http://repub.eur.nl/res/aut/13830/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/26260/ 2011-09-01T00:00:00Z Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/-mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR VΒ gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B-as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/-mice using lentiviral SIN vectors. http://repub.eur.nl/res/pub/34512/ 2011-04-12T00:00:00Z To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements. http://repub.eur.nl/res/pub/20311/ 2010-09-01T00:00:00Z X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.Leukemia advance online publication, 24 June 2010; doi:10.1038/leu.2010.140. http://repub.eur.nl/res/pub/15560/ 2009-01-01T00:00:00Z Hematopoietic stem cells (HSC) are rare with estimated frequencies of 1 in 10,000 bone marrow cells and 1 in every 100,000 blood cells. The most important characteristic of HSC is their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation. Therefore they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene therapy. In adult mice and men, the main stem cell source is the bone marrow. For clinical applications HSC derived from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (PB) have been demonstrated to have several advantages compared to bone marrow; therefore, they are slowly replacing BM as alternative source of stem cells. The mouse is the model organism of choice for immunological and hematological research; therefore, studies of murine HSC are an important research topic. Here we described the most often used protocols and methods to isolate human and mouse HSC to high purity. http://repub.eur.nl/res/pub/14548/ 2008-10-23T00:00:00Z In clinical gene therapy trials for X-linked severe combined immunodeficiency, the development of leukemia has come up as a severe adverse effect. In all five cases, T-cell acute lymphoblastic leukemia (T-ALL) occurred as a direct consequence of insertional mutagenesis by the retrovirus used to deliver the therapeutic gene. Here, we review the mechanisms of insertional mutagenesis, the fuction of the Il2RG gene and the future developments in the field. New lentiviral and γ retroviral vectors can significantly improve the safety profile of the tools used but still carry the risk of insertional mutagenesis, as shown in this issue of Leukemia. Finally, the unfortunate side effects of gene therapy have given more insight into the development of human T-ALL. http://repub.eur.nl/res/pub/32328/ 2008-09-02T00:00:00Z X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design. http://repub.eur.nl/res/pub/10651/ 2007-11-21T00:00:00Z Severe combined immunodeficiency (SCID) is a rare class of primary, inherited, immunodeficiency causing infants to suffer from persistent diarrhea, opportunistic infections and a failure to thrive. RAG proteins play a crucial role in the initiation of V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) gene segments. Inactivating mutations in RAG1 or RAG2 lead to a developmental block at early checkpoints in B- and T-cell development. SCID can be cured by hematopoietic stem cell transplantation, but an HLA-matched donor is not always available. For two types of SCID, ADA- SCID and X-linked SCID, gammaretrovirus-based gene therapy has proven a successful treatment. For my thesis, several aspects of T-cell development and gene therapy were studied. A feature all forms of SCID have in common, is the occurrence of a block in T-cell development. Therefore, one of the studies in my thesis aimed at gaining more detailed knowledge of normal human T-cell development. Subsets corresponding to consecutive stages of human T- cell development were isolated and their gene expression profile was correlated to the T-cell receptor (TCR) gene rearrangement status. This analysis resulted in the identification of candidate factors involved in regulation of TCR gene rearrangements. In one of the X-linked SCID gene therapy trials using hematopoietic stem cells, lymphoproliferations were observed in four patients. In two cases, insertion of the therapeutic vector was found near the T- ALL oncogene LMO2. We found that gammaretrovirus-driven overexpression LMO2, but not the therapeutic IL2RG gene resulted in a block in human T-cell development. HIV-based vectors have been shown to have a more favorable integration pattern then gammaretroviruses, and self-inactivating (SIN) versions of this transfer system have been developed. We tested the feasibility of RAG gene therapy using HIV-based SIN vectors. We evaluated RAG1 and RAG2 gene transfer into hematopoietic stem cells using murine models for RAG-SCID. Correction of both T- and B-cell development was established when using a RAG2 transfer vector in Rag2-/- mice, showing proof-of-principle for the use of lentiviral SIN vectors for RAG gene transfer. 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/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. http://repub.eur.nl/res/pub/36332/ 2007-02-01T00:00:00Z Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34+CD38-human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34+CD38-CD7+cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34+CD38-CD7+cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34+CD38-CD7+and CD34+CD38-CD7-cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7+population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before. http://repub.eur.nl/res/pub/8406/ 2005-01-01T00:00:00Z To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.