期刊
STEM CELLS
卷 26, 期 12, 页码 3257-3266出版社
WILEY
DOI: 10.1634/stemcells.2008-0258
关键词
Self-inactivating retroviral vectors; Insertional mutagenesis; Genotoxicity; Enhance blocking elements; CCCTC-binding factor
资金
- NIH [R01-HL65519, R01-HL66305, R24-RR1620]
- Elaine H. Snyder Cancer Research Award
- The George Washington University Medical Center
- NATIONAL CENTER FOR RESEARCH RESOURCES [R24RR016209] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL065519, R01HL066305] Funding Source: NIH RePORTER
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 beta-globin 5'HS4 insulator and a homologous region from the human T-cell receptor alpha/delta 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 enhancerblocking modification is a promising approach to dramatically improve the safety of retroviral vectors for therapeutic gene transfer. STEM CELLS 2008; 26: 3257- 3266
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