Report from the 2nd Stem Cell Clonality and Genotoxicity Retreat
Authors: Robert M. Frederickson
Journal: Molecular Therapy (2005)

The 2nd Stem Cell Clonality and Genotoxicity Retreat was held June 1, 2005, on the eve of the 8th annual meeting of the American Society of Gene Therapy in St. Louis. The meeting was organized by David Williams, Chris Baum and Christof von Kalle of Cincinnati Children's Hospital. The immense popularity of the meeting was a testament to the strong interest on the part of the gene therapy community in the topic of insertional mutagenesis and the effects of retroviral insertion on hematopoiesis. Of course, the impetus for this retreat was the need and desire to gain a better understanding of the mechanism behind the cases of insertional mutagenesis leading to leukemogenesis in patients with X-linked severe combined immunodeficiency (X-SCID) who were treated with hematopoietic stem cells (HSC) genetically modified by retroviral transduction [1Kohn D.B. et al.American Society of Gene Therapy (ASGT) Ad Hoc sub-committee on retroviral-mediated gene transfer to hematopoietic stem cells.Mol. Ther. 2003; 8: 180-187Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 2Baum C. et al.Chance or Necessity? Insertional mutagenesis in gene therapy and its consequences.Mol. Ther. 2004; 9: 9-13Abstract Full Text Full Text PDF Scopus (193) Google Scholar]. As discussed recently in an editorial in these pages [3Baum C. et al.A Balanced Decision? Regulatory Reaction to the “Third Case”.Mol. Ther. 2005; 11: 819-820Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar], the field is rapidly gaining a better understanding of why a minority of patients in the French X-SCID trial developed lymphoproliferative disorders linked to the gene transfer vector. However, work in this area goes beyond this more narrow aim, as the reaction to these adverse events has stimulated fascinating new avenues of research that seem likely to have a strong impact on the broader field of gene transfer technology and research. The results have laid bare our poor understanding of the mechanism of viral integration into the genome and how much we have yet to learn before we can fully exploit this phenomenon for therapeutic purposes. We are gaining important new information on the insertional preferences of various viral and nonviral vector systems, as well as a better understanding of how integration of gene transfer vectors affects the genomic neighborhoods into which they land. Although the vector used in the French X-SCID study was an early-generation retroviral vector with intact LTRs, diverse vectors in preclinical and clinical studies have been shown to possess the capacity, at least under certain circumstances, to affect nearby genes following insertion into the genome. Clearly, the nature of the vector and its components will dictate any potential effects upon the genome of transduced cells. Moreover, the human genome is clearly not a wasteland of so-called “junk DNA” thinly populated by scarce genes, and insertion of foreign genetic material can have both long-distance and long-term consequences that are unpredicted. This issue is exacerbated by the now well-established preference of some viral vectors to insert at or around the 5’ regulatory regions of active genes. Importantly, it has also become clear that appropriately designed preclinical model systems are necessary to evaluate and predict the safety of gene transfer agents in human clinical application. These models will need to incorporate screens for both genotoxicity of the vector backbone and components and the phenotoxicity of the transgene. The first session, chaired by Cynthia Dunbar (NIH), included presentations of studies assessing the insertional potential of several gene transfer vectors, including discussion of animal models that can assess genotoxicity and more basic studies that aim to understand the mechanism of integration. The first speaker was Art Nienhuis (St. Jude Children's Research Hospital), who presented data examining the impact of integrated globin or LTR-containing lentivectors on expression of nearby genes in clonal populations of erythroid cells. The investigators observed local examples of increased expression due to insertion close to gene promoters as well as gene inactivation due to intragenic insertion. Global patterns of gene expression appeared largely unaffected, however. Interestingly, there was increased expression of a gene located more than 300 kb from a globin vector insertion. However, most insertions, even when in a gene-rich area, did not influence gene expression. The results imply that globin vectors have little impact on global gene expression in erythroid cells, although Nienhuis cautioned that the significance of gene activation with respect to oncogenic potential requires assessment in appropriately designed mouse models. Utpal Davé (NIH) presented interesting results of an analysis of retroviral insertion in a mouse model of leukemia, providing clear evidence that the proto-oncogene, LMO2, and the IL-2 common γ chain receptor gene (IL2γr) cooperate to induce leukemia in mouse hematopoietic cells [4Davé U.P. et al.Gene Therapy Insertional Mutagenesis Insights.Science. 