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

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 MDS1EVI1 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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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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