Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production

Abstract

The expression of endogenous genes as well as transgenes depends on regulatory elements within and surrounding genes as well as their epigenetic modifications. Members of a cloned cell population often show pronounced cell-to-cell heterogeneity with respect to the expression of a certain gene. To investigate the heterogeneity of recombinant protein expression we targeted cassettes into two preselected chromosomal hot-spots in Chinese hamster ovary (CHO) cells. Depending on the gene of interest and the design of the expression cassette, we found strong expression variability that could be reduced by epigenetic modifiers, but not by site-specific recruitment of the modulator dCas9-VPR. In particular, the implementation of ubiquitous chromatin opening elements (UCOEs) reduced cell-to-cell heterogeneity and concomitantly increased expression. The application of this method to recombinant antibody expression confirmed that rational design of cell lines for production of transgenes with predictable and high titers is a promising approach.

Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9‐mediated knock‐in in mammalian cells

“…This strategy can mitigate integration of fragmented transgene and unpredictable transgene silencing that were observed in recombinant cell lines generated by traditional RI method, and exploit the well‐established and simple RI‐based CLD process. Given that the recombinase‐mediated cassette exchange (RMCE)‐based CLD is increasingly being used for the development of mammalian cell lines that produce therapeutic proteins [ 25 , 26 ], these KI constructs provide relatively high and stable transgene expression sites and streamlined CRISPR/Cas9‐mediated KI without the need for the selection and screening of sgRNA target sequences, suggesting as an alternative to RMCE. These artificial KI constructs can be improved by adjusting the cell‐type independent promoter and customizing the combination of elements.…”

Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology

“…UCOEs have also been tested following site-specific integration into a predetermined genomic site. The Rps3 UCOE and 1.5 kb A2UCOE flanking an antibody light chain transgene stabilised its expression over six weeks of continuous culture at two distinct chromosomal locations [54] . Furthermore, GOI expression was highly homogeneous between individual cells, suggesting that UCOEs allow replicable expression from a single gene copy.…”

A compendium of stable hotspots in the CHO genome

“…The rarity of stable high‐producing cells within these pools necessitated the isolation and screening of hundreds or thousands of clones during a time‐ and resource‐intensive procedure to identify a clonally‐derived cell line with stable and acceptable productivity and product quality attributes, creating a significant bottleneck in the drug development pipeline (Jostock, 2011). Targeted integration platforms, particularly those that employ recombinase‐mediated cassette exchange (RMCE), can accelerate material generation for early product testing by offering unparalleled control over genotype and phenotype (Cain et al, 2013; Carver et al, 2020; Feary et al, 2021; Gödecke et al, 2021; Rajendra et al, 2017; Scarcelli et al, 2017; Shenet al, 2017; Stuible et al, 2018). However, the development of this type of CLD platform first requires knowledge of a precise location within the genome defined as a “hotspot” by its ability to facilitate stable and high transgene expression.…”