Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements ( i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.