2004; 303: 333Crossref PubMed Scopus (222) Google Scholar]. Clonal integrations were noted in both genes, and clones were even identified where both genes received an inserted retrovirus—an event that would be predicted to occur with an extremely low probability unless it provided some selective growth advantage. Each mouse leukemia had many insertions in a myriad of disease-related genes and analysis of proviral insertion sites combined with human microarray data showed that humans and mice mutate similar genes and pathways. The results provide strong evidence in favor of the idea that LMO2 and ILγR cooperated with additional genetic mutations to cause leukemogenesis in the human patients, and highlight the potential utility for clinical studies of mouse models that can predict geno- and phenotoxicity. However, a member of the audience did note that cooperation between LMO2 and IL2γR leading to leukemogenesis has not been detected following direct vector-mediated delivery of these genes to mouse HSC. Frederic Bushman (University of Pennsylvania) presented more basic studies aimed at elucidating the mechanisms by which lentiviruses target genomic integration in the human genome. Previous genome-wide studies of HIV DNA integration revealed that transcription units were favored targets. However, other lentiviruses have been shown to favor different integration sites—suggesting that the preintegration complex formed by each virus interacts specifically and differently with chromosomes to guide site selection. Finally, the results showed that targeting of integration appeared to be mediated, at least in part, by interactions between components of preintegration complexes and cellular transcriptional control proteins (see [5Mitchell R.S. et al.Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.PLoS Biol. 2004; 2: e234Crossref PubMed Scopus (761) Google Scholar]). The implication of the results is that there are distinct preferences based on the composition of the particular vector and that a better understanding of these preferences should allow better manipulation of targeting in vivo. Of course, integration of genetic elements into the genome is not a characteristic solely of retroviruses, but is also mediated by other promiscuous DNA elements such as transposons. Mark Kay's (Stanford University) talk introduced the concept of DNA transposition in mammalian cells, focusing on the Sleeping Beauty (SB) system (see [6Izsvák Z. Ivics Z. Sleeping Beauty transposition: Biology and applications for molecular therapy.Mol. Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar] for a recent review). Using an adenovirus-SB vector engineered by his group, Kay showed that transposition-mediated integration of trasgenes in mice could provide stable long-term levels of the protein product. When sites of integration were isolated, it was found that there was a strong preference for integration in microsatellite regions and a much weaker preference for integration close to or in genes than has been observed with retroviral vectors [7Yant S.R. et al.High-resolution genome-wide mapping of transposon integration in mammals.Mol. Cell. Biol. 2005; 25: 2085-2094Crossref PubMed Scopus (257) Google Scholar]. Kay also summarized data on the integration of rAAV genomes in the livers of mice. He showed that integration was relatively inefficient and randomly distributed throughout the genome. While no evidence for integration in known oncogenes was found, a strong preference for integration near genes was noted [8Nakai H. et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J. Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (138) Google Scholar]. As mentioned earlier, an important aim is the development of animal models that can predict genotoxicity of viral vectors used in clinical studies. Luigi Naldini (San Raffaele Telethon Institute for Gene Therapy, Milan, Italy) presented data on insertional mutagenesis by retroviral and lentiviral vectors in hematopoietic stem cells from a Cdk2 knockout mouse—a tumor-prone mouse model. Transplantation of transduced Cdk2a-/- HSC demonstrated early tumor onset with increasing frequency of retroviral insertion accomