Abstract

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

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Acknowledgements

We thank D. Weissman for advice on mRNA production and the whole Naldini laboratory for discussion, F. Benedicenti for help with MiSeq sequencing, L. Sergi Sergi, T. Plati, V. Valtolina, B. Camisa and A. Ranghetti for technical help. SR1 was provided by T. Boitano and M. Cooke under an MTA with the Genomics Institute of the Novartis Research Foundation. This work was supported by grants to L.N. from Telethon (TIGET grant D2) EU (FP7 222878 PERSIST, FP7 601958 SUPERSIST, ERC Advanced Grant 249845 TARGETINGGENETHERAPY) and the Italian Ministry of Health.

Author information

Author notes

    • Davide Moi
    •  & Roberta Mazzieri

    Present address: The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland 4102, Australia.

    • Angelo Lombardo
    •  & Luigi Naldini

    These authors contributed equally to this work.

Affiliations

  1. TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy

    • Pietro Genovese
    • , Giulia Schiroli
    • , Giulia Escobar
    • , Tiziano Di Tomaso
    • , Claudia Firrito
    • , Andrea Calabria
    • , Davide Moi
    • , Roberta Mazzieri
    • , Bernhard Gentner
    • , Eugenio Montini
    • , Angelo Lombardo
    •  & Luigi Naldini
  2. Vita Salute San Raffaele University, 20132 Milan, Italy

    • Giulia Schiroli
    • , Giulia Escobar
    • , Bernhard Gentner
    • , Angelo Lombardo
    •  & Luigi Naldini
  3. Experimental Hematology Unit, San Raffaele Scientific Institute, 20132 Milan, Italy

    • Chiara Bonini
  4. Sangamo BioSciences Inc., Richmond, California 94804, USA

    • Michael C. Holmes
    •  & Philip D. Gregory
  5. Department of Immunology Erasmus MC, University Medical Center, 3015 Rotterdam, The Netherlands

    • Mirjam van der Burg

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Contributions

P.G. designed experiments, performed research, interpreted data and wrote the manuscript. G.S. and G.E. performed research and interpreted data. T.D.T. performed mRNA production. C.F. characterized the corrective cDNA. A.C. and E.M. performed bioinformatics analysis of ZFN specificity. R.M. and D.M. developed the NSG human tumour rejection model. C.B. contributed to the T-cell studies. M.v.d.B. provided SCID-X1 patient cells. M.C.H. and P.D.G. provided ZFNs, interpreted data and edited the manuscript. B.G. set up culture conditions for HSC maintenance. A.L. and L.N. designed and supervised research, interpreted data and wrote the manuscript. L.N. coordinated the study. G.S. and G.E. contributed equally to this work. A.L. and L.N. share senior authorship.

Competing interests

M.C.H. and P.D.G. are employees of Sangamo BioSciences Inc. P.G., A.L., L.N., M.C.H. and P.D.G. filed a patent application on the protocol for targeted integration in human HSPC.

Corresponding author

Correspondence to Luigi Naldini.

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    Supplementary Information

    This file contains Supplementary Text, Supplementary Figures, Supplementary Tables and a list of primers.

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DOI

https://doi.org/10.1038/nature13420

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