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...

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (␣-and ␤2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit 2. Phosphorylation of 2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for 2, ϳ25 min for ␤2, and ϳ70 min for ␣. We were also able to purify a phosphatase that dephosphorylates the 2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic C␤ subunit, a scaffolding A␤ subunit, and a regulatory B␣ subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.

The large-scale chromatin organization of retrovirus and retroviral gene vector integration loci has attracted little attention so far. We compared the nuclear organization of transcribed integration loci with the corresponding loci on the homologous chromosomes. Loci containing gamma-retroviral gene transfer vectors in mouse hematopoietic precursor cells showed small but significant repositioning of the integration loci towards the nuclear interior. HIV integration loci in human cells showed a significant repositioning towards the nuclear interior in two out of five cases. Notably, repositioned HIV integration loci also showed chromatin decondensation. Transcriptional activation of HIV by sodium butyrate treatment did not lead to a further enhancement of the differences between integration and homologous loci. The positioning relative to splicing speckles was indistinguishable for integration and homologous control loci. Our data show that stable retroviral integration can lead to alterations of the nuclear chromatin organization, and has the potential to modulate chromatin structure of the host cell. We thus present an example where a few kb of exogenous DNA are sufficient to significantly alter the large-scale chromatin organization of an endogenous locus.

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Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.