Overexpression of YTHDF3 increases the specific productivity of the recombinant protein in CHO cells by promoting the translation process.

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

Apilimod enhances specific productivity in recombinant CHO cells through cell cycle arrest and mediation of autophagy.

Chinese hamster ovary (CHO) cells are expected to acquire the ability to produce higher recombinant therapeutic protein levels using various strategies. Genetic engineering targeting the cell cycle and autophagy pathways in the regulation of cell death in CHO cell cultures has received attention for enhancing the production of therapeutic proteins. In this study, we examined the small-molecule compound apilimod, which was found to have a positive influence on recombinant protein expression in CHO cells. This was confirmed by selective blocking of the cell cycle at the G0/G1 phase. Apilimod treatment resulted in decreased expression of cyclin-dependent kinase 3 (CDK3) and Cyclin C and increased expression of cyclin-dependent kinase suppressor p27Kip1, which are critical regulators of G1 cell cycle progression and important targets controlling cell proliferation. Furthermore, total transcription factor EB (TFEB) was lower in apilimod-treated CHO cells than in control cells, resulting in decreased lysosome biogenesis and autophagy with apilimod treatment. These multiple effects demonstrate the potential of apilimod for development as a novel enhancer for the production of recombinant proteins in CHO cell engineering.

Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells.

Matrix attachment regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.

CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability.

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a-deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a-deficent CHO cell line based on Dnmt3a KO displayed an enhanced long-term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a-deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a-deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.