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The Wiskott–Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients’ clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.)
The Wiskott–Aldrich syndrome (WAS) is a complex primary immunodeficiency disorder that is characterized by recurrent infections, thrombocytopenia, eczema, and autoimmunity1,2 and caused by mutations in WAS.3 Its gene product, WAS protein (WASP), is a key regulator of actin polymerization in hematopoietic cells, with domains involved in signaling, cell locomotion, and immunologic-synapse formation.4 The complex biologic features of this disease result from multiple dysfunctions in different subgroups of leukocytes, including defective function of T and B cells, disturbed formation of the NK-cell immunologic synapse, and impaired migratory responses in all leukocyte subgroups.4,5 Severe WAS leads to an early death from infection or bleeding.6 Currently, the only curative therapy consists of allogeneic HSC transplantation, which may be associated with considerable risk of death or complications related to transplantation.7
HSC gene therapy has emerged as an innovative therapeutic strategy for various primary immunodeficiency disorders.8–12 WAS is a promising candidate disease for gene-therapy approaches, since WASP expression is restricted to cells of the hematopoietic system, which grants a proliferative advantage over WASP-negative cells.13,14 Preclinical studies have shown efficient reconstitution of cellular function in vitro and in vivo.13 On the basis of our own preclinical in vitro and in vivo studies,15,16 we developed a clinical gene-transfer protocol.13 Here we report the results in two young boys who were treated with HSC gene therapy.
The clinical gene-therapy protocol was reviewed by local and federal authorities, in accordance with current regulations of the European Community. Inclusion criteria for the study included an age of at least 12 months, a diagnosis of severe WAS (as documented by molecular and clinical phenotype), no indication of malignant disease before gene therapy, documentation of informed consent or informed assent, and isolation of a sufficient number of vital CD34+ cells from bone marrow or leukapheresis. Two 3-year-old boys who had been referred for gene therapy ful-filled the inclusion criteria, and their parents provided written informed consent.
The trial was conducted in compliance with the Good Clinical Practice guidelines of the European Clinical Trials Directive and the provisions of the Declaration of Helsinki. A detailed description of the technical methods that were used in the study is provided in the Supplementary Appendix, available with the full text of this article at NEJM.org.
Patient 1 presented with hematoma and petechiae after birth, and within the first year of life, hemorrhagic colitis, eczema, and therapy-refractory autoimmune hemolytic anemia developed. In addition, he had autoimmune neutropenia and recurrent infections (bronchitis, otitis media, and enterocolitis). Molecular analysis revealed a splice site mutation in WAS, IVS6+1. No residual protein or somatic chimerism was detectable (Fig. 1 in the Supplementary Appendix).
Patient 2 presented with thrombocytopenia and hemorrhagic diathesis soon after birth. He had recurrent episodes of upper respiratory tract infection, life-threatening salmonella septicemia, and eczema that was not responsive to local and systemic therapy. Molecular studies revealed a known mutation in WAS (c. G257A, p. Arg86His).3,6 No residual protein or somatic chimerism was detectable (Fig. 1 in the Supplementary Appendix).
We collected autologous CD34+ HSCs by leukapheresis. The cells were then transduced with WASP-expressing retroviral vectors that were created with backbone vector CMMP and pseudotyped with gibbon ape leukemia virus (GALV) envelope protein; the cells were then reinfused 4 days later (Table 1 in the Supplementary Appendix). Before reinfusion of CD34+ cells, busulfan was administered at a dose of 4 mg per kilogram of body weight per day on days 3 and 2 before the procedure. Transient myelosuppression and partial alopecia were observed as therapy-associated side effects. For details regarding the vector that was used and the clinical gene-therapy protocol, see the Supplementary Appendix.
After gene therapy, WASP-positive cells in various leukocyte subgroups were detected on flow cytometry (Fig. 1A and 1B). The frequency of WASP-positive monocytes, initially detected soon after gene therapy, has remained stable at 7 to 28%. An increased percentage of lymphoid cells were WASP-positive, consistent with a known proliferative advantage conferred by WASP expression in that lineage.14 The level of WASP-positive NK cells ranged from 25 to 90% over time. An increase in the percentage of WASP-positive CD4+ and CD8+ T cells was seen 6 to 12 months after gene therapy and has remained stable ever since. Restoration of WASP expression in peripheral-blood mononuclear cells was confirmed on Western blot analyses (Fig. 1C and 1D).
At the level of hematopoietic progenitor cells, a stable chimerism was found in the bone marrow of both patients, with 9% of CD34+ cells expressing the WASP transgene in Patient 1 and 20% in Patient 2 (Fig. 2 in the Supplementary Appendix). The presence of vector-positive cells in all leukocyte subgroups at different time points after gene therapy was confirmed on quantitative polymerase-chain-reaction (PCR) assay (Fig. 3 in the Supplementary Appendix). PCR analysis of colony-forming units from both patients confirmed vector integrations in 3 of 101 colonies in Patient 1 and in 23 of 103 colonies in Patient 2 (data not shown).
An increase in platelet counts was noted, starting 6 to 9 months after gene therapy, which marked the end of episodes of clinical bleeding (Fig. 1E and 1F). On the most recent follow-up, 2.5 years after gene therapy, platelet counts were 256,000 for Patient 1 and 88,000 for Patient 2. WASP expression in platelet-rich plasma from both patients was confirmed on Western blot and flow cytometric analysis (Fig. 1G and 1H, and Fig. 4 in the Supplementary Appendix).
We next analyzed whether restored WASP expression would lead to improved leukocyte function. In NK cells, WASP deficiency leads to a decreased accumulation of F-actin and perforin at the NK-cell immunologic synapse,17 which reduces NK-cell–mediated cytotoxicity. After gene therapy, substantial proportions of the NK-cell immunologic synapses were restored in both patients (Fig. 2A, and Fig. 5 and 6 in the Supplementary Appendix), and NK-cell lytic activity was reconstituted (Fig. 2B). WASP-deficient monocytes and dendritic cells are characterized by the inability to form podosomes.4 The fraction of podosome-positive monocytes after gene transfer ranged from 8 to 34% (Fig. 2C, and Fig. 7 in the Supplementary Appendix), indicating that restored WASP expression reconstituted the capacity to rearrange the cytoskeleton in myeloid cells.
T-cell abnormalities in patients with WAS include diminished T-cell proliferative responses and a skewed antigen-recognition repertoire for T-cell receptors.4 After gene therapy, T-cell proliferative responses were reconstituted at multiple time points in both patients (Fig. 2D, and Fig. 8 in the Supplementary Appendix). Vβ gene spectratyping of T-cell receptors showed increasing polyclonality of TCR usage in both patients after gene therapy, as shown by a distribution pattern that was consistent with decreasing oligoclonal T-cell receptor Vβ rearrangements (Fig. 2E, and Fig. 9 in the Supplementary Appendix).
The percentage of WASP-expressing B cells increased over time after gene therapy, with proportions of WASP+CD19+ cells of 57% in Patient 1 and 69% in Patient 2 2.5 years after gene therapy. Abnormal immunoglobulin levels (increased IgM in Patient 1 and decreased IgG in Patient 2) normalized after gene therapy. Although normal immunoglobulin levels have remained stable in Patient 2, IgG levels have not remained at a protective level in Patient 1, who had undergone earlier splenectomy (Table 2 in the Supplementary Appendix). Both patients were immunized with hexavalent vaccine 12 months after gene therapy and successfully produced specific antibodies to tetanus, diphtheria, and Haemophilus influenzae (Table 3 in the Supplementary Appendix).
After gene therapy, the frequency and severity of infections decreased in both patients. However, 2 years after gene therapy, Patient 1 had an episode of pneumococcal meningitis, which was responsive to intravenous antibiotic treatment. Contributing factors may have been his previous splenectomy or insufficient B-cell reconstitution.
