Abstract

Aim: The A2UCOE, a well-characterized chromatin-modifying element originating from a constitutively expressed human gene locus, promotes consistent and durable gene activity, even when foreign DNA sequences are inserted into densely packed heterochromatin regions. The aim of this study is to examine the Daedalus fragment in A2UCOE to identify key sub-regions responsible for its prominent chromatin opening activity. This would enable the discovery of a minimal yet fully functional element, allowing for more efficient utilization of vector capacity to accommodate therapeutic transgenes when incorporated into lentiviral vectors (LVs).

Method: To assess the chromatin-opening activity of the Daedalus sub-region within the A2UCOE, lentiviral vectors carrying eGFP reporter constructs were generated and transduced into murine embryonal carcinoma cell lines (P19 and F9). Transgene expression stability was assessed across both pluripotent cells and those differentiated with retinoic acid. HEK293T cells were used for lentiviral packaging. Plasmid propagation was performed in E. coli DH5α, and standard molecular cloning techniques were applied. Flow cytometry was used to measure eGFP expression levels, and significance was assessed via Student’s t-test (p < 0.05).

Results: Flow cytometry and RT-qPCR analyses revealed that the positive control vector (1.5A2UCOE-SEW) maintained stable eGFP expression in both differentiated states of P19 and F9 cells, whereas the UCOE vectors named Daedalus-F and Daedalus-R were unable to prevent transgene silencing. In undifferentiated cultures, eGFP expression from these vectors decreased by 40–50% within two weeks, with a similar decline observed in differentiated cells. The average vector copy numbers remained stable, indicating transcriptional silencing rather than vector loss.

Conclusion: The Daedalus sub-region of A2UCOE alone could not prevent transgene silencing in pluripotent or differentiated cells, indicating additional A2UCOE elements are needed for stable long-term expression.

Keywords: chromatin structure modulation, daedalus vector system, lentiviral gene delivery, neuroectodermal and endodermal differentiation, transcriptional silencing, ubiquitous chromatin opening element

Copyright and license

Copyright © 2025 The Author(s). This is an open-access article published by Bolu İzzet Baysal Training and Research Hospital under the terms of the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

How to cite

1.
Arslan AO, Anakök ÖF. Daedalus fragment from the CBX3 ubiquitous chromatin opening element as an optimum UCOE candidate. Northwestern Med J. 2025;5(4):222-33. https://doi.org/10.54307/2025.NWMJ.174

