Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

Abstract

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation.

Disruption of the gene C12orf35 leads to increased productivities in recombinant CHO cell lines

“…Moreover, it needs to be considered that transcriptional activity is also dependent on chromatin structure and structural accessibility of DNA to allow binding of RNA polymerase or regulating proteins and can be altered by epigenetic regulation. The CMV promoter driving transgene expression is susceptible to gene silencing in CHO cells, most likely due to DNA methylation and histone modifications (Mariati et al, ,; Moritz et al, ; Paredes et al, ). It is possible that C12orf35 impacts transcriptional activity of the exogenes by epigenetic regulation.…”

Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity

“…Additional CMV promoter research supports the conclusion that DNA methylation level does not correlate with expression stability and that histone modification may be more important for transgene stability . Recombinant fusions of a CpG Island to human CMVie, mouse CMVie and human EF‐1α promoters demonstrated variable results for transgene expression stability, suggesting epigenetic regulation may have a promoter‐specific component in this context. Mutations of CpG sites in the CMVie promoter were shown to stabilize expression indicating sequence context is also important .…”

Low concentrations of cadmium chloride promotes protein translation and improve cell line productivity

“…Unexpectedly, BI‐D1870 promoted transcription from the CMV promoter in an off‐target manner. A possible mechanism for the effect of BI‐D1870 may be negating epigenetic regulation, as the immediate early CMV promoter is sensitive to epigenetic silencing in CHO cells (Mariati, Koh, Ho, & Yang, ). When single‐cell clones were analyzed, each probably represent a different integration site of the transgene, we observed a large variability in the response to BI‐D1870.…”