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Query: UNIPROT:P06889 (Mol)
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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 IL2Rgamma 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.
Mol Ther 2007 Nov
PMID:New insights and unresolved issues regarding insertional mutagenesis in X-linked SCID gene therapy. 1772 55

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.
Mol Ther 2008 Mar
PMID:Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency. 1818 Jul 72

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.
Mol Ther 2008 Mar
PMID:Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells. 1819 19

Gene therapy trials have been performed with virus-based vectors that have the ability to integrate permanently into genomic DNA and thus allow prolonged expression of corrective genes after transduction of hematopoietic stem and progenitor cells. Adverse events observed during the X-linked severe combined immunodeficiency gene therapy trial revealed a significant risk of genotoxicity related to retrovirus vector integration and activation of adjacent proto-oncogenes, with several cases of T-cell leukemia linked to vector activation of the LMO2 gene. In patients with chronic granulomatous disease (CGD), rhesus macaques, and mice receiving hematopoietic stem and progenitor cells transduced with retrovirus vectors, a highly non-random pattern of vector integration has been reported. The most striking finding has been overrepresentation of integrations in one specific genomic locus, a complex containing the MDS1 and the EVI1 genes. Most evidence suggests that this overrepresentation is primarily due to a modification of primitive myeloid cell behavior by overexpression of EVI1 or MDS1-EVI1, as opposed to a specific predilection for integration at this site. Three different proteins can be produced from this complex locus: MDS1, MDS1-EVI1, and EVI1. This review will summarize current knowledge regarding this locus and its gene products, with specific focus on issues with relevance to gene therapy, leukemogenesis, and hematopoiesis. Insights into the mechanisms that result in altered hematopoiesis and leukemogenesis when this locus is dysregulated could improve the safety of gene therapy in the future.
Mol Ther 2008 Mar
PMID:The MDS1-EVI1 gene complex as a retrovirus integration site: impact on behavior of hematopoietic cells and implications for gene therapy. 1822 42

The recent development of leukemia in gene therapy patients with X-linked severe combined immunodeficiency disease because of retroviral vector insertional mutagenesis has prompted reassessment of the genotoxic potential of integrating vector systems. In this chapter, various strategies are described to reduce the associated risks of retroviral genomic integration. These include deletion of strong transcriptional enhancer-promoter elements in the retroviral long terminal repeats, flanking the retroviral transcriptional unit with enhancer blocking sequences and designing vectors with improved RNA 3' end processing. Protocols are provided to evaluate the relative biosafety of the modified vectors based on their ability to immortalize hematopoietic progenitor cells and propensity to trigger clonal hematopoiesis or leukemogenesis following hematopoietic stem cell transplantation.
Methods Mol Biol 2008
PMID:Reducing the genotoxic potential of retroviral vectors. 1847 Jun 46

Successful retroviral gene transfer into hematopoietic cells has been demonstrated in a number of small and large animal models and clinical trials. However, severe adverse events related to insertional muta-genesis in a recent clinical trial for X-linked severe combined immunodeficiency reinforced the need to develop novel retroviral vectors with improved biosafety. Improvements include the use of self-inactivating (SIN) vectors as well as improvements in vector design. This chapter describes the basic design of gamma-retroviral and lentiviral SIN vectors that utilize a split-packaging system and includes a description of the various cloning modules frequently used in the design of such vectors that impact biosafety, titer, and transgene expression. In addition, this chapter describes the methods used for high titer vector production using calcium phosphate transfection both at research scale and at large scale for clinical application using a closed system bioreactor.
Methods Mol Biol 2009
PMID:Design and production of retro- and lentiviral vectors for gene expression in hematopoietic cells. 1911 Jun 28

Large animal models have played a crucial role in the development of gene therapy protocols. A significant advantage of large animal models over rodent models includes the ability to more easily translate protocols developed in large animals to humans. For gene therapy applications, nonhuman primates and canines have been the main large animal models. Canines have the advantage that there are disease models available, e.g., hemolytic anemia (pyruvate kinase deficiency), leukocyte adhesion deficiency, severe combined immunodeficiency (XSCID), storage diseases, and others. In addition, all three major integrating virus systems, i.e., gammaretrovirus-, HIV-derived lenti- and foamy virus vectors are able to efficiently transduce canine hematopoietic cells. Here we describe protocols developed for efficient transduction of canine hematopoietic repopulating cells.
Methods Mol Biol 2009
PMID:Canine models of gene-modified hematopoiesis. 1911 Jun 37

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).
Mol Ther 2009 Jun
PMID:Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients. 1964 96

The emergence of leukemia following gene transfer to restore common cytokine receptor gamma chain (gammaC) function in X-linked severe combined immunodeficiency (SCID-X1) has raised important questions with respect to gene therapy safety. To explore the risk factors involved, we tested the oncogenic potential of human gammaC in new strains of transgenic mice expressing the gene under the control of the CD2 promoter and locus control region (LCR). These mice demonstrated mildly perturbed T-cell development, with an increased proportion of thymic CD8 cells, but showed no predisposition to tumor development even on highly tumor prone backgrounds or after gamma-retrovirus infection. The human CD2-gammaC transgene rescued T and B-cell development in gammaC(-/-) mice but with an age-related delay, mimicking postnatal reconstitution in SCID-X1 gene therapy subjects. However, we noted that gammaC(-/-) mice are acutely susceptible to murine leukemia virus (MLV) leukemogenesis, and that this trait was not corrected by the gammaC transgene. We conclude that the SCID-X1 phenotype can be corrected safely by stable ectopic expression of gammaC and that the transgene is not significantly oncogenic when expressed in this context. However, an underlying predisposition conferred by the SCID-X1 background appears to collaborate with insertional mutagenesis to increase the risk of tumor development.
Mol Ther 2009 Jun
PMID:A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but no evidence of gammaC gene oncogenicity. 1933 36

Insertional mutagenesis leading to insurgence of leukemia has been shown as a consequence of retroviral (RV)-mediated gene transfer in animal models and in clinical trials of gene therapy for X-linked severe combined immunodeficiency. Aberrant expression of oncogenes neighboring the gamma-RV vector insertion site via induction by the enhancer element of the viral long terminal repeats (LTRs) is thought to have played a role in leukemogenesis. Consequently, RV vectors devoid of LTR enhancer elements could prove as safer tools for gene transfer. To test this hypothesis, we evaluated the immortalization ability of two RV vectors: one carrying the full-length Moloney leukemia virus (MLV) LTR and one with the same LTR in which the enhancer element was deleted [MLV self-inactivating (SIN)]. Unexpectedly, transduction with MLV SIN resulted in an only slightly and not significant decreased immortalization frequency of primary bone marrow (BM) cultures (about 37%) compared to transduction with MLV (about 48%). Similar to MLV, immortalization by MLV SIN is likely caused by insertional activation of oncogenes including Evi1, Mds1, Mef2c, and Hoxa7. Our results indicate that the MLV SIN, devoid of the LTR enhancer element, was still able to immortalize BM cells by activating nearby gene expression, indicating the need of an accurate selection of the internal promoter to obtain safer SIN RV vectors.
Mol Ther 2009 Nov
PMID:Self-inactivating retroviral vector-mediated gene transfer induces oncogene activation and immortalization of primary murine bone marrow cells. 1963 58


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