The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function

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.

Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells

“…Also in the context of lentiviral vectors, the MRP8 promoter shows a high abundance of repressive histone marks and low transgene expression in iPSC-derived myeloid cells 34 . The inclusion of insulators or ubiquitous chromatin opening elements (UCOE), such as the 1.8 kb element or a shortened CBX3 version, could help to overcome transgene silencing 21,[34][35][36] . Insulators are DNA sequences that block the spread of heterochromatin and thus protect against silencing and DNA methylation 37 .…”

A Novel Chromatin-Opening Element for Stable Long-term Transgene Expression

“…Soon after, the 1.5 kb core region was identified, along with the discovery that a 1.2 kb sub-fragment of that element was equally effective in promoting stable expression (14,17). Since then, a variety of individual studies have found efficacy from variable lengths of the core sequence, but those additional truncations, up to 600 bp, have not been proven out further than the one specific application in each study (16,23,24). Thus, there remains a need for a single modular sequence under 1 kb that can predictably stabilize a broad diversity of gene expression constructs.…”

Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production

“…As a second strategy, we exploited targetable CHO cell lines to adjust the cassette design to the requirements of the particular integration sites. We focused on UCOEs since they have been shown to convey an open chromatin status that was also maintained upon differentiation of blood as well as pluripotent stem cells, thereby preventing the silencing of transgenes. Moreover, UCOEs have been shown to improve expression of various recombinant genes including antibodies. − So far, however, UCOEs have been mainly characterized upon random chromosomal integration of the vectors, by comparing clones with UCOE-free and UCOE-supplemented vectors.…”

“…integration sites. We focused on UCOEs since they have been shown to convey an open chromatin status that was also maintained upon differentiation of blood71 as well as pluripotent72 stem cells, thereby preventing the silencing of transgenes. Moreover, UCOEs have been shown to improve expression of various recombinant genes including antibodies.73−76 So far, however, UCOEs have been mainly…”