Chromatin Accessibility Plays an Important Epigenetic Role on Antibody Expression From CMV Promoter and DNA Elements Flanking the CHO TI Host Landing-Pad.

Abstract

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines.