title: Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20.
abstract: 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.
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Pieper
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Strotbek
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co.KG, Birkendorfer Str. 65, 88400 Biberach an der Riß, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Wenger
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co.KG, Birkendorfer Str. 65, 88400 Biberach an der Riß, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Gamer
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Germany; Stuttgart Research Center Systems Biology, University of Stuttgart, Germany. Electronic address: monilola.olayioye@izi.uni-stuttgart.de.
authors.forename: Monilola A
authors.initials: MA
authors.lastname: Olayioye
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Germany; Stuttgart Research Center Systems Biology, University of Stuttgart, Germany. Electronic address: angelika.hausser@izi.uni-stuttgart.de.
authors.forename: Angelika
authors.initials: A
authors.lastname: Hausser
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D024861:Genetic Enhancement / Q000379:methods*; D054629:Genome, Mitochondrial; D008565:Membrane Proteins / Q000235:genetics*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D012313:RNA / Q000235:genetics; D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering / Q000235:genetics*; D055571:Secretory Pathway / Q000502:physiology*; D051049:Sphingosine N-Acyltransferase / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D020761:rab1 GTP-Binding Proteins / Q000235:genetics*
chemical_list: D008565:Membrane Proteins; D035683:MicroRNAs; D000077278:RNA, Mitochondrial; D034741:RNA, Small Interfering; C526344:TBC1D20 protein, human; D025521:Tumor Suppressor Proteins; D012313:RNA; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase; D020761:rab1 GTP-Binding Proteins
keywords: Cell line engineering; Fed-batch culture; MicroRNA; Next generation sequencing; Non-coding RNA; RNA interference; Recombinant antibody; Secretory pathway; mitosRNA