Signs and symptoms of autoimmunity disappeared in both patients within the first year after gene therapy. In Patient 1, severe autoimmune hemolytic anemia (with polyspecific anti-Jka antibodies), autoimmune thrombocytopenia (with antibodies directed against membrane glycoproteins IIb/IIIa, Ib/IX, and V), and autoimmune neutropenia (with antibodies against HNA-1a) disappeared (Table 4 in the Supplementary Appendix), and severe eczema resolved in Patient 2 (Fig. 2F). Since recent studies have highlighted the role of regulatory T cells (Tregs) in controlling autoimmunity in WASP deficiency,18 we measured the relative proportion of WASP-expressing regulatory T cells. The percentage of WASP-positive cells in CD4+CD25+FOXP3+ cells was higher than in the nonregulatory T-cell population (CD4+CD25−FOXP3−) in both patients (Fig. 11 in the Supplementary Appendix).
No morphologic or cytogenetic aberrations were seen in bone marrow aspirates after gene therapy. The clonal distribution and fate of gene-corrected cells in vivo were monitored by large-scale analyses of retroviral vector insertion sites with linear-amplification– mediated PCR (LAM-PCR),19 followed by 454 pyrosequencing. In both patients, a highly polyclonal reconstitution pattern was seen in bone marrow cells, primary blood leukocytes, lymph-node cells (analyzed in Patient 1), sorted lymphoid CD3+ T cells, and myeloid granulocytes (Fig. 3A and 3B). Of approximately 400,000 raw LAM-PCR amplicon sequence reads, 5709 unique retroviral insertion sites (in Patient 1) and 9538 sites (in Patient 2) were retrieved from bone marrow, lymph nodes, and primary blood leukocytes and could be mapped to a single position in the human genome. Vector integration occurred preferentially in the vicinity of transcription start sites and into gene coding regions of the human genome, reflecting a typical gammaretroviral target-site distribution (Fig. 12 in the Supplementary Appendix).
The abundance of individual gene-corrected cells and their clonal progeny in the hematopoietic system was estimated by the retrieval frequency of individual retroviral insertion site sequences generated by deep sequencing (Fig. 3C and 3D). In both patients, several clones appeared repeatedly within the 10 most abundant clones in each analyzed time point, indicating that clonal skewing had emerged among active hematopoietic gene-corrected clones. Ingenuity pathway analyses in both patients revealed a substantial enrichment of genes that are involved in biologic processes regulating cellular development, growth, and proliferation, as well as hematopoiesis. More specifically, a substantial enrichment of vector insertions in or near genes regulating immune and autoimmune responses was observed in both patients (Fig. 13 in the Supplementary Appendix).
We further monitored the genomic distribution of insertion sites that were clustered in subgenomic regions and associated with particular reference sequence (RefSeq) genes over time as an indicator of in vivo selection of gene-corrected cells. In both patients, the same multiple vector targeted genes that were previously found to expand clones in gene-corrected myelopoiesis (MDS1/EVI1, PRDM16, and SETBP1) and lymphopoiesis (LMO2, CCND2, and BMI1) were noted12,20–23 (Fig. 3E and 3F). The most active cell clone in Patient 1 showed a vector integrant upstream of CCND2, and in Patient 2, the site of insertion was MDS1/EVI1 (Fig. 4A and 4B, and Table 5 in the Supplementary Appendix).
Insertion-site analyses in sorted CD3+ lymphocytes and myeloid granulocytes revealed that the clonal contribution of cells with insertions in coincident insertion sites that have been found to trigger clonal expansion in gene-corrected lymphopoiesis and myelopoiesis was almost exclusively related to either lymphoid cells or myeloid cells (Fig. 4C and 4D). In contrast, retroviral insertion sites that were identical between sorted lymphoid or myeloid cell fractions were also retrieved from both patients, at least 9000 times as frequently as expected by statistical chance alone. This marking indicates the initial transduction of cells with multilineage capacity (Table 6 in the Supplementary Appendix). Prospective monitoring for signs of clonal dominance in gene-corrected hematopoiesis will continue in both patients.
