Testing of novel CpG chromatin fragments as UCOE candidates for improved gene therapy vectors

Aim: The A2UCOE sequence, positioned between the HNRPA2B1 and CBX3 gene promoters in the human genome, supports durable and consistent expression of integrated transgenes, even within compact heterochromatin domains such as centromeres. This project focuses on evaluating the dual-component hypothesis of A2UCOE function by analyzing alternative DNA elements that possess CpG-rich content and divergent promoter features. Method: To investigate expression stability, lentiviral vectors carrying eGFP reporter constructs driven by novel UCOE candidates and various A2UCOE subregions were introduced into P19 and F9 mouse embryonal carcinoma cells. Expression was tracked over time, both before and after lineage-specific differentiation toward neuroectoderm and endoderm. To examine the proposed bipartite model of UCOE function, we employed two types of CpG-rich, bidirectionally transcribed elements: the endogenous SETD3–CCNK housekeeping gene pair, and a synthetically arranged divergent configuration composed of RPS11 and HNRPA2B1 promoters. Results: Placing these regulatory elements in either orientation upstream of the SFFV-eGFP reporter gene—known for its susceptibility to transcriptional silencing—conferred a noticeable, though incomplete, resistance to silencing when compared to the full activity of the reference 1.5A2UCOE-SFFV-eGFP construct. This partial protective effect was consistently observed in both P19 and F9 cell lines, prior to and following their differentiation. In conclusion, we successfully identified a naturally occurring (SETD3–CCNK) and synthetically engineered (RPS11–HNRPA2B1) pair of divergent promoters that exhibited measurable but incomplete UCOE-like activity relative to the established HNRPA2B1–CBX3 core element. Conclusion: This study demonstrates that natural and synthetic divergent promoter pairs confer significant, though partial, resistance to transgene silencing. This finding directly supports the A2UCOE's dual-component hypothesis, confirming that CpG-rich bidirectional architecture is key for sustaining stable expression through differentiation and in challenging genomic contexts.