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Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

Nature Medicine volume 16, pages 198–204 (2010)Cite this article

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Abstract

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

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Figure 1: Hematopoietic reconstitution, gene marking and superoxide production in subjects 1 and 2 after gene therapy.
Figure 2: Proviral DNA methylation, clonal distribution of gene-marked cells and MDS1–EVI1 activation in blood samples of subjects 1 and 2.
Figure 3: Monosomy 7 in dominant clones of subjects 1 and 2.
Figure 4: Genomic instability in MDS1−EVI1-expressing cells.
Figure 5: EVI1 induces centrosomal aberrations in human cells.

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Acknowledgements

We are indebted to K. Bleuel and S. Wehner for contributions and advice during the study; to H. Holtgreve-Grez and B. Schoell for I-FISH and M-FISH analysis, respectively; to H. Konrad Müller-Hermelink for reference pathology; to B. Staehle for quantitative PCR; to M.C. Dinauer (Indiana University School of Medicine) for plasmid gp91phox(P415H) and to H. Kunkel, E. Rudolf, A. Dillmann, R. Kramer, B. Moreno, I. Vogler and L. Chen-Wichmann for assistance during this work. This work was supported by the Bundesministerium für Bildung und Forschung (grant 01GU0507, TP6b), the Chronic Granulomatous Disorder Research Trust, London (grant J4G/04B/GT), the Georg-Speyer-Haus (T300131), the EU (VIth Framework Program, CONSERT), the Research Priority Program 1230 from the Deutsche Forschungsgemeinschaft and, in part, by a grant of the Tumorzentrum Heidelberg/Mannheim to A.J. A.J.T. is also supported by the Wellcome Trust and the Department of Health (HTH/011/025/004). A. Krämer is supported by the Deutsche Krebshilfe (grant 108560). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.

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Author notes

  1. Stefan Stein, Marion G Ott, Dieter Hoelzer, Reinhard Seger, Christof von Kalle and Manuel Grez: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany

    Stefan Stein, Stephan Schultze-Strasser, Andrea Kinner, Carolin Preiss & Manuel Grez

  2. Department of Hematology/Oncology, University Medical School, Frankfurt, Germany

    Marion G Ott, Joachim Schwäble, Hans Martin & Dieter Hoelzer

  3. Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany

    Anna Jauch

  4. Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany

    Barbara Burwinkel, Sandrine Tchatchou & Rongxi Yang

  5. Division Molecular Biology of Breast Cancer, University Women's Clinic, Heidelberg, Germany

    Barbara Burwinkel, Sandrine Tchatchou & Rongxi Yang

  6. Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany

    Manfred Schmidt, Hanno Glimm, Claudia Ball, Kerstin Schwarzwaelder & Christof von Kalle

  7. German Cancer Research Center and Department of Internal Medicine V, Clinical Cooperation Unit Molecular Hematology/Oncology, University of Heidelberg, Heidelberg, Germany

    Alwin Krämer & Kadin Karakaya

  8. Pediatric Hematology, Oncology and Hemostaseology, University Medical School, Frankfurt, Germany

    Ulrike Koehl

  9. Department of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany

    Gudrun Göhring & Brigitte Schlegelberger

  10. Department of Hematology, Oncology and Transfusion Medicine, Charité, Campus Benjamin Franklin, Berlin, Germany

    Wolf-Karsten Hofmann

  11. Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany

    Petra Reinecke

  12. EUFETS AG, Idar-Oberstein, Germany

    Klaus Kühlcke

  13. Centre for Immunodeficiency, UCL Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust London, UK

    Adrian J Thrasher

  14. Division of Immunology/Hematology, University Children's Hospital Zurich, Zurich, Switzerland

    Reinhard Seger

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  1. Stefan Stein
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  2. Marion G Ott
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Contributions

S.S., M.G.O. and M.G. conceived, organized and supervised the study; S.S., A. Kinner, S.S.-S., C.P. and K. Kühlcke performed experiments; B.B., S.T. and R.Y. performed the SNP and methylation arrays and analyzed data; A. Kinner, A. Krämer, C.B., K. Karakaya. and H.G. generated and analyzed data on genomic instability; A.J. performed the I-FISH and M-FISH analysis; S.S.-S. performed the CpG methylation studies; M.G.O., J.S., H.M., G.G., W.-K.H., P.R., B.S., U.K., R.S. and D.H. were responsible for patient care, analyzed materials from the subjects and provided data on these samples; M.S., K.S. and C.v.K. provided data on retroviral integrations sites; A.J.T., R.S., U.K., M.S., C.v.K. and M.G. prepared and wrote the manuscript.

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Correspondence to Manuel Grez.

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Stein, S., Ott, M., Schultze-Strasser, S. et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med 16, 198–204 (2010). https://doi.org/10.1038/nm.2088

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