Combinatorial Incorporation of Enhancer-Blocking Components of the Chicken β-Globin 5′HS4 and Human T-Cell Receptor α/δ BEAD-1 Insulators in Self-Inactivating Retroviral Vectors Reduces Their Genotoxic Potential

Abstract

Insertional mutagenesis by retroviral vectors has emerged as a serious impediment to the widespread application of hematopoietic stem cell gene transfer for the treatment of hematologic diseases. Here we report the development of a 77-base pair element, FII/BEAD-A (FB), which contains the minimal enhancer-blocking components of the chicken ␤-globin 5HS4 insulator and a homologous region from the human T-cell receptor ␣/␦ BEAD-1 insulator. With a new flow cytometry-based assay, we show that the FB element is as effective in enhancer-blocking activity as the prototypical 1.2-kilobase 5HS4 insulator fragment. When incorporated into the residual U3 region of the 3 long terminal repeat (LTR) of a self-inactivating (SIN) gammaretroviral vector, the FB element was stably transferred to the 5 LTR during reverse transcription, flanking the integrated transgene expression cassette. Notably, using a recently established in vitro insertional mutagenesis assay involving primary murine hematopoietic cells, we found that SIN gammaretroviral vectors, as well as SIN lentiviral vectors, containing the FB element exhibited greatly reduced transforming potential-to background levels under the experimental conditions used-compared with their unshielded counterparts. These results suggest that the FB element-mediated enhancer-blocking modification is a promising approach to dramatically improve the safety of retroviral vectors for therapeutic gene transfer. STEM CELLS 2008;26:3257-3266 Disclosure of potential conflicts of interest is found at the end of this article.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…Here, our results introduce novel insulator elements suitable for implementation in viral vectors, endorsing previous reports on insulator elements able to reduce insertional genotoxicity. 9,26,44,45 The barrier activity of the novel insulating elements was probed in the context of random transgene chromosomal integration upon stable transfection. Flanking the transgene with CTCF binding sites led to a decrease in its expression, stably propagated over cell division.…”

Insulated Foamy Viral Vectors

“…6xCTCF and 12xCTCF contain repeats of either the CTCF-binding sites from cHS4 and the blocking element a/d (BEAD) insulator from the human a/d T cell locus (6xCTCF) or the consensus CTCF-binding site (12xCTCF). 51,52 The 7xCTF/NF1 insulator is not based on CTCF binding but has seven repeats of the binding site for CTF/NF1. This insulator was originally described as a barrier insulator but may have blocking activity as well when repeated in tandem.…”

Gene therapy for hemoglobin disorders - a mini-review

“…Though gene dysregulation was seen in the vicinity of the integrated vector, no protooncogene upregulation was noted 34 . Use of cHS4 insulators decreased the transforming potential further, along with decreasing position dependent variable gene expression and methylation-associated silencing 35 .…”