Assessing the Risk of T-cell Malignancies in Mouse Models of SCID-X1
Authors: Brian Sorrentino
Journal: Molecular Therapy (2010)

It has long been recognized that severe combined immunodeficiency disorders (SCIDs) are favorable settings for the application of stem cell gene therapy. This genetically heterogeneous group of disorders includes adenosine deaminase (ADA) deficiency and X-linked SCID (SCID-X1),1Buckley RH Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution.Annu Rev Immunol. 2004; 22: 625-655Crossref PubMed Scopus (433) Google Scholar the latter caused by mutations in the common gamma chain (γc) gene, which is required for a broad range of cell signaling events in lymphocytes and their precursors. The strong selective advantage for genetically corrected cells during development circumvents the requirement for high levels of transduction and engraftment, which remains a significant barrier for other blood diseases that lack an in vivo selection mechanism for transduced cells. Pioneering gene therapy trials for SCID-X1 have taken place in France,2Hacein-Bey-Abina S Le Deist F Carlier F Bouneaud C Hue C De Villartay JP et al.Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.N Engl J Med. 2002; 346: 1185-1193Crossref PubMed Scopus (996) Google Scholar,3Cavazzana-Calvo M Hacein-Bey S de Saint Basile G Gross F Yvon E Nusbaum P et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease [see comments].Science. 2000; 288: 669-672Crossref PubMed Scopus (2256) Google Scholar the United Kingdom,4Gaspar HB Parsley KL Howe S King D Gilmour KC Sinclair J et al.Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.Lancet. 2004; 364: 2181-2187Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar and—for ADA deficiency—Italy5Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (826) Google Scholar; these have clearly demonstrated the curative potential of this approach. Retroviral vectors derived from the murine leukemia virus (MLV) were used to transfer a normal copy of the diseased gene to autologous CD34+ cells from the infant patient's bone marrow. After reinfusion of these modified cells, most of the more than 30 treated patients have had full reconstitution of T-cell–mediated immunity, with restoration of B-cell function in fewer, but significant numbers of, cases. However, in both SCID-X1 trials, some patients developed T-cell malignancies that were clearly related to insertional mutagenesis from the integrated vector.6Hacein-Bey-Abina S Von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (3034) Google Scholar,7Hacein-Bey-Abina S von Kalle C Schmidt M Le Deist F Wulffraat N McIntyre E et al.A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2003; 348: 255-256Crossref PubMed Scopus (1626) Google Scholar,8Howe SJ Mansour MR Schwarzwaelder K Bartholomae C Hubank M Kempski H et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J Clin Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (969) Google Scholar Molecular analyses of these five cases have shown that the tumors were caused by activation of known proto-oncogenes due to the action of the MLV enhancer element on those adjacent cellular promoters. Strikingly, four of the five cases showed activation of the LMO2 gene, a known T-cell oncogene involved in a number of T-cell acute lymphoblastic leukemia (T-ALL) chromosomal translocations.9McCormack MP Rabbitts TH Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2004; 350: 913-922Crossref PubMed Scopus (243) Google Scholar This unexpected adverse event has been a major limitation to this approach and has led to a new field within gene therapy focusing on the mechanisms of genotoxicity associated with integrating vectors.10Nienhuis AW Dunbar CE Sorrentino BP Genotoxicity of retroviral integration in hematopoietic cells.Mol Ther. 2006; 13: 1031-1049Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar Much has been learned from these studies, yet important questions remain. One question is why the leukemia cases have been seen only in the SCID-X1 trials and not in the trials involving ADA deficiency. Intriguingly, insertion-site analyses in ADA-deficient patients treated with MLV-derived retroviral vectors have shown minor clones with insertions into the LMO2 locus, but no cases of T-cell malignancies or other hematologic abnormalities have been noted.11Aiuti A Cassani B Andolfi G Mirolo M Biasco L Recchia A et al.Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.J Clin Invest. 