A Novel Chromatin-Opening Element for Stable Long-term Transgene Expression

Abstract

Long-term stable expression of transgenes in mammalian cells is a challenge in gene therapy, recombinant protein production, and mammalian synthetic biology due to epigenetic silencing and position effect variegation. While multiple classes of regulatory elements have been discovered and proposed to help stabilize expression, the most efficacious has been the Ubiquitous Chromatin Opening Element (UCOE), and in particular, the prototypical A2UCOE from the HNRPA2B1-CBX3 locus. We developed a feature-driven bioinformatics algorithm to discover other putative UCOEs from the human genome, and identified a novel UCOE (SRF-UCOE) that can resist transgene silencing in the methylation-prone P19 cell line. We demonstrate that a 767 bp core sequence of SRF-UCOE is modular to four common mammalian promoters. Notably, SRF-UCOE stabilizes gene expression in transduced P19 cells up to 2.4-fold better over 26 days than the existing A2UCOE by resisting constructs susceptibility to DNA methylation and histone deacetylation. Unlike existing UCOEs, SRF-UCOE lacks inherent transcriptional initiation activity, which can bolster its safe and predictable use in gene therapy constructs. We expect that expanding the set of UCOEs available will expand their utility to novel applications in gene therapy, synthetic biology, and biomanufacturing, as well as contribute to understanding their molecular mechanism.