CHO genome mining for synthetic promoter design

Abstract

Bioinformatic analysis of endogenous CHO promoter sequences.  Linking transcriptomic and genomic datasets for bioprocess-directed promoter design.  In silico DOE-based design method for synthetic promoter construction.  Design promoters exhibited 2.5-fold increase in activity over CMV-IE promoter.  Promoters designed to function in context of a biphasic fed-batch production process.

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

“…We previously devised a methodical, comprehensive approach for bioprocess‐directed design of synthetic promoters based on genomic sequence information (Johari et al, 2019). The work flow enables identification of TFREs associated with endogenous gene promoters with specific characteristics, thus allowing de novo design of synthetic promoters with relevant functional features.…”

“…Importantly, given that a TF can act as a transcriptional activator or repressor (or both), we designed a set of promoters (promoter 1/02–1/10) containing specific combinations of the endogenous TFREs selected in the bioinformatic analysis to assess elements that primarily function as repressors in RPE cells. For each promoter, 6 copies of HELT/MITF were included to provide a promoter basal expression, and the other 27 specific TFREs (4 copies each) were randomly distributed using R software with the following design rules: (i) each specific TFRE occurred twice in different promoters, (ii) no two specific TFREs re‐occurred in another promoter, and (iii) the relative order of constituent TFREs was random, separated by minimal spacers (Brown et al, 2017; Johari et al, 2019). Additionally, to test whether the selected endogenous elements from the bioinformatics analysis could generally act as positive effectors in heterotypic constructs, we constructed a promoter (1/11) containing two repeat copies of each element that present in upstream of BEST1 promoter (–900 to –1 relative to the TSS).…”

Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

“…For DTE proteins, very low yielding transient expression systems can be an early indication of reduced stable production (Mason et al, 2012), where particular engineering strategies may be required to obtain stable cells with desirable production characteristics. To generate CHO cells stably expressing recombinant spike trimer, we tested two in‐house CHO synthetic promoters, namely 40 RPU (~90% CMV activity) and 100 RPU (~220% CMV activity) promoters (see Brown et al, 2017; Johari et al, 2019). Although the use of extremely strong promoters may be counterintuitive for DTE proteins, we reasoned that only those transfectants harboring a sub‐UPR threshold productivity, and thus capable of proliferation would survive.…”

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

“…preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 13, 2020. ; https://doi.org/10.1101/2020.08.07.20169441 doi: medRxiv preprint For DTE proteins, very low yielding transient expression systems can be an early indication of reduced stable production (Mason et al, 2012), where particular engineering strategies may be required to obtain stable cells with desirable production characteristics. To generate CHO cells stably expressing recombinant spike trimer, we tested two in-house CHO synthetic promoters, namely 40RPU (~90% CMV activity) and 100RPU (~220% CMV activity) promoters (see Brown et al, 2017;Johari et al, 2019). Although the use of extremely strong promoters may be counterintuitive for DTE proteins, we reasoned that only those transfectants harboring a sub-UPR threshold productivity, and thus capable of proliferation would survive.…”

“…Of course we also expect that co-expression of product-specific combinations of ER chaperones/UPR regulators may also be a useful strategy to minimise the impact of spike expression on the cell, e.g. by effectively raising the cellular threshold for UPR induction (Johari et al 2015;Brown et al 2019;Cartwright et al 2020).…”