Minimal CBX3-Derived UCOE Confers Long-Term Resistance to Transgene Silencing in Human iPSCs During Neuronal Differentiation

Long-term transgene silencing remains a major challenge in lentiviral gene delivery, particularly in pluripotent stem cells undergoing lineage-specific differentiation. Universal chromatin opening elements (UCOEs) have emerged as effective regulatory elements for protecting transgene expression against epigenetic silencing; however, their relatively large-size limits vector design flexibility. In the present study, we evaluated the anti-silencing activity of three next-generation UCOE constructs (1.7 kb, 1.2 kb, and a newly developed minimal 0.5 kb fragment) during long-term culture and neuronal differentiation of human induced pluripotent stem cells (iPSCs). UCOE fragments were cloned into self-inactivating lentiviral vectors and validated by restriction enzyme analysis and agarose gel electrophoresis. Lentiviral particles produced in HEK293T cells were used to transduce human iPSCs, followed by neuronal differentiation. Transgene expression was monitored for up to 60 days using fluorescence microscopy, confocal immunofluorescence, flow cytometry, and vector copy number analysis by quantitative PCR. All UCOE-containing vectors demonstrated significantly improved transgene stability compared with the UCOE-less control throughout both the undifferentiated and differentiated stages. The UCOE-containing constructs maintained substantially greater expression stability than the UCOE-less control, with the minimal 0.5 kb UCOE showing long-term performance comparable to the larger UCOE constructs. These findings demonstrate that considerable UCOE minimization can be achieved without markedly compromising anti-silencing activity, providing a potential vector-design advantage by reducing the regulatory cassette footprint and increasing available vector capacity. The compact 0.5 kb UCOE therefore represents a promising regulatory element for sustained transgene expression in stem cell engineering, disease modeling, regenerative medicine, and future gene therapy applications. However, the present study did not directly assess genomic safety, insertional effects, genotoxicity, or cellular transformation, and dedicated preclinical studies will be required to determine the long-term safety and in vivo performance of this compact UCOE configuration.