Novel Insulators that can Enhance and Sustain Transgene Expression

Biopharmaceuticals are important for the treatment of many diseases including multiple types of cancer. Their production involves a complex multistep process that typically begins with the random integration of a recombinant protein transgene into the host cell genome. However, this random integration often occurs in epigenetically variable regions, leading to inconsistent and unstable transgene expression. To address this issue, biomanufacturing organizations frequently employ genetic elements that improve expression, with the most common being the ubiquitous chromatin opening element (UCOE). Nonetheless, prior research indicates that the effectiveness of UCOEs varies depending on the expression system.

This thesis examined the barrier and insulator properties of various genetic elements. The barrier activity of the UCOE was compared to an alternative barrier, tRNA genes. Using an eGFP reporter assay in CHO cells, I demonstrate that some tRNA gene clusters serve as a more effective barrier compared to the Rps3 UCOE and the A2UCOE. Molecular analysis shows that a tRNA gene barrier can reduce H3K9me3, a marker of transcriptional repression, and sustain H3K27ac, associated with transcriptional activation, in long-term culture.

Industrial testing revealed that tRNA gene barriers improved the expression of an Fc-fusion protein, and increased the specific productivity of cells expressing an anti-CD20 mAb in an Ambr® 15 bioreactor.