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Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1


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 gp91phox. 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.

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Figure 1: Hematopoietic reconstitution and gene marking in P1 and P2 after gene therapy.
Figure 2: Polyclonal hematopoietic repopulation and non-random distribution of RIS in P1 and P2.
Figure 3: Insertions in MDS1-EVI1, PRDM16 and SETBP1 dominate gene-modified long-term myelopoiesis.
Figure 4: Functional reconstitution of NADPH oxidase activity in peripheral blood leukocytes (PBLs) and isolated granulocytes of P1 and P2 as revealed by oxidation of dihydrorhodamine (DHR) 123 (af) and NBT reduction (gj).
Figure 5: Antimicrobial activity of gene corrected neutrophils.
Figure 6: Fused PET scans of P1 (a,b) and fused PET-CT scans of P2 (c,d) before (a,c) and 50 (b) or 53 d (d) after gene therapy.


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We are indebted to families of the subjects for their continuous support and to the medical and nursing staff of the bone marrow transplantation unit of the Department of Hematology at the University Hospital in Frankfurt. We thank E. Karaus, M. Rutishauser and C. Wenk (University Children's Hospital, Zurich) for technical assistance with the granulocyte function tests, L. Chen (Georg-Speyer-Haus, Frankfurt) for valuable help during monitoring of the subjects, S. Wehner, R. Quaritsch, S. Grohal, R. el Kaláoui and C. Kramm (University Children's Hospital, Frankfurt) for assistance with granulocyte tests and immunophenotyping, and S. Schmidt, S. Fessler, C. Prinz, M. Wissler, S. Braun and R. Cziumplik (University of Freiburg) for technical assistance with the molecular analysis. Special thanks to D. Pfeifer (University Hospital Freiburg) for performing the microarray analysis. We are also grateful to T. Bächi (Central laboratory for electron microscopy, University of Zurich) for electron microscopic analysis, to H. Steinert (Nuclear Medicine Clinic, University Hospital Zurich, Switzerland) for PET-CT scans and D. Roos (Sanquin, Department of Experimental Hematology, The Netherlands) for advice with the E. coli killing assays. We also thank C. Baum (Hannover Medical School) and K. Cichutek (Paul-Ehrlich-Institute) for the gift of materials and discussions during this work. RetroNectin (CH-296) was provided by Takara Bio Inc. This work was supported by the Swiss National Science Foundation (National Research Program on Somatic Gene Therapy NFP 37), by the German Ministry of Education and Research (grants 01GE9634/2 and 01GE9904), by the CGD Research Trust, London (grant J4G/01/01), by the European Union (Sixth Framework Program, CONSERT) and by Deutsche Forschungsgemeinschaft grants Ka976/5-3 and Ka976/6-2. A.J.T. is supported by the Wellcome Trust. The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.

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Correspondence to Christof von Kalle or Manuel Grez.

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Supplementary information

Supplementary Fig. 1

RIS distribution of retroviral vector insertions within or near RefSeq genes in P1 and P2. (PDF 73 kb)

Supplementary Fig. 2

Long-term follow up of individual clones contributing to hematopoeisis at different time points after transplantation in P1 and P2. (PDF 79 kb)

Supplementary Fig. 3

Transcriptional activation of CIS genes by retroviral insertion. (PDF 72 kb)

Supplementary Fig. 4

Expression of gp91phox protein on transduced cells. (PDF 74 kb)

Supplementary Fig. 5

Superoxide anion production by granulocytes obtained from a healthy control, P1 at day +193 and P2 at day +50 as revealed by cytochrome c reduction after stimulation with 0.1 μg/ml PMA plus 1 μM fMLP. (PDF 139 kb)

Supplementary Fig. 6

Killing of A. fumigatus hyphae by gene-modified granulocytes as revealed by mitochondrial MTT reduction and transmission electron microscopy. (PDF 188 kb)

Supplementary Fig. 7

Immortalized bone marrow cells (SF-1 cells) containing a Setbp1 integration can engraft and induce myeloid leukemia with minimal to mild maturation. (PDF 255 kb)

Supplementary Table 1

Proviral integration site sequences detected by LAM-PCR. (PDF 122 kb)

Supplementary Table 2

Detection of vector integrants in the CIS genes MDS1/EVI1, PRDM16 and SETBP1. (PDF 25 kb)

Supplementary Table 3

Primers used for specific tracking of individual CIS clones and generation of clone specific internal standard. (PDF 20 kb)

Supplementary Methods (PDF 46 kb)

Supplementary Note (PDF 44 kb)

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Ott, M., Schmidt, M., Schwarzwaelder, K. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12, 401–409 (2006).

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