2007; 117: 2233-2240Crossref PubMed Scopus (224) Google Scholar This and other observations raise the question of whether the disease background influences the vector insertion patterns and risk of oncogenesis. Two patients in a German trial for chronic granulomatous disease (CGD) developed a severe form of early leukemia, called myelodysplasia, due to insertions into the MDS1/EVI1 gene,12Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (647) Google Scholar,13Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (988) Google Scholar,14Modlich U Schambach A Brugman MH Wicke DC Knoess S Li Z et al.Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16.Leukemia. 2008; 22: 1519-1528Crossref PubMed Scopus (86) Google Scholar a locus known to be preferentially targeted by MLV vectors in other contexts unrelated to SCID gene therapy.15Calmels B Ferguson C Laukkanen MO Adler R Faulhaber M Kim HJ et al.Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells.Blood. 2005; 106: 2530-2533Crossref PubMed Scopus (136) Google Scholar,16Metais JY Dunbar CE The MDS1-EVI1 gene complex as a retrovirus integration site: impact on behavior of hematopoietic cells and implications for gene therapy.Mol Ther. 2008; 16: 439-449Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar These observation suggest that the genetic profile for vector toxicity may differ with the underlying disease. There are many potential reasons for these differences; however, we still do not understand this phenomenon in great detail. In this context, the new report from Ginn et al. in this issue of Molecular Therapy is of particular interest.17Ginn SL Liao SHY Dane AP Hu M Hyman J Finnie JW et al.Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and γc overexpression.Mol Ther. 2010; 18: 965-976Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar These investigators used a SCID-X1 mouse model to show that the SCID-X1 background itself is an important risk factor for T-cell malignancies, independent of vector-related perturbations of proto-oncogene expression. These results suggest that the increased susceptibility of SCID-X1 to transformational events will be an important consideration in future gene therapy efforts; they also underscore the need for safer approaches that retain the proven efficacy of such treatment but eliminate or reduce this serious complication. This report describes a series of self-inactivating lentiviral vectors that were tested in a mouse model of SCID-X1 gene therapy. Various human cellular promoters were tested, including the now widely utilized elongation factor (EF)-1α promoter, along with a phosphoglycerate kinase (PGK) promoter and the promoter from the WASP gene. The EF-1α promoter was most effective and restored full immune function in transplanted mice, the PGK promoter gave intermediate results, and the WASP endogenous promoter had little activity with regard to reconstitution. These results are important in considering promoter choice not only for SCID-X1 but also potentially for other lymphodeficiency disorders that require expression of the therapeutic gene throughout T-, B-, and natural killer–cell compartments. The mice in these studies were followed for more than 1 year, and 4 of 14 mice treated with the EF-1α-hγc vector developed malignancy. No tumors were seen in a smaller cohort of mice transplanted with untransduced SCID-X1 cells or in mice transplanted with wild-type cells transduced with an EF-1α-GFP vector. At first impression, it seemed that the EF-1α-hγc vector was inducing oncogenesis specifically on the SCID-X1 background. This was a surprising result, because the EF-1α promoter contains little to no enhancer activity and was considered to be unlikely to activate cellular proto-oncogenes adjacent to the insertion site.18Zychlinski D Schambach A Modlich U Maetzig T Meyer J Grassman E et al.Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol Ther. 2008; 16: 718-725Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar Consistent with this prediction, further molecular analyses of the integrated provirus in all four malignant clones showed no evidence of vector-induced oncogene activation. In two cases, the integration sites were not located within 150 kb of any cellular genes. In the other two cases, microarray expression analyses showed no evidence of vector-induced gene expression within a 300-kb window of the insertion site. These results suggest that the requirement for the EF-1α-hγc vector for inducing T-cell malignancies was not related to insertional mutagenesis but instead reflects the requirement for restoration of T-cell development via gene replacement. The absence of insertional mutagenesis in this model stands in marked contrast to the clinical results from the SCID-X1 and CGD trials, which invariably show that