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

doi:10.1016/j.biomaterials.2018.11.006

Cited by 9 publications

(9 citation statements)

References 40 publications

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“…This observation was further confirmed in rtTA rescue experiments in polyclonal cells as well as in clonally selected lines wherein rtTA overexpression rescued P TET -expression in a dose-dependent and less chromatin context-dependent manner. Consistent with our results, Cullmann and colleagues reported that re-expression of rtTA using gammaretroviral vector partially rescued P TET -driven expression in previously silenced cells . Intriguingly, we found that a stable and sufficient supply of rtTA alone rescued inducibility without the need to specifically overwrite the epigenetic signature as previously reported. , As clonal cells in this study were mostly affected by histone deacetylation-mediated silencing, which, in contrast to DNA methylation, is quickly reverted upon induction of transcription, the degree of silencing could explain this discrepancy.…”

Section: Discussionsupporting

confidence: 91%

“…For delivering Tet-On all-in-one constructs and other genes in vivo , the use of lentiviral vectors is among the most widely used strategies . However, a progressive loss of inducibility has been previously reported for lentivirus-mediated gene expression and for Tet-On all-in-one vectors, − which is often attributed to silencing in the promoter regions . Isolation of stable clones is routinely used to overcome this obstacle, yet this procedure is laborious and not viable for low proliferative or highly sensitive cells, such as primary cells .…”

Section: Introductionmentioning

confidence: 99%

“…Isolation of stable clones is routinely used to overcome this obstacle, yet this procedure is laborious and not viable for low proliferative or highly sensitive cells, such as primary cells . Other approaches to improve construct stability include the use of human housekeeping gene promoters and the incorporation of gene insulator elementsDNA sequences that can shield genes from other regulatory domains in their proximityor ubiquitous chromatin opening elements . Although these elements can attenuate silencing, they often do not completely prevent it, suggesting that the Tet-On all-in-one approach itself could be an additional contributor to the impaired control of transgene expression over time.…”

Section: Introductionmentioning

confidence: 99%

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Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems

et al. 2022

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MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs’ expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs’ expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs.

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Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems

et al. 2022

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“…In contrast to the reported beneficial effect on various constitutive promoters upon integration in hematopoietic cells, ,, the larger UCOE fragments HNRPA2B1-CBX3 and A2UCOE reduced the overall expression level and frequency of P tet -controlled expression in the Rosa26 locus in ES cells. This observation is in contrast to a previous study in which the A2UCOE did not significantly reduce the expression of the P tet promoter after random integration in mouse pluripotent stem cells . This suggests that the effect of the 1,5kb spanning A2UCOE on the P tet promoter is negatively modulated in the context of the chromosomal environment of the Rosa26 locus.…”

Section: Discussioncontrasting

confidence: 99%

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice

et al. 2023

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Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten− eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.

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“…Of potential note is that the 3’UCOE-CORE element, which possesses both HNRPA2B1 and CBX3 promoters appears to provide somewhat greater stability of expression (Fig. 4b) than fragments encompassing just the CBX3 half of the 1.5UCOE [22, 27, 48]. This suggests that the dual divergently transcribed promoter structure of the UCOE-CORE element may confer a more potent anti-transcriptional silencing capability.…”

Section: Discussionmentioning

confidence: 99%

Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

et al. 2019

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BackgroundDNA transposon-based vectors are effective nonviral tools for gene therapy and genetic engineering of cells. However, promoter DNA methylation and a near-random integration profile, which can result in transgene integration into heterochromatin, renders such vectors vulnerable to transcriptional repression. Therefore, to secure persistent transgene expression it may be necessary to protect transposon-embedded transgenes with anti-transcriptional silencing elements.ResultsWe compare four different protective strategies in CHO-K1 cells. Our findings show robust protection from silencing of transgene cassettes mediated by the ubiquitous chromatin-opening element (UCOE) derived from the HNRPA2B1-CBX3 locus. Using a bioinformatic approach, we define a shorter HNRPA2B1-CBX3 UCOE core fragment and demonstrate that this can robustly maintain transgene expression after extended passaging of CHO-K1 cells carrying DNA transposon vectors equipped with this protective feature.ConclusionsOur findings contribute to the understanding of the mechanism of HNRPA2B1-CBX3 UCOE-based transgene protection and support the use of a correctly oriented core fragment of this UCOE for DNA transposon vector-based production of recombinant proteins in CHO-K1 cells.

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

Cited by 9 publications

References 40 publications

Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems

Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice

Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

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