Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors

• Published in: Journal of biotechnology 2009-08, Vol.143 (2), p.85-94
• Main Authors: Sgourou, Argyro, Routledge, Samantha, Spathas, Dionysios, Athanassiadou, Aglaia, Antoniou, Michael N.
• Format: Article
• Language: English
• Subjects: Animals; beta-Globins - biosynthesis; beta-Globins - genetics; beta-Globins - metabolism; Biological and medical sciences; Biotechnology; Cell Line, Tumor; Cloning, Molecular - methods; Episomal vector; Fundamental and applied biological sciences. Psychology; Gene Dosage; Genetic Vectors - genetics; Genetic Vectors - metabolism; Haemoglobinopathies; HBB; Humans; In Situ Hybridization, Fluorescence; Interferon-beta - genetics; Interferon-beta - metabolism; K562 Cells; Matrix Attachment Regions; Mice; Plasmids - genetics; Plasmids - metabolism; S/MAR; Transcription, Genetic; Transgenes
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2009.06.018

Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5′ of the human β-interferon gene ( IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid β-globin locus control region-β-globin gene (βLCR- HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The βLCR- HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the βLCR- HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from βLCR- HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.