Engineering synthetic G-quadruplexes as regulatory elements for biopharmaceutical gene expression

Precise regulation of gene expression is a critical requirement in biopharmaceutical applications to balance high productivity with cell viability and product quality. Although approaches to modulate gene expression exist, they often fail to meet desired criteria. Secondary structure-based control elements present a promising avenue for achieving precise regulation. Among them, G-quadruplexes show strong potential for broad applications in recombinant gene expression control.
In this study, the application of synthetic G-quadruplexes within biopharmaceutical production was explored. Initial experiments with endogenous motifs demonstrated the ability of G-quadruplexes to function as regulatory elements and helped identify suitable positions within an industry-relevant expression construct. A toolbox of 67 distinct motifs was systematically designed, constituting the largest collection of synthetic G-quadruplexes reported to date. In total, 134 putative DNA and RNA G-quadruplexes were evaluated in CHO cells, the predominant host cell line used for recombinant therapeutic protein production. The synthetic motifs modulated gene expression over a broad range (4-110%), confirming G-quadruplexes as promising elements for gene expression control. A variety of biological and biophysical analyses were undertaken to confirm G-quadruplex formation and to provide insight into their function within a cellular context. Engineering dual controllers by combining DNA and RNA G-quadruplexes revealed a synergistic interaction. To corroborate their universal applicability, selected synthetic motifs were tested across additional relevant cell types and products. Furthermore, adjusting the heavy and light chain ratio of a monoclonal antibody using combinations of synthetic G-quadruplexes resulted in an almost 20% increase in titre, demonstrating a potential application in biopharmaceutical processes. Overall, this study highlights the potential of synthetic G-quadruplexes as versatile elements for next-generation expression systems to achieve predictable and tunable expression profiles. The here created library of synthetic G-quadruplexes represents a toolbox of regulatory elements that can be readily implemented to advance biopharmaceutical research and production.