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TRANSLATIONAL AND CLINICAL RESEARCH

Combinatorial Incorporation of Enhancer-Blocking Components of the Chicken β-Globin 5'HS4 and Human T-Cell Receptor / BEAD-1 Insulators in Self-Inactivating Retroviral Vectors Reduces Their Genotoxic Potential

^aDepartment of Anatomy and Regenerative Biology and
^bFlow Cytometry Core Facility, The George Washington University Medical Center, Washington, DC, USA

Key Words. Self-inactivating retroviral vectors • Insertional mutagenesis • Genotoxicity • Enhancer-blocking elements • CCCTC-binding factor

Correspondence: Correspondence: Robert G. Hawley, Ph.D., Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037, USA. Telephone: 202-994-3511; Fax: 202-994-8885; e-mail: rghawley{at}gwu.edu

Received on March 18, 2008; accepted for publication on September 1, 2008.

First published online in STEM CELLS EXPRESS  September 11, 2008.

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 β-globin 5'HS4 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 5'HS4 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.

Disclosure of potential conflicts of interest is found at the end of this article.