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Adeno-associated virus inverted terminal repeats stimulate gene editing

Gene Therapy volume 22, pages 190–195 (2015)Cite this article

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Abstract

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

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Acknowledgements

I thank Drs C Yin, C Hewitt, K Lobachev, and R J Samulski for molecular biology expertise, reagents and helpful discussions. This work was supported by the Northwest Genome Engineering Consortium, the Jain Foundation, and the NIH (RO1AI072176-06A1, RO1AR064369-01A1). Funding was also provided in part through a departmental Unrestricted Grant from Research to Prevent Blindness, New York, NY, USA.

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  1. Gene Therapy Center, University of North Carolina at Chapel, Chapel Hill, NC, USA

    M L Hirsch

  2. Department of Ophthalmology, University of North Carolina, Chapel Hill, NC, USA

    M L Hirsch

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Correspondence to M L Hirsch.

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Hirsch, M. Adeno-associated virus inverted terminal repeats stimulate gene editing. Gene Ther 22, 190–195 (2015). https://doi.org/10.1038/gt.2014.109

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