Transfection of glycoprotein encoding mRNA for swift evaluation of N-glycan engineering strategies.

N-glycosylation is defined as a key quality attribute for the majority of complex biological therapeutics. Despite many N-glycan engineering efforts, the demand to generate desired N-glycan profiles that may vary for different proteins in a reproducible manner is still difficult to fulfill in many cases. Stable production of homogenous structures with a more demanding level of processing, for instance high degrees of branching and terminal sialylation, is particularly challenging. Among many other influential factors, the level of productivity can steer N-glycosylation towards less mature N-glycan structures. Recently, we introduced an mRNA transfection system capable of elucidating bottlenecks in the secretory pathway by stepwise increase of intracellular model protein mRNA load. Here, this system was applied to evaluate engineering strategies for enhanced N-glycan processing. The tool proves to indeed be valuable for a quick assessment of engineering approaches on the cellular N-glycosylation capacity at high productivity. The gene editing approaches tested include overexpression of key Golgi-resident glycosyltransferases, partially coupled with multiple gene deletions. Changes in galactosylation, sialylation, and branching potential as well as N-acetyllactosamine formation were evaluated.

mRNA Transfection into CHO-Cells Reveals Production Bottlenecks.

Obtaining highly productive Chinese hamster ovary (CHO)-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbor potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product may therefore prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. The aim of the study is to reach specific productivities approaching those of industrial production cell lines by transfection of in vitro transcribed mRNA. The system is characterized with respect to transfection efficacy (by quantitative PCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturates at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remain linearly correlated to the amount of mRNA taken up. Nevertheless, MS shows a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization.

Transient manipulation of the expression level of selected growth rate correlating microRNAs does not increase growth rate in CHO-K1 cells.

Engineering of Chinese Hamster Ovary cells by manipulating microRNA (miRNA) expression levels has been shown to induce advantageous, desired phenotypes. Most of these studies so far were concerned with increasing productivity or reducing growth rate (with the implied intention of thus freeing cellular resources to also increase productivity). Here we evaluated the ability of growth correlating miRNAs to increase the growth rate of CHO-K1 cells by transient overexpression or knock down, respectively. Candidates were selected based on the correlation between growth rate and miRNA expression levels as observed in previous studies. These candidates were then up- or downregulated initially by transfection of mimics or inhibitors and subsequently by transfection of plasmids bearing the corresponding miRNAs or sponges. None of the 40 selected candidates was able to induce a better growth phenotype under these conditions. Overlap between miRNAs identified to correlate to growth in published miRNA expression studies and those identified to actively increase growth rate in a functional screen is minimal, indicating that the here selected approach of traditional overexpression/knock down engineering of miRNAs may not be a suitable strategy for the purpose of increasing growth rate.

The contributions of individual galactosyltransferases to protein specific N-glycan processing in Chinese Hamster Ovary cells.

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.

CRISPR-Based Targeted Epigenetic Editing Enables Gene Expression Modulation of the Silenced Beta-Galactoside Alpha-2,6-Sialyltransferase 1 in CHO Cells.

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.

Enhanced Genome Editing Tools For Multi-Gene Deletion Knock-Out Approaches Using Paired CRISPR sgRNAs in CHO Cells.

Since the establishment of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged. Nevertheless, there is still room to expand the scope of the CRISPR tool box for further applications to improve CHO cell factories. Here, the authors demonstrate activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9 and Cpf1, can be effectively used for multi-gene engineering with a strategy based on paired single guide RNAs (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by removing the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. FUT8 also served as model to verify that deletion efficiency is size-independent (2-150 kb). To test the suitability for multi-gene approaches in combination with gene deletion, clones harboring triple deletions in ß-1,4-Galactosyltransferase (B4GALT) isozymes are identified using solely conventional PCR/qPCR. In addition, two bicistronic transcription strategies are implemented to enable unequivocal pairing of sgRNAs: a CHO-derived tRNA linker that works for both, Cas9 and Cpf1, as well as paired sgRNAs in an array format, which can be used with Cpf1 due to its RNA processing ability. These strategies broaden the range of application of CRISPR for novel gene editing approaches in CHO cells and also enable the efficient realization of a genome-wide deletion library.

The 6th International Conference on Analysis of Microbial Cells at the Single Cell Level, Retz, Austria, 19-22 July 2015.

The 6th International Conference on Analysis of Microbial Cells at the Single Cell Level, held in Retz, Austria from 19 to 22 July 2015, brought together experts from different areas working with bacterial, yeast and mammalian cell systems. The conference highlighted the importance of dissecting cell behaviour down to the single cell level, as analysis of mixed populations can obscure crucial cell-to-cell variations. The sessions covered advances in the fields of image analysis and microscopy, flow cytometry and cell sorting as well as bioinformatics, including recent developments and new applications of existing tools. In addition, a high speed poster slam session contributed to the lively discussions and exchange of expertise among academic and industrial researchers.