CHO Cells; Cricetulus; Fed-batch culture; Genetic Enhancement - methods; Genome, Mitochondrial; Membrane Proteins - genetics; Metabolic Networks and Pathways - genetics; MicroRNA; MicroRNAs - genetics; mitosRNA; Next generation sequencing; Non-coding RNA; rab1 GTP-Binding Proteins - genetics; Recombinant antibody; RNA - genetics; RNA interference; RNA, Small Interfering - genetics; Secretory pathway; Secretory Pathway - physiology; Sphingosine N-Acyltransferase - genetics; Tumor Suppressor Proteins - genetics
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2017.01.003

Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins. •Mitochondrial genome-encoded small RNA mitosRNA-1978 improves CHO-IgG productivity.•CerS2 and Tbc1D20 are target genes of mitosRNA-1978.•Combined knockdown of CerS2 and Tbc1D20 boosts productivity of CHO-IgG cells.