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Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells

Gene Therapy volume 20, pages 201–214 (2013)Cite this article

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Abstract

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

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Acknowledgements

This study was supported by NIH grants R21HL094994 (AL), R01 HLA078836 (AL), R01CA136487 (AL), R01CA141018 (KB), R01DK083310 (KB), R37DK45978 (KB) and NIH/NIDDK DK grant R37-45978. We thank Drs. Linden and Trempe for providing Rep78 antibodies. We are grateful to Carol Ware and the members of the Ellison Stem Cell Core Laboratory of the Institute of Stem Cell and Regenerative Medicine.

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Authors and Affiliations

  1. Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA, USA

    R van Rensburg, I Beyer, X-Y Yao, H Wang, Z-Y Li & A Lieber

  2. UW Medicine Lake Union, University of Washington, Seattle, WA, USA

    O Denisenko & K Bomsztyk

  3. Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA

    D W Russell

  4. Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA, USA

    D G Miller

  5. Sangamo BioSciences, Inc., Richmond, CA, USA

    P Gregory & M Holmes

  6. Department of Pathology, University of Washington, Seattle, WA, USA

    A Lieber

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  1. R van Rensburg
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van Rensburg, R., Beyer, I., Yao, XY. et al. Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther 20, 201–214 (2013). https://doi.org/10.1038/gt.2012.25

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