Manipulating gene expression levels in mammalian cell factories: An outline of synthetic molecular toolboxes to achieve multiplexed control.

Mammalian cells have developed dedicated molecular mechanisms to tightly control expression levels of their genes where the specific transcriptomic signature across all genes eventually determines the cell's phenotype. Modulating cellular phenotypes is of major interest to study their role in disease or to reprogram cells for the manufacturing of recombinant products, such as biopharmaceuticals. Cells of mammalian origin, for example Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Early genetic engineering approaches to alter their phenotype have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, highlight that rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much more precision than such "uncontrolled" approaches. To this end, synthetic biology tools have been generated that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools used in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. We discuss the advantages and disadvantages of using these tools for each cell regulatory layer and with respect to cell line engineering approaches. This review highlights the plethora of synthetic toolboxes that could be employed, alone or in combination, to optimize cellular systems and eventually gain enhanced control over the cellular phenotype to equip mammalian cell factories with the tools required for efficient production of emerging, more difficult-to-express biologics formats.

Single-cell RNA sequencing reveals homogeneous transcriptome patterns and low variance in a suspension CHO-K1 and an adherent HEK293FT cell line in culture conditions.

Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.