The Chicken Hypersensitivity Site 4 Core Insulator Blocks Promoter Interference in Lentiviral Vectors

Abstract

Lentiviral vectors, including double internal promoters, can be used to express two transgenes in a single vector construct; however, transcriptional activities from double internal promoters are often inhibited by promoter interference. To determine whether the chicken hypersensitivity site 4 insulator (cHS4) could block promoter interference, lentiviral vectors including an MSCV-U3 promoter (Mp) and an EF1a promoter (Ep) were generated, and transgene expression was evaluated among transduced cells. In the Ep-Mp configuration, transcriptional activity from Mp was much lower, while Mp-Ep had similar transcription levels from both promoters. The cHS4 core insulator increased expression levels from Mp in HeLa cells, hematopoietic cell lines, and mouse peripheral blood cells following hematopoietic stem cell transplantation transduced with the Mp-Ep configured vector. This blocking function was mainly mediated by barrier activity regions in the insulator but not by CCCTC-binding factor (CTCF) binding sites. Cytosine-phosphate-guanine (CpG) methylation did not contribute to this barrier activity. In summary, combining the cHS4 insulator in double promoter vectors can improve transgene expression levels in various cell lines and mouse hematopoietic repopulating cells. These findings are useful for developing hematopoietic stem cell gene therapy.

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.…”

Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model

“…20,21 The GFP-expressing vector contained a GFP expression cassette under the control of a murine stem cell virus promoter (pCL20cMpGFP). 22,23 The empty vector contained a multiple cloning site instead of the GFP gene, which did not express a transgene. The GFP/WPRE vector contained a WPRE element between GFP and 3′LTR.…”

3.4 Rational Approaches for Transgene Expression: Targeted Integration and Episomal Maintenance

“…To shield transgene cassettes from the neighboring chromosomal environment, insulators have been integrated into expression cassettes. In particular, they were employed in lentiviral and γ-retroviral vectors aiming at improvement of safety and performance (e.g., [40][41][42][43]. In general, the insulators seem to increase the likelihood and/or consistency of transgene expression ( [44,45] for review).…”