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Sustained and regulated gene expression by Tet-inducible “all-in-one” retroviral vectors containing the HNRPA2B1-CBX3 UCOE®

Cullmann, Katharina, Blokland, Kaj E.C., Sebe, Attila, Schenk, Franziska, Ivics, Zoltán, Heinz, Niels, Modlich, Ute
Biomaterials 2019 v.192 pp. 486-499
cell growth, cell lines, chromatin, doxycycline, epigenetics, gene expression, genetic engineering, humans, mice, promoter regions, retroviral vectors, thrombopoietin, transactivators, transcription (genetics), transgenes
Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site.In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.