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

Ali Ramezani, Teresa S. Hawley, Robert G. Hawley

Stem Cells Express, first published online September 11, 2008; doi:10.1634/stemcells.2008-0258

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-bp 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 α/δ 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-kb 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 to 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.

Abstract |  References |  Full Text: HTML, PDF (475K)

Problems getting Full Text?

Comments (0)

Subscribe to this comment's feed