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Targeted gene therapy into a safe harbor site in human hematopoietic progenitor cells

Gene Therapy volume 27, pages 435–450 (2020)Cite this article

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Abstract

Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34+ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34+ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.

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Fig. 1: Integration of a Green Matrix in the SH6 safe harbor site mediated by SH6-meganucleases in HEK-293H cells.
Fig. 2: Comparative analysis of TALE nucleases versus SH6v5-MNs to mediate homologous recombination in the SH6 safe harbor site in HEK-293H cells.
Fig. 3: SH6v5-meganuclease and TALE nuclease mediated homologous recombination in EGFP+ sorted CD34+ hematopoietic progenitor cells.
Fig. 4: SH6v5-meganuclease and TALE nuclease mediated homologous recombination in nonsorted CD34+ hematopoietic progenitor cells.
Fig. 5: SH6 TALE nuclease mediated homologous recombination in CD34+ hematopoietic progenitor cells.
Fig. 6: Reconstitution potential of CD34+ cells subjected to SH6 TALE nuclease mediated homologous recombination protocol.
Fig. 7: Integration of a therapeutic matrix in the SH6 safe harbor site mediated by SH6-meganucleases and TALEN.

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Acknowledgements

The authors would like to thank Agnes Gouble, Roman Galetto and Laurent Poirot from CELLECTIS S.A (Paris) for their help providing us with reagents, tools, and their contribution with ideas, Miguel A. Martin for the careful maintenance of NSG mice, Rebeca Sánchez and Omaira Alberquilla for their technical assistance in flow cytometry and Centro de Transfusiones de la Comunidad de Madrid for the cord blood availability. The authors also thank Fundación Botín for promoting translational research at the Hematopoietic Innovative Therapies Division of the CIEMAT. CIBERER is an initiative of the Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER).

Funding

This work was supported by grants from the 7th Framework Program European Commission (HEALTH-F5-2012-305421; EUROFANCOLEN), Ministerio de Ciencia, Innovación y Universidades y Fondo Europeo de Desarrollo Regional (FEDER) (SAF2015-68073-R, SAF2017-84248-P and RTI2018-097125-B-I00) and Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (RD16/0011/0011).

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  1. Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)/Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), 28040, Madrid, Spain

    Fatima Rodriguez-Fornes, Oscar Quintana-Bustamante, M. Luz Lozano, Jose C. Segovia, Juan A. Bueren & Guillermo Guenechea

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  1. Fatima Rodriguez-Fornes
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Correspondence to Guillermo Guenechea.

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Rodriguez-Fornes, F., Quintana-Bustamante, O., Lozano, M.L. et al. Targeted gene therapy into a safe harbor site in human hematopoietic progenitor cells. Gene Ther 27, 435–450 (2020). https://doi.org/10.1038/s41434-020-0144-x

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