N Biotechnol. 2023 Nov 29. pii: S1871-6784(23)00066-3. [Epub ahead of print]
Cells, both of prokaryotic and eukaryotic origin, 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 phenotype. Modulating cellular phenotypes is of major interest, either to study their role in disease or to reprogram cells for the manufacture of recombinant products, such as biopharmaceuticals. For the latter, cells of mammalian origin, such as Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Altering their phenotype is often achieved randomly by subcloning and selection of appropriate behavior or by genetic engineering. In both cases, the objective is to obtain expression systems that generate the desired product with the highest possible quality and quantity. Early genetic engineering approaches have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, have highlighted that a controlled manipulation of transgene expression, by rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much greater precision than such "uncontrolled" approaches. To control and (fine-)tune the expression level of one or multiple transgenes or endogenous genes, synthetic biology tools inspired by naturally occurring gene regulation mechanisms have been generated to develop novel, molecular toolboxes that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools that have been established in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. Major emphasis is placed on studies in which such tools were employed to engineer recombinant protein production in CHO or other mammalian cell factories. 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 existence of a 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.
Keywords: CHO cells; Cell line engineering; Gene expression; Synthetic Biology