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

Abstract

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.

Context-aware synthetic biology by controller design: Engineering the mammalian cell

“…The rates of each of these processes are shaped by the values of physical parameters that can be used for design (Figure 3C). For example, the transcription rate can be tuned by the choice of promoter (Ede et al, 2016;Haberle and Stark, 2018;Ponjavic et al, 2006) and terminator (Cheng et al, 2019;Proudfoot, 2016), while the translation rate can be tuned by the sequence in the 5 0 and 3 0 UTRs (De Nijs et al, 2020) (such as with the addition of binding sites for endogenous miRNAs (Gam et al, 2018;Michaels et al, 2019)). Similarly, protein degradation can be tuned by adding protein degradation domains (Trauth et al, 2019).…”

Data-Driven and in Silico-Assisted Design of Broad Host-Range Minimal Intrinsic Terminators Adapted for Bacteria

“…Tremendous efforts have been made in the forward engineering of RNA tools with predefined performance. , However, it is still a huge challenge to design intrinsic terminators because of the complicated relationship between sequence and efficiency, although some terminator models are able to predict the TE for a given sequence. , Typically, the synthetic terminators are generated by screening random sequences or by re-engineering endogenous terminators . These strategies require tremendous, tedious, and repetitive screening work.…”

Rational design and construction of multi-copy biomanufacturing islands in mammalian cells

“…To evaluate whether mRNA secondary structure in the 3′-UTR region may directly impact protein expression, for instance by occluding the target sites required for binding by the cleavage and polyadenylation protein complex, minimum folding energies for the synthetic 3′-UTR library components were calculated using the RNAfold software ( 31 ). Only a weak-to-moderate positive correlation between folding propensity and reporter expression was found (Pearson's r = 0.34, P -value < 0.005) ( Supplementary Figure S5 ), indicating that while weak RNA structure in the 3′-UTR and polyadenylation sequence may to a certain extent favor expression ( 50 ), other factors are likely to be implicated in determining final expression levels, including, for instance, the impact of the 3′-UTR sequences on overall mRNA stability and half-life ( 51 ).…”