Previous clinical studies of hematopoietic gene therapy have reported sustained or temporary correction of lymphocytes in patients with severe combined immunodeficiency or adrenoleukodystrophy8,9,24 and of myeloid cells in patients with chronic granulomatous disease or adrenoleukodystrophy. 12,24 Our study shows that gene therapy for the Wiskott–Aldrich syndrome is feasible and has not been associated with treatment-limiting adverse events up to 3 years after gene therapy. These findings indicate the successful correction of early hematopoietic progenitor or stem cells that contributed quantitatively to all cell lineages of blood. We also confirmed correction of platelets on transplantation of retrovirus-transduced HSCs in humans.
In line with a proliferative advantage of WASP-positive cells,14 our results show increasing proportions of corrected lymphocytes over time. Since the reconstitution of the lymphoid system reaches a plateau, there continues to be homeostatic control. Engraftment of progenitor cells was stable, similar to the results achieved in a recent trial of lentivirus-based gene therapy for adrenoleukodystrophy, 24 in which a completely myeloablative conditioning regimen was used.
The pathophysiology of thrombocytopenia in WAS is complex and involves decreased production of platelets and increased immune and nonimmune mechanisms of platelet destruction.25 This study provides evidence that gene transfer can correct thrombocytopenia in humans, a major cause of death and complications in patients with WAS.
The assessment of the clonal inventory in patients whose disease has been successfully corrected uncovers vector influence on key regulators of hematopoiesis. In addition, we observed preferential targeting of genes implicated in autoimmunity control, suggesting an unexpected, disease-related gene targeting. Most interestingly, insertion-site recovery in sorted lymphoid and myeloid fractions provides novel molecular evidence that the molecular signatures contributing to long-term production of hematologic cells are distinct in both cell lineages of WAS patients. These data suggest that distinctive mechanisms control selective proliferative advantage or differentiation of myeloid and lymphoid cells.
Despite targeting potential oncogenes, no persistent clonal imbalance has yet been observed. Deep sequencing revealed a fluctuating pattern of molecularly defined hematopoietic clones and evidence of initial transduction of HSCs with multilineage capacity, suggesting that hematopoiesis continues to follow a cyclic pattern. Clinically, both patients had marked improvement in their susceptibility to infection, autoimmunity, and bleeding. Longer follow-up and in-depth analyses in additional patients are warranted in light of the considerable clinical benefit derived from genetic correction of this disease. Hematopoietic stem-cell gene transfer may achieve complete immunologic correction without producing the toxic effects implicit in the concept of retrovirus-based stem-cell gene transfer.
Supported by grants from the Deutsche Forschungsgemeinschaft (DFG KliFo 110-2 and SPP1230) and the Bundesministerium für Bildung und Forschung (BMBF TreatID, BMBF PID-Net, and BMBF iGENE), the Sixth and Seventh Framework European Economic Community Program (CONSERT, PERSIST, and CLINIGENE), a grant (AI-067946) from the National Institutes of Health, TÁMOP (4.2.2-08/1-2008-0015), and a fellowship from the Else Kröner-Fresenius Stiftung (to Dr. Boztug).
We thank the patients and their families for their participation in this study and their openness to share the scientific data resulting from this clinical trial; all the physicians and nurses involved in this study; Paul Fisch of the University of Freiburg for help with TCR Vβ spectratyping; Roland Jacobs of Hannover Medical School for help with cytotoxicity assays; Anna Paruzynski and Christina Lulay of the National Center for Tumor Diseases (NCT) and the German Cancer Research Center (DKFZ), Heidelberg, for molecular analyses and data mining; Anne Arens of the NCT and DKFZ, Heidelberg, for bioinformatical analyses; Dr. Hans Ochs of the University of Washington, Seattle, for providing rabbit anti–WASP antibody; Ulrike Köhl of the University Hospital Frankfurt am Main for help with CD34+ cell purification; Jana Diestelhorst of Hannover Medical School for expert technical help; the Hannover Clinical Trial Center for monitoring the clinical trial; and the Care-for-Rare Foundation for its support.
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.