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. 1 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany. 2 Department of Hematology/Oncology, University Medical School, Frankfurt, Germany. 3 Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany. 4 Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany. 5 University Women's Clinic, Division Molecular Biology of Breast Cancer, Heidelberg, Germany. 6 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany. 7 Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center and Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany. 7 Pediatric Hematology, Oncology and Hemostaseology, University Medical School, Frankfurt, Germany. 8 Department of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany. 9 Department of Hematology, Oncology and Transfusion Medicine, Charité, Campus Benjamin Franklin, Berlin, Germany. 10 Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany. 11 EUFETS AG, Idar-Oberstein, Germany. 12 Centre for Immunodeficiency, UCL Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust London, UK. 13 Division of Immunology/Hematology, University Children's Hospital Zurich, Zurich, Switzerland. 15 These authors contributed equally to this work. a r t i c l e sThe subject received daily granulocyte colonystimulating factor (G-CSF) support (5 µg per kg body weight per day) from months 18 to 20 and months 24 to 26, as well as multiple red blood and platelet transfusions. Following a dental abscess and a febrile episode requiring antibiotic and antimycotic treatment, subject 1 was noted to have extensive splenomegaly and underwent splenectomy at month 25 to avoid spontaneous rupture. Histopathological examination of the spleen revealed extramedullary hematopoiesis and siderosis in the red pulp, without signs of dys...

De novo acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): a clinicopathologic and cytogenetic study of an entity recently added to the WHO classification

“…However, one of our cases showed monosomy 7 present as a subclone, suggesting that inv(3)/t(3;3) is an initial event. This is supported by the observation that insertional activation of EVI1 in human cells induces genomic instability and monosomy 7 reported by Stein et al 17 None of these additional abnormalities seemed to affect overall survival, suggesting that the presence of inv(3)/t(3;3) alone predicts a poor prognosis. It is possible that additional molecular genetic events that are not readily detectable by conventional cytogenetic analysis are also implicated in cases of apparently isolated inv(3)/t(3;3).…”

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

“…23 This vector is, thus, predicted to carry a considerably reduced risk of insertional mutagenesis, compared with more ubiquitously active gene transfer systems, such as ones recently used in a CGD gene therapy clinical trial. 3,4 Natural regulation of gp91phox may be advantageous for gene therapy in X-CGD than using the recently described synthetic promoter, created by fusion of the myeloid specific proto-oncogene c-fes and Cathepsin G 5¢-flanking regions. 24 Transcriptional targeting is increasingly sought in gene therapy, whether to prevent toxic effects of the transgene product in nonphysiological cellular environments, to limit immune responses linked to its expression in antigen-presenting cells or to avoid the activation of proto-oncogenes in early precursors by insertional mutagenesis in the case of integrating vectors.…”

“…2,3 Furthermore, both subjects showed silencing of the transgene 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). 4 The spleen focus-forming virus promoter is strong and ubiquitously active, notably inducing high levels of expression in HSCs. 5 Such property is completely dispensable for CGD, in which the functional correction of the underlying defect is required only in differentiated myeloid cells, which have a low proliferative capacity and a short half-life, hence a minimal likelihood of leukemic degeneration.…”

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

“…Lentiviral and retroviral vectors integrate the transgene into the host cell’s genome and could possibly express the transgene for the life of the host cells. However, the preference of lentiviral and retroviral vectors to integrate into transcriptionally active genomic regions is associated with a high risk of vector-associated insertional mutagenesis, which could be harmful to the host cell and to the patient 12, 13, 14, 15, 16, 17, 18, 19…”