vector insertions lead to transcriptional activation of cellular proto-oncogenes. Furthermore, the clinical trials have shown a remarkable preference for—and/or selection of—integration into a few particular proto-oncogenes, a feature not seen in this mouse model. In this sense, the mouse model does not recapitulate several key findings seen in the clinical trials, namely, preferred integration into the LMO2 locus and transcriptional activation due to the vector-encoded enhancer. This discrepancy has also been noted in other mouse model studies, in which no LMO2 insertions were seen in tumor cells.19Shou Y Ma Z Lu T Sorrentino BP Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy.Proc Natl Acad Sci USA. 2006; 103: 11730-11735Crossref PubMed Scopus (96) Google Scholar,20Scobie L Hector RD Grant L Bell M Nielsen AA Meikle S et al.A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but no evidence of gammaC gene oncogenicity.Mol Ther. 2009; 17: 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar These considerations argue that this mouse model will not be helpful in testing the relative safety of newer vectors designed to avoid the activation of proto-oncogenes. Other good alternatives now exist for vector safety testing that do not rely on mouse transplantation assays.21Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (278) Google Scholar,22Ryu BY Evans-Galea MV Gray JT Bodine DM Persons DA Nienhuis AW An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation.Blood. 2008; 111: 1866-1875Crossref PubMed Scopus (69) Google Scholar Another problem with mouse transplant assays is that they are generally associated with a high background of spontaneous tumors due to irradiation and other factors.23Will E Bailey J Schuesler T Modlich U Balcik B Burzynski B et al.Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials.Mol Ther. 2007; 15: 782-791Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Therefore an important feature of this study is that it shows how mouse models and human trials differ with regard to retroviral vector–related insertional mutagenesis and that it illustrates the limitations of using mouse models for making safety determinations. Newer retroviral vectors that are less prone to the activation of flanking cellular genes are just now becoming available. One important design feature is the use of self-inactivating vectors that are completely devoid of viral transcriptional elements, particularly enhancers (see the Editorial in this issue24Persons DA Lentiviral vector gene therapy: effective and safe? (2010).Mol Ther. 2010; 18: 861-862Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These vectors, instead, contain cellular promoters that allow for adequate expression of the therapeutic gene but lack the ability to activate proto-oncogenes dominantly. The EF-1α-hγc vector used in this study is an example of such a design. Another safety feature being employed in the use of genetic enhancer blocking elements is the cHS4 insulator fragment from the chicken β-globin locus,25Li CL Emery DW The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus.Gene Ther. 2008; 15: 49-53Crossref PubMed Scopus (36) Google Scholar,26Arumugam PI Scholes J Perelman N Xia P Yee JK Malik P Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element.Mol Ther. 2007; 15: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar which has been shown to attenuate the effects of viral enhancers on cellular oncogenes.22Ryu BY Evans-Galea MV Gray JT Bodine DM Persons DA Nienhuis AW An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation.Blood. 2008; 111: 1866-1875Crossref PubMed Scopus (69) Google Scholar Other recent studies suggest that a lentiviral vector may be less prone than MLV vectors to induce transformation, even when relative enhancer strength is controlled for.27Montini E Cesana D Schmidt M Sanvito F Bartholomae CC Ranzani M et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (467) Google Scholar It is still a challenge to define the best safety measures for evaluating these newer lentiviral vectors. At this time, there are no preclinical assays that can “guarantee” the safety of any retroviral vector for use in a clinical trial. Instead, investigators must use an appropriate battery of preclinical assays to make the case for a potentially increased safety profile and then design a definitive safety evaluation in an appropriately designed clinical trial where the potential risks are fully disclosed. This position is supported by reflecting on the unique toxicity data that have arisen from the SCID-X1, ADA deficiency, and CGD trials and the fact that none of these risks were prospectively identified in numerous preclinical studies. The potential mechanisms of the increased susceptibility in this mouse model are unknown. Further genetic analyses of the four tumors have shown that these clones had acquired activating mutations in Notch1. This feature has also been seen in the SCID-X1 T-cell malignancies8Howe SJ Mansour MR Schwarzwaelder K Bartholomae C Hubank M Kempski H et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J Clin Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (969) Google Scholar,28Deichmann A Hacein-Bey-Abina S Schmidt M Garrigue A Brugman MH Hu J et al.Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.J Clin Invest. 2007; 117: 2225-2232Crossref PubMed Scopus (220) Google Scholar and is thought to be a secondary acquired mutation that collaborates with vector-induced LMO2 activation. Other acquired mutations have been recurrently noted in human SCID-X1 transformation and have been assumed to occur during the 24- to 68-month latency period associated with these cases. For instance, many of the human SCID-X1 cases have displayed biallelic deletions of the tumor suppressor gene CDKN2a. These deletions can occur when aberrant recombinase activity results in gene deletion by engaging cryptic recombinase recognition sites, which are known to be present in the CDKN2a locus. This genetic mutation occurs frequently in “naturally” occurring cases of human T-ALL29Mullighan CG Miller CB Radtke I Phillips LA Dalton J Ma J et al.BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros.Nature. 2008; 453: 110-114Crossref PubMed Scopus (821) Google Scholar,30Ferrando AA Neuberg DS Staunton J Loh ML Huard C Raimondi SC et al.Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia.Cancer Cell. 2002; 1: 75-87Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar that are not associated with gene therapy. The authors discuss several possible mechanisms for these genetic events and the generally increased susceptibility. One possibility could be increased signaling due to deregulated expression of the vector-encoded γc gene, as others have argued,31Woods NB Bottero V Schmidt M von Kalle C Verma IM Gene therapy: therapeutic gene causing lymphoma.Nature. 2006; 440: 1123Crossref PubMed Scopus (250) Google Scholar but this study found no evidence for autonomous signaling in vector-transduced cells. Another possibility is that the “stalled” pool of γc-deficient lymphoid progenitors may have increased susceptibility to mutations during the rapid clonal expansion induced by the sudden acquisition of cytokine signaling. Associated high levels of proliferation could lead to subsequent DNA damage and mutations during a period of “proliferative stress.” It would be interesting to determine whether the mutations in Notch1 and/or CDKN2a were actually acquired and present prior to vector transduction and subsequent proliferation. Another possibility is that the lack of tumor immune surveillance in the SCID-X1 transplant setting could predispose to T-cell malignancies. In this regard, it is interesting to note that control animals receiving wild-type donor cells did not develop leukemia, most likely because the adoptive transfer of mature immune cells, most particularly T cells, would be predicted to result in rapid immune reconstitution. This is known to occur both in mouse transplants using wild-type bone marrow cells and in human allogeneic transplants using matched sibling donors. In contrast, immune reconstitution is often delayed for 3 to 4 months in the gene therapy setting using corrected autologous cells and in the haploidentical allogeneic transplants that are commonly used for patients lacking a matched sibling donor. Such patients require high-grade T-cell depletion of the graft, thereby potentially leading to prolonged periods of inadequate surveillance. There are certainly other possibilities to be considered; this paper provides new information and systems for evaluating this susceptibility process. It remains important to keep in mind that, unless future human cases provide new information, all cases of retroviral vector-induced leukemia and myelodysplasia have demonstrated clear evidence of vector-induced oncogene activation due to enhancer elements within the vector as a “smoking gun” feature. We think that this is a solvable problem, and it will be interesting to see whether elimination of this specific vector property will eliminate or reduce the oncogenic features of SCID-X1 gene therapy. Less likely, but still not ruled out, is the possibility that new mechanisms of transformation will emerge as limiting features.