Abstract
Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.
Highlights
The design of lentiviral vectors has improved substantially over the last decade, even modern SIN-lentiviral vectors remain subject to positional effects that can lead to silenced or variegated transgene expression1, 2
Cells transduced with the vectors C(1-508)-SG, C(85-508)-SG, C(170-508)-SG, C(340-508)-SG, or C(503-679)-SG maintained transgene expression at levels comparable to the positive controls ranging from 71% to 80% in mouse embryonic stem cells (mESCs) and 61% to 68% in miPSCs (Fig. 2A,B)
The transgene expression stabilized and stable eGFP-expression was observed for at least 9 passages i.e. until day 31 (Figs 2C,D, S1B,C). These results indicate the CpG-rich central and 3′ region of the CBX3 region to be involved in ubiquitous chromatin opening elements (UCOEs)-mediated transgene stabilization in pluripotent cells
Summary
The design of lentiviral vectors has improved substantially over the last decade, even modern SIN-lentiviral vectors remain subject to positional effects that can lead to silenced or variegated transgene expression . While mechanistically the anti-silencing function of the UCOEs has been attributed to the presence of non-methylated CGIs, a feature shared by all known UCOEs, up to now the anti-silencing properties of the UCOE have not been linked to specific structural features To potentially identify such features within the CBX3 structure we have generated a number of CBX3-derived subfragments and analysed the functionality of these subfragments in the context of lentiviral vectors and a broad range of integration sites within murine ESCs and iPSCs as well as their differentiated progeny. All investigations assessing UCOE function have been performed in bulk cell populations with varying integration sites while a direct comparison of expression cassettes with and without UCOE in a defined integration site and chromosomal architecture has not yet been performed It is not known if UCOEs can protect from silencing irrespective of the nature of the chromosomal integration site. We have examined the anti-silencing ability of the CBX3 at two defined chromosomal loci, the ROSA2616 and the TIGRE locus T117, in single-cell derived mESC clones utilising recombinase mediated cassette exchange (RMCE)
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