NF‐κB, CRE and YY1 elements are key functional regulators of CMV promoter‐driven transient gene expression in CHO cells

Abstract

Transient gene expression (TGE) in CHO cells is utilized to produce material for use in early stage drug development. These systems typically utilize the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. In this study, we have mechanistically dissected CMV-mediated TGE in CHO cells in order to identify the key regulators of this process. An in silico analysis of the promoter composition of transcription factor regulatory elements (TFREs) and the CHO cell repertoire of transcription factors identified eight TFREs as likely effectors of CMV activity. We determined the regulatory function of these elements by preventing their cognate transcription factors from binding at the CMV promoter. This was achieved by both scrambling promoter binding site sequences and using decoy molecules to sequester intracellular transcription factors. We determined that the vast majority of CMV activity is mediated by just two discrete TFREs, showing that simultaneous inhibition of NF-κB and CRE-mediated transactivation reduced CMV-driven transient secreted alkaline phosphatase (SEAP) production by over 75%. Further, we identified a mechanism by which CMV-mediated TGE is negatively regulated in CHO cells, showing that inhibition of YY1-mediated transrepression increased SEAP production 1.5-fold. This work enables optimization and control of CMV-mediated TGE in CHO cells, in order to improve transient protein production yields.

A transgenic pig model expressing a ZsGreen1 reporter across an extensive array of tissues

“…Elk-1 and CCAAT/enhancer-binding protein [13]. Thus, it is apparent that the CMV promoter has adapted to respond to a variety of cell signals in order to allow for the promiscuous infection of host cells [12].…”

“…Gene products of this initial interaction mediate the activation of other promoters 55 within the CMV genome that facilitate virus replication and invasion [13]. Therefore, the CMV promoter has evolved to contain binding sites for many ubiquitous transcription factors in order to replicate in a broad range of host cells [12]. This discovery has had a major impact on the scientific community; the CMV promoter is now the most commonly used ubiquitous promoter and it is also considered the most robust [14].…”

“…The human cytomegalovirus (CMV) immediate early promoter is one example of a 50 ubiquitous promoter. It was discovered by Boshart et al [11] and has since been heavily utilized to drive recombinant gene expression due to its constitutive and promiscuous nature [12]. These characteristics are directly related to its role in viral infection.…”

Overexpression of the regulatory subunit of glutamate‐cysteine ligase enhances monoclonal antibody production in CHO cells

“…Further investigations need to study the optimal Gclm:Gclc ratio and expression level for increased mAb productivity. This can be achieved using new precise genome editing tools, such as CRISPR/Cas9 (Lee et al, 2015), in combination with an inducible promoter or by regulating the typically used CMV promoter to manipulate expression levels (Brown et al, 2015).…”

“…All rights reserved tools, such as CRISPR/Cas9 (Lee et al 2015), in combination with an inducible promoter or by regulating the typically used CMV promoter to manipulate expression levels (Brown et al 2015). …”