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Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

Gene Therapy volume 20, pages 35–42 (2013)Cite this article

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Abstract

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

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Acknowledgements

We thank Kelley Ellis for her helpful review and comments. This work was funded by the Northwest Genome Engineering Consortium (NGEC) to support MH. Work in the Porteus lab is supported by funds from the state of Texas, a career development award from the Burroughs-Wellcome fund, the Amon Carter Foundation and grant PN2EY018244 supporting a Nanomedicine Development Center through the Director's Common Fund.

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  1. B L Ellis and M L Hirsch: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    B L Ellis

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

    M L Hirsch & R J Samulski

  3. Department of Pediatrics, Stanford Medical School, Stanford, CA, USA

    S N Porter & M H Porteus

  4. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R J Samulski

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  1. B L Ellis
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Correspondence to M H Porteus.

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Ellis, B., Hirsch, M., Porter, S. et al. Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs. Gene Ther 20, 35–42 (2013). https://doi.org/10.1038/gt.2011.211

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