Impact of Promoter-Independent CpG-Rich A2UCOE Subregions on Transgene Expression

Aim: This study aimed to evaluate the ability of CpG-rich subregions within the A2UCOE element, excluding the HNRPA2B1 and CBX3 promoters, to sustain long-term transgene expression in lentiviral systems. These regions are cloned upstream of the spleen focus-forming virus (SFFV) promoter and tested in P19 and F9 cells. Material and Methods: The study assessed the ubiquitous chromatin opening element (UCOE) activity of three A2UCOE subregions that lack promoters. Using P19 and F9 murine embryonal carcinoma cells, enhanced green fluorescent protein (eGFP) expression stability and durability were examined in undifferentiated and differentiated states. Lentiviral vectors (LVs) containing CpG-rich, promoterless fragments, 455UCOE (OFA1), 527UCOE (OFA2), and 945UCOE (OFA3), were transduced into the cells, and sustained transcriptional activity was analyzed via flow cytometry. Results: Some vectors sustained eGFP expression, while others exhibited a rapid decline in both undifferentiated and differentiated states. Specifically, the OFA1, OFA2, and OFA3 vectors showed trends similar to the SEW vector, with significant reductions in eGFP expression over time. None of the CBX3-derived fragments provided stable expression, suggesting no UCOE activity. The rates of expression decline were similar between the SEW vector with the SFFV promoter and the LVs containing OFA1, OFA2, and OFA3. Conclusion: CpG-rich, promoterless fragments failed to support stable transgene expression in murine embryonal carcinoma cells, indicating a lack of UCOE functionality. These findings highlight the role of promoter and regulatory elements in maintaining open chromatin for gene therapy. Future studies should investigate other CpG-rich regions or minimal promoters to improve transgene stability.