http://repub.eur.nl/res/aut/55712/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/39808/ 2013-04-10T00:00:00Z http://repub.eur.nl/res/pub/39956/ 2013-04-09T00:00:00Z Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation. http://repub.eur.nl/res/pub/37463/ 2012-11-01T00:00:00Z Purpose: 5-Androstene-3β,17β-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. Methods and Materials: BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. Results: BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unit (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. Conclusions: 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO. http://repub.eur.nl/res/pub/37203/ 2012-01-01T00:00:00Z Deficient thymopoiesis and retarded recovery of naive CD4+T cells are important determinants of insufficient immune-competence following hematopoietic stem cell transplantation (HSCT). Although keratinocyte growth factor (KGF) may protect the thymic epithelium, stem cell factor (SCF) is involved in early thymopoiesis. We evaluated whether KGF alone or combined with SCF would affect thymopoiesis and hematologic recovery following myeloablative autologous HSCT into rhesus macaques. Purpose-bred adult rhesus macaques received 106autologous CD34+-selected mononuclear bone marrow cells (BMC)/kg after 9 Gy myeloablative conditioning. Animals were treated with phosphate-buffered saline (PBS) (n = 2), KGF alone (n = 2), or KGF combined with SCF (n = 2). KGF-treated animals showed accelerated hematologic recovery, improved thymopoiesis, and enhanced naive T-cell recovery following transplantation. Improved Tcell recovery was not associated with protection against cytomegalovirus reactivation nor with improved antibody response to tetanus toxoid vaccination. Animals treated with KGF and SCF experienced severe adverse events that precluded evaluation of thymopoiesis and T cell recovery. Collectively, our data confirm that KGF may enhance thymopoiesis. http://repub.eur.nl/res/pub/36968/ 2007-11-01T00:00:00Z The oncogenic potential of retrovirus-mediated gene therapy has been re-emphasized because four patients developed T-cell acute lymphoblastic leukemia (T-ALL)-like disease from an otherwise successful gene therapy trial for X-linked severe combined immunodeficiency (X-linked SCID). X-linked SCID, a disease caused by inactivating mutations in the IL2Rγ gene, is part of a heterogeneous group of SCIDs characterized by the lack of T cells in conjunction with the absence of B and/or natural killer (NK) cells. Gene therapy approaches are being developed for this group of diseases. In this review we discuss the various forms of SCID in relation to normal T-cell development. In addition, we consider the possible role of LMO2 and other T-ALL oncogenes in the development of adverse effects as seen in the X-linked SCID gene therapy trial. Furthermore, we debate whether the integration near the LMO2 locus is sufficient to result in T-ALL-like proliferations or whether the gamma-retroviral viral expression of the therapeutic IL2RG gene contributes to leukemogenesis. Finally, we review some newly developed murine models that may have added value for gene therapy safety studies. http://repub.eur.nl/res/pub/36578/ 2007-10-01T00:00:00Z Viruses are the most commonly used vectors for clinical gene therapy. The risk of dissemination of a viral vector into the environment via excreta from the treated patient, a phenomenon called shedding, is a major safety concern for the environment. Despite the significant number of clinical gene therapy trials that have been conducted worldwide, there is currently no overview of actual shedding data available. In this article, an inventory of shedding data obtained from a total of 100 publications on clinical gene therapy trials using retroviral, adenoviral, adeno-associated viral and pox viral vectors is presented. In addition, the experimental set-up for shedding analysis including the assays used and biological materials tested is summarized. The collected data based on the analysis of 1619 patients in total demonstrate that shedding of these vectors occurs in practice, mainly determined by the type of vector and the route of vector administration. Due to the use of non-quantitative assays, the lack of information on assay sensitivity in most publications, and the fact that assay sensitivity is expressed in various ways, general conclusions cannot be made as to the level of vector shedding. The evaluation of the potential impact and consequences of the observations is complicated by the high degree of variety in the experimental design of shedding analysis between trials. This inventory can be supportive to clinical gene therapy investigators for the establishment of an evidence-based risk assessment to be included in a clinical protocol application, as well as to national regulatory authorities for the ongoing development of regulatory guidelines regarding gene therapy. Copyright http://repub.eur.nl/res/pub/35269/ 2007-08-01T00:00:00Z We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+progenitors during transduction. In contrast to those in CD34+cells, RISs recovered from engrafted CD3+T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation. http://repub.eur.nl/res/pub/35291/ 2007-08-01T00:00:00Z Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34+progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1. http://repub.eur.nl/res/pub/36301/ 2007-04-01T00:00:00Z The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rγ), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rγ had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rγ may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth. http://repub.eur.nl/res/pub/35566/ 2007-03-01T00:00:00Z Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major "stemness" pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.