Site-specific methylation of the CMV promoter alters transcriptional
factor-transgene interactions and demethylation leads to enhanced
specific productivity of a recombinant monoclonal antibody in CHO
Chinese hamster ovary (CHO) cells are the industry standard cell line
for high yield expression of biopharmaceuticals. However, leveraging the
influencers of specific productivity to expedite generation of high
productivity clones remains a challenge. One factor impacting specific
productivity is the strong, constitutive activity of the CMV promoter
used in most vectors for transgene applications. In this study, we
sought to understand the influence of DNA methylation along the CMV
promoter in driving transcription in higher vs lower productivity cell
lines. Bisulfite pyrosequencing was used to characterize the CMV
methylation pattern in parental (lower productivity) and DHFR-amplified
(higher productivity) monoclonal antibody-expressing cell lines.
In-silico analysis of promoter sequence revealed a large CpG island
covering the transcription factor binding sites for CREB1 and NFκB.
Variable methylation of specific CpG sites appears correlated with
transcription factor binding. Treatment of the cells with
5’-azacytidine, a known DNA hypomethylation agent, led to methylation
reduction in the CMV promoter, particularly upstream of the NFκB binding
consensus region. This induced DNA hypomethylation correlated with cell
line-specific increases in RNA expression and specific productivity.
These encouraging findings suggest that site-specific methylation along
the CMV promoter plays an underutilized, and important role in improving
transgene transcription using the CMV promoter and de-bottlenecking the
production capacity of CHO cell lines.