References

  1. Antoniou MN, Skipper KA, Anakok O. Optimizing retroviral gene expression for effective therapies. Hum Gene Ther. 2013; 24(4): 363-74. https://doi.org/10.1089/hum.2013.062
  2. Griesenbach U, Alton EW. Moving forward: cystic fibrosis gene therapy. Hum Mol Genet. 2013; 22(R1): R52-8. https://doi.org/10.1093/hmg/ddt372
  3. Neville JJ, Orlando J, Mann K, McCloskey B, Antoniou MN. Ubiquitous Chromatin-opening Elements (UCOEs): applications in biomanufacturing and gene therapy. Biotechnol Adv. 2017; 35(5): 557-64. https://doi.org/10.1016/j.biotechadv.2017.05.004
  4. Sizer RE, White RJ. Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology. Comput Struct Biotechnol J. 2022; 21: 275-83. https://doi.org/10.1016/j.csbj.2022.11.059
  5. Fu Y, Han Z, Cheng W, Niu S, Wang T, Wang X. Improvement strategies for transient gene expression in mammalian cells. Appl Microbiol Biotechnol. 2024; 108(1): 480. https://doi.org/10.1007/s00253-024-13315-y
  6. Naldini L, Blömer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259): 263-7. https://doi.org/10.1126/science.272.5259.263
  7. Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol. 2009; 157(2): 153-65. https://doi.org/10.1038/bjp.2008.349
  8. Gurumoorthy N, Nordin F, Tye GJ, Wan Kamarul Zaman WS, Ng MH. Non-integrating lentiviral vectors in clinical applications: a glance through. Biomedicines. 2022; 10(1): 107. https://doi.org/10.3390/biomedicines10010107
  9. Teich NM, Weiss RA, Martin GR, Lowy DR. Virus infection of murine teratocarcinoma stem cell lines. Cell. 1977; 12(4): 973-82. https://doi.org/10.1016/0092-8674(77)90162-3
  10. Speers WC, Gautsch JW, Dixon FJ. Silent infection of murine embryonal carcinoma cells by Moloney murine leukemia virus. Virology. 1980; 105(1): 241-4. https://doi.org/10.1016/0042-6822(80)90171-3
  11. Razin A. CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J. 1998; 17(17): 4905-8. https://doi.org/10.1093/emboj/17.17.4905
  12. Fuks F. DNA methylation and histone modifications: teaming up to silence genes. Curr Opin Genet Dev. 2005; 15(5): 490-5. https://doi.org/10.1016/j.gde.2005.08.002
  13. Benton T, Chen T, McEntee M, et al. The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein. Cytotechnology. 2002; 38(1-3): 43-6. https://doi.org/10.1023/A
  14. Ellis J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum Gene Ther. 2005; 16(11): 1241-6. https://doi.org/10.1089/hum.2005.16.1241
  15. Zhang F, Thornhill SI, Howe SJ, et al. Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells. Blood. 2007; 110(5): 1448-57. https://doi.org/10.1182/blood-2006-12-060814
  16. Zhang F, Frost AR, Blundell MP, Bales O, Antoniou MN, Thrasher AJ. A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors. Mol Ther. 2010; 18(9): 1640-9. https://doi.org/10.1038/mt.2010.132
  17. Cavazzana-Calvo M, Fischer A, Hacein-Bey-Abina S, Aiuti A. Gene therapy for primary immunodeficiencies: part 1. Curr Opin Immunol. 2012; 24(5): 580-4. https://doi.org/10.1016/j.coi.2012.08.008
  18. Cavazza A, Moiani A, Mavilio F. Mechanisms of retroviral integration and mutagenesis. Hum Gene Ther. 2013; 24(2): 119-31. https://doi.org/10.1089/hum.2012.203
  19. Ott MG, Seger R, Stein S, Siler U, Hoelzer D, Grez M. Advances in the treatment of chronic granulomatous disease by gene therapy. Curr Gene Ther. 2007; 7(3): 155-61. https://doi.org/10.2174/156652307780859044
  20. Aiuti A, Bacchetta R, Seger R, Villa A, Cavazzana-Calvo M. Gene therapy for primary immunodeficiencies: part 2. Curr Opin Immunol. 2012; 24(5): 585-91. https://doi.org/10.1016/j.coi.2012.07.012
  21. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009; 326(5954): 818-23. https://doi.org/10.1126/science.1171242
  22. Biffi A, Montini E, Lorioli L, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science. 2013; 341(6148): 1233158. https://doi.org/10.1126/science.1233158
  23. Bosticardo M, Ferrua F, Cavazzana M, Aiuti A. Gene therapy for Wiskott-Aldrich Syndrome. Curr Gene Ther. 2014; 14(6): 413-21. https://doi.org/10.2174/1566523214666140918103731
  24. Bandaranayake AD, Correnti C, Ryu BY, Brault M, Strong RK, Rawlings DJ. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res. 2011; 39(21): e143. https://doi.org/10.1093/nar/gkr706
  25. Lienert F, Wirbelauer C, Som I, Dean A, Mohn F, Schübeler D. Identification of genetic elements that autonomously determine DNA methylation states. Nat Genet. 2011; 43(11): 1091-7. https://doi.org/10.1038/ng.946
  26. Lindahl Allen M, Antoniou M. Correlation of DNA methylation with histone modifications across the HNRPA2B1-CBX3 ubiquitously-acting chromatin open element (UCOE). Epigenetics. 2007; 2(4): 227-36. https://doi.org/10.4161/epi.2.4.5231