Application of microRNA Targeted 3′UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools

Abstract

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

Comprehensive characterization of dihydrofolate reductase‐mediated gene amplification for the establishment of recombinant human embryonic kidney 293 cells producing monoclonal antibodies

“…Alternatively, the DHFR selection stringency and system in KO-HEK cells can be improved by attenuating or reducing DHFR expression, as in CHO cells. [38,39] In conclusion, a KO HEK cell line, generated by disrupting both dhfr and dhfrl1 using CRISPR technology, was successfully used as a host cell line for establishing a mAb-producing rHEK293 cell line. Due to the improved selection stringency by KO of dhfr and dhfrl1, mAb-producing cell pools could be generated in the absence of MTX using the KO-HEK host cell line, followed by DHFR/MTX-mediated gene amplification at increasing levels of MTX.…”

A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

“…As outlined in the introduction, selection marker technologies are routinely employed to generate recombinant mammalian cell lines producing a range of biotherapeutic protein molecules of interest. After a lack of developments over the last several decades there has been an upsurge of interest with a number of groups reporting the recent development of alternatives to GS and DHFR metabolic selection systems for use in CHO cells ( Kaufman and Sharp, 1982a ; Jossé et al, 2018 ; Cockett et al, 1990 ; Birch et al, 2008 ). Additional selection systems also allow for the engineering and maintenance of host cell systems to improve, or introduce new, cellular attributes whilst leaving, for example, the traditional GS and DHFR systems available for the subsequent introduction of a target biopharmaceutical.…”

“…An alternative approach to antibiotic selection is to use metabolic selection markers. Dihydrofolate reductase (DHFR) ( Kaufman and Sharp, 1982a ; Jossé et al, 2018 ) and glutamine synthetase (GS) ( Cockett et al, 1990 ; Birch et al, 2008 ) are well established examples of metabolic selection. DHFR reduces dihydrofolate to produce tetrahydrofolate, a vitamin required as a precursor for de novo synthesis of essential molecules such as hypoxanthine and thymidine ( Kaufman and Sharp, 1982b ).…”

Defining lncRNAs Correlated with CHO Cell Growth and IgG Productivity by RNA-Seq

“…Despite advancements in the ability of CHO cells to reach higher cell concentrations and generate increasing amounts of target biotherapeutic proteins, particularly monoclonal antibodies (mAbs), there remains a desire to further understand the limitations upon CHO cell phenotypes and to engineer cells for the production of more difficult-to-express products (Godfrey et al, 2017;Jossé et al, 2018;Mead et al, 2009). One approach that has been applied toward improving our understanding of the limitations on CHO cell growth and recombinant protein production is the field of transcriptomics (Tamo saitis and Smales, 2018).…”