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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 EF1α 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.

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