Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Abstract

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.

Small molecule epigenetic modulators for enhancing recombinant antibody production in CHO cell cultures

“…We found that treatment of rCHO cell cultures with selected iDNMTs failed to induce any significant increase in MMC, which can probably be attributed to the fact that these iDNMTs did not induce a significant reduction in the global 5-mC ratio (Figure S3). Previously, the knockdown of DNMT in rCHO cells based on RNA interference enabled the sorting of rCHO cells with improved productivity (Weinguny et al, 2020). Thus, if DNA methylation in rCHO cells is appropriately reduced by the addition of iDNMTs, mAb production in rCHO cell culture is likely to increase.…”

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

“…( 40 ). Similarly, short hairpin RNA regulation elements (RgE), previously evaluated siRNAs sequences for Rad21, Chd4 ( 41 ), Dnmt1 and Dnmt3a ( 42 ) were used as RgEs (see Supplementary Figure S8b for sequences). Noteworthy, the ordered oligos contained only the required overhang sequences and not the full restriction enzyme recognition sequence.…”

Key Challenges in Designing CHO Chassis Platforms

“…Recent developments now allow the controlled manipulation of such epigenetic marks at specific sites using CRISPR/dCas9 tools [192,193]. Finally, it was recently shown that random changes in a population's DNA-methylation pattern induce higher phenotypic diversity and thus enable a more efficient isolation of "outliers" with enhanced performance [194]. A more detailed understanding of these mechanisms is still required, but the new tools recently developed will contribute to generate such understanding while at the same time providing the possibility of more sophisticated and reliable control to researchers.…”