Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.

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...

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Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/ AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.