Influence of cytoskeleton organization on recombinant protein expression by CHO cells

Abstract

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.

Methylthioadenosine (MTA) boosts cell‐specific productivities of Chinese hamster ovary cultures: dosage effects on proliferation, cell cycle and gene expression

“…Dinnis et al ., [66] observed that actin, tubulin, or the actin‐binding cofilin demonstrated an important role in protein transport and secretion of high producers. Selection procedures for high producers revealed according to data with enhanced gene expression of actin‐related proteins [67]. Recently, Berger et al ., [55] identified DEGs involved in cytoskeleton organization and vesicular trafficking as Rassf9 that is linked to endosome recycling and is a trafficking regulator [68] in high producers.…”

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

“…We also identified the pioneering transcription factor Foxa1 as a candidate gene of interest for cell metabolism engineering, as it increases cell viability, VCD, specific productivity, and the production of both easy-to-express and difficult-to-express therapeutic proteins when overexpressed. This effect may result in part from the Foxa1- Moreover, Tagap and the cardiac muscle actin alpha (ACTC1) were found to be upregulated in vitamin B5-selected cells producing therapeutic proteins at very high levels, and Tagap overexpression was shown to increase the expression of ACTC1, which in turn increases the production of various therapeutic proteins (see accompanying paper, Pourcel, Buron, Arib, et al, 2020). Interestingly, spherical integrin clustering, as well as an increase in actin content and formation of a spherical actin sheath, was observed in suspension-adapted CHO cells (Walther, Whitfield, & James, 2016).…”

“…RNA-seq analysis of the B5-selected interferon β high producer clones, of the puromycin-selected Tras polyclonal cell populations and of the parental CHO-M cells were as described by Pourcel, Buron, Arib, et al (2020) in the accompanying paper. These cells and the high producer clones expressing the Tras or the bevacizumab antibodies were grown for 4 days in spin tubes in batch culture.…”

High‐resolution three‐dimensional chromatin profiling of the Chinese hamster ovary cell genome

“…Recent exemplar research has uncovered the positive effects of ACTC1 (Pourcel et al, 2020) and SCD1 (Budge et al, 2020) overexpression on not only transgene production but also in reducing the toxic effects of lactate metabolism and expanding the endoplasmic reticulum, respectively. A key part of this study is our production of a CHOK1SV 10E9 3D genome interactome, identifying the active distal regulatory elements that interact in 3D space with, and influence the transcription of, specific target genes.…”

“…Aside from the requirements for transgene integration sites, flexibility in terms of recombinant cell line phenotypes, for example, cell lines with an increased capacity for protein translation, increased bioreactor cell viability, or tailored protein glycosylation patterns, would also be highly beneficial for the biopharmaceutical sector. Recent exemplar research has uncovered the positive effects of ACTC1 (Pourcel et al, 2020) and SCD1 (Budge et al, 2020) overexpression on not only transgene production but also in reducing the toxic effects of lactate metabolism and expanding the endoplasmic reticulum, respectively. A key part of this study is our production of a CHOK1SV 10E9 3D genome interactome, identifying the active distal regulatory elements that interact in 3D space with, and influence the transcription of, specific target genes.…”