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Genomic discovery of potent chromatin insulators for human gene therapy

Abstract

Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

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Figure 1: Identification of putative insulators by genomic profiling.
Figure 2: Functional identification of enhancer-blocking insulators.
Figure 3: Assessing the enhancer-blocking insulators for silencer activity and effects on lentiviral vector titers.
Figure 4: Genotoxicity assay.

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Acknowledgements

This research was supported by US National Institutes of Health (NIH) grants PO1 HL53750 and R90 HG004152 to G.S., grants U54 HG007010 and UO1 ES017156 to J.A.S., and fellowship F31 MH 094073 to M.T.M.

Author information

Authors and Affiliations

Authors

Contributions

G.S. designed the experimental strategies and supervised the study. M.L., C.-Z.S., H.Q. and D.W.E. performed the cell biology and virology experiments. M.T.M., H.W. and J.A.S. developed the genomics approaches. H.W. performed the ChIP-seq experiments. G.S., D.W.E., M.T.M., H.W., P.A.N. and J.A.S. analyzed the data. G.S., D.W.E. and M.T.M. wrote the paper. All authors edited and approved the manuscript.

Corresponding author

Correspondence to George Stamatoyannopoulos.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–7 (PDF 8965 kb)

Supplementary Dataset 1

Ranking of the CTCF motifs of the human genome according to CTCF occupancy. (XLSX 368 kb)

Supplementary Dataset 2

Chromosomal locations, hg18 coordinates and CTCF sequences of all CTCF motifs of ranks 1 to 1000 of Supplementary Table 1 (XLSX 3920 kb)

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Liu, M., Maurano, M., Wang, H. et al. Genomic discovery of potent chromatin insulators for human gene therapy. Nat Biotechnol 33, 198–203 (2015). https://doi.org/10.1038/nbt.3062

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