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Lentiviral gene therapy and vitamin B3 treatment enable granulocytic differentiation of G6PC3-deficient induced pluripotent stem cells

Gene Therapy volume 27, pages 297–306 (2020)Cite this article

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Abstract

Induced pluripotent stem cells (iPSCs) from patients with genetic disorders are a valuable source for in vitro disease models, which enable drug testing and validation of gene and cell therapies. We generated iPSCs from a severe congenital neutropenia (SCN) patient, who presented with a nonsense mutation in the glucose-6-phosphatase catalytic subunit 3 (G6PC3) gene causing profound defects in granulopoiesis, associated with increased susceptibility of neutrophils to apoptosis. Generated SCN iPSC clones exhibited the capacity to differentiate into hematopoietic cells of the myeloid lineage and we identified two cytokine conditions, i.e., using granulocyte-colony stimulating factor or granulocyte-macrophage colony stimulating factor in combination with interleukin-3, to model the SCN phenotype in vitro. Reduced numbers of granulocytes were produced by SCN iPSCs compared with control iPSCs in both settings, which reflected the phenotype in patients. Interestingly, our model showed increased monocyte/macrophage production from the SCN iPSCs. Most importantly, lentiviral genetic correction of SCN iPSCs with a codon-optimized G6PC3 transgene restored granulopoiesis and reduced apoptosis of in vitro differentiated myeloid cells. Moreover, addition of vitamin B3 clearly induced granulocytic differentiation of SCN iPSCs and increased the number of neutrophils to levels comparable with those obtained from healthy control iPSCs. In summary, we established an iPSC-derived in vitro disease model, which will serve as a tool to test the potency of alternative treatment options for SCN patients, such as small molecules and gene therapeutic vectors.

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Fig. 1: Defective granulocyte production from G6PC3-deficient iPSCs under different differentiation conditions.
Fig. 2: Lentiviral vector mediated gene transfer reconstituted granulocytic differentiation in G6PC3-deficient iPSCs.
Fig. 3: Stimulation of G6PC3-deficient myeloid differentiation cultures with vitamin B3 increases the production of granulocytes.

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Data availability

Datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by grants from the Bundesministerium für Bildung und Forschung (PID-NET FK2016M1517F; MyPred), the Deutsche Forschungsgemeinschaft (Cluster of Excellence REBIRTH (EXC 62/2) and SFB738), and the Deutsche Akademische Austauschdienst (DAAD). We thank Gerald Draeger and Thomas Scheper for providing Rock inhibitor Y-27632 and bFGF (Leibniz University Hannover, Hannover, Germany) and Malte Sgodda and Tobias Cantz for H9 ESC RNA (Hannover Medical School, Hannover, Germany), and Robert E. Campbell (University of Alberta, Alberta, Canada) for providing EBFP2.

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Authors and Affiliations

  1. Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany

    Dirk Hoffmann, Johannes Kuehle, Daniela Lenz, Friederike Philipp, Daniela Zychlinski, Nico Lachmann, Thomas Moritz, Michael Morgan & Axel Schambach

  2. REBIRTH – Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany

    Dirk Hoffmann, Johannes Kuehle, Friederike Philipp, Nico Lachmann, Thomas Moritz, Michael Morgan & Axel Schambach

  3. Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany

    Doris Steinemann

  4. Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tuebingen, Tuebingen, Germany

    Julia Skokowa

  5. Department of Pediatrics, Dr. von Hauner Children’s Hospital University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany

    Christoph Klein

  6. Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Axel Schambach

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Correspondence to Axel Schambach.

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Hoffmann, D., Kuehle, J., Lenz, D. et al. Lentiviral gene therapy and vitamin B3 treatment enable granulocytic differentiation of G6PC3-deficient induced pluripotent stem cells. Gene Ther 27, 297–306 (2020). https://doi.org/10.1038/s41434-020-0127-y

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