Epigenetic Approaches to Enhance Productivity

Biologics are used to treat diseases including different cancers. They often require production in mammalian cell systems such as Chinese hamster ovary (CHO) cells owing to their complexity. However, epigenetic silencing mechanisms within the CHO genome can lead to loss of expression of the integrated biologic transgene, compromising stability and delaying production timelines. Promoter epigenetic state is a key determinant of expression of transgenes and host cell genes which support protein synthesis. Furthermore, transfer RNA (tRNA) gene regulation is an important consideration as cellular tRNA availability determines translational capacity. tRNA genes are also utilised as barrier elements which maintain an epigenetic environment conducive to high expression. This thesis explores epigenetic approaches to enhance productivity in novel contexts through elevating expression of transgenes, endogenous host genes and tRNA genes. Chapter 2 applies CRISPR-Cas9 technology to target chromatin modifying enzymes to a transgene promoter in CHO cells, showing that elevated histone acetylation can double productivity. Chapter 3 explores the role of the pioneer factor FOXA1 which activated expression of a mAb transgene and host cell genes alongside recruitment of epigenetic modifiers and receptors, ultimately elevating productivity. Chapter 4 examines the ability of FOXA1 to activate expression of tRNA genes which are important for a cell’s translational capacity and can be used as barriers to protect epigenetic silencing of transgenes. Chapter 5 investigates histone acetylation at tRNA genes through the lens of tRNA gene dysregulation in cancer, which can drive metastatic or proliferative phenotypes. Further understanding the epigenetic regulation of tRNA genes offers valuable insight for optimising translational efficiency in CHO cells. Overall, this thesis contributes novel insights into approaches to alter epigenetic states at the promoters of transgenes, host genes and tRNA genes to those conducive to higher expression. Ultimately this can lead to beneficial effects on titre and productivity.