MiRNAs negatively regulate gene expression at post-transcriptional level, and consequently play an important role in the control of many cellular pathways. The use of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Here, we describe the effect of transient and stable miRNA overexpression on CHO cell phenotype. Using an established transient miRNA screening protocol, the effects of miR-17, miR-92a and cluster miR17-92a on CHO growth and protein productivity were studied and followed by analysis of cell pools with stable overexpression of these miRNAs. CHO cells stably engineered with miR-17 exhibited both enhanced growth performance and a 2-fold increase in specific productivity, which resulted in a 3-fold overall increase in EpoFc titer. While further studies of miRNA-mRNA interactions will be necessary to understand the molecular basis of this effect, these data provide valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity.

Despite great efforts to control and modify gene expression of Chinese Hamster Ovary (CHO) cells by conventional genetic engineering approaches, i.e. overexpression or knockdown/‐out, subclonal variation, induced unknown regulatory effects as well as overexpression stress are still a major hurdle for efficient cell line engineering and for unequivocal characterization of gene function. The use of epigenetic modulators – key players in CHO clonal heterogeneity – has only been marginally addressed so far. Here, we present the application of an alternative engineering strategy in CHO cells by utilizing targeted epigenetic editing tools that enable the turning‐on or ‐off of genes without altering the genomic sequence. The present, but silent beta‐galactoside alpha‐2,6‐sialyltransferase 1 (ST6GAL1) gene is activated by targeting the catalytic domain (CD) of Ten‐Eleven Translocation methylcytosine dioxygenase 1 (TET1) via deactivated Cas9 (dCas9) to its methylated promoter. Stable upregulation in up to 60% of transfected cells is achieved over a time span of more than 80 days. No difference in growth and recombinant protein productivity is observed between activated and control cultures. Re‐silencing by targeted methylation via DNA methyltransferase (DNMT) 3A‐CD resulted in an up to 5.4‐fold reduction of ST6GAL1 mRNA expression in ST6GAL1 expressing cells. This proof‐of‐concept demonstrates the feasibility of using epigenetic editing tools to efficiently modulate gene expression and provide a promising complement to conventional genetic engineering in CHO cells.

The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell‐line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9‐day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12‐ and 24‐hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription‐ and promoter‐specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non‐coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA–DNA–DNA triplexes. Such triplex‐forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex‐forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA‐triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes.

Galactosylation as part of N-glycan processing is conducted by a set of beta-1,4-galactosyltransferases (B4GALTs), with B4GALT1 as the dominant isoenzyme for this reaction. Nevertheless, the exact contributions of this key-player as well as of the other isoenzymes involved in N-glycosylation, B4GALT2, B4GALT3 and B4GALT4, have not been studied in-depth. To increase the understanding of the protein- and site-specific activities of individual galactosyltransferases in Chinese Hamster Ovary cells, a panel of triple deletion cell lines was generated that expressed only one isoform of B4GALT each. Two model proteins were selected for this study to cover a large spectrum of possible N-glycan structures: erythropoietin and deamine-oxidase. They were expressed as Fc-fusion constructs (EPO-Fc and Fc-DAO) and their N-glycan processing status was analyzed by site-specific mass spectrometry. The sole activity of B4GALT1 resulted in a decrease of 15-21 % of fully galactosylated structures for erythropoietin, emphasizing the involvement of other isoenzymes. Interestingly, the contributions of B4GALT2 and B4GALT3 differed for the two model proteins. Unexpectedly, removal of galactosyltransferases influenced the overall process of N-glycan maturation, with the result of a higher occurrence of poorly processed oligosaccharides. In the context of high productivity cell lines, which can push N-glycan maturation towards incomplete galactosylation, galactosyltransferases are potential targets to ensure stable product quality. In view of our results, specifically engineered "designer" cell lines may be required for different proteins.

By repeatedly selecting and pooling the most rapidly outgrowing clones during multiple single cell subcloning rounds, the time required for subcloning was reduced from 3 to 2 weeks and the cloning efficiency significantly increased. Transcriptome analyses of the resulting host cell lines revealed a diverse set of pathways differentially enriched in each host cell line treated, with the only shared DE pathway related to changes in extracellular matrix. This indicates that cells struggle with the lack of cell-to-cell communication in isolation during subcloning.

Chinese Hamster Ovary (CHO) cells are the preferred cell line for production of biopharmaceuticals. These cells are capable to grow without serum supplementation, but drastic changes in their phenotype occur during adaptation to protein-free growth, which typically include the change to a suspension phenotype with reduced growth rate. A possible approach to understand this transformation, with the intention to counteract the reduction in growth by targeted supplementation of protein-free media, is gene expression profiling. The increasing availability of genome-scale data for CHO now facilitates quests for a better understanding of metabolic pathways and gene networks. So far, systematic large-scale expression profiling in CHO cells by microarray was limited due to lack of publicly available array designs and limitations of alternative approaches. Based on the recent release of CHO and Chinese Hamster genome sequences, including an annotated RefSeq genome, we have constructed a publicly available microarray design for effective genome-scale expression profiling. The design employed microarray probes optimized for uniformity, sensitivity, and specificity, with probe properties computed using the latest thermodynamic models. We validated the platform in an analysis of gene expression changes in response to serum-free adaptation. The observed effects on the lipid metabolism as well as on nucleotide synthesis were used to successfully select media supplements that were able to increase growth rate.