No 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.

The VAPYRIN (VPY) gene in Medicago truncatula and Petunia hybrida is required for arbuscular mycorrhizal (AM) symbiosis. The moss Physcomitrella patens has a close homologue (VPY-like, VPYL), although it does not form AM. Here, we explore the phylogeny of VPY and VPYL in land plants, and we study the expression and developmental function of VPYL in P. patens. We show that PpVPYL is expressed primarily in the protonema, the early filamentous stage of moss development, and later in rhizoids arising from the leafy gametophores and in adult phyllids. Knockout mutants have specific phenotypes in branching of the protonema and in cell division of the leaves (phyllids) in gametophores. The mutants are responsive to auxin and strigolactone, which are involved in the regulation of protonemal branching, indicating that the mutants are not affected in hormonal signaling. Taken together, these results suggest that PpVPYL exerts negative regulation of protonemal branching and of cell division in phyllids. We discuss VPY and VPYL phylogeny and function in land plants in the context of AM symbiosis in angiosperms, and of development in the moss.

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.

The cover image is based on the Original Article Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release by PIERRE‐OLIVIER DUROY et al., https://doi.org/10.1002/bit.27200.