The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

Abstract

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish

“…This observation is quite consistent with the most frequent use of cHS4 insulators in mouse genetics: to increase transgene expression and reduce silencing of transgenes (Emery et al 2000; Rivella et al 2000; Yannaki et al 2002; Yao et al 2003; Rincón-Arano et al 2007; Yahata et al 2007; Ochiai et al 2011; Sharma et al 2012; Emery et al 2013; Uchida et al 2013). Insulators are most often tested for this activity in cell culture-based assays and our observation of increased transgene expression is completely consistent with the outcomes of these experiments.…”

“…Insulators have been demonstrated to shield transgenes from surrounding repressive chromatin (Chung et al 1993; Emery et al 2000; Rivella et al 2000; Rincón-Arano et al 2007; Yahata et al 2007; Ochiai et al 2011; Sharma et al 2012; Uchida et al 2013). Therefore, we decided to test if insulator-flanked transgenes display stable expression of mRFP and GFP in zebrafish.…”

Gene Therapy with the Sleeping Beauty Transposon System

“…Thus, it appears that SB transposon vectors have the capacity to provide long-term expression of transgenes both ex vivo and in vivo, suggesting that incorporating insulator sequences in SB vectors is not necessary to protect the transgene from silencing. The use of insulators was demonstrated to effectively shield the promoter activity of the transgene cassette at the integration locus [39,125]. However, although insulators could prevent transactivation of oncogenes, they could also have undesired effects on genome structure and function.…”

Nonviral Gene Therapy—The Challenge of Mobilizing DNA

“…Hyperactive transposase variants SB100X and HyPBase have been shown to increase in vivo efficacy even further Doherty et al 2012) and currently represent the prime choices for therapeutic mobilization of transgenes. However, although high efficiency is certainly attractive in hard-to-transfect cell types or tissues that are difficult to reach with plasmid DNA, it should be kept in mind that the most efficacious transposases hold the potential to insert several transposons into the same cell (Sharma et al 2012), leading in many cases to an increased risk of insertional mutagenesis. Therefore, the amount of transfected plasmid (transposon donor as well as transposase-encoding plasmid) should be optimized for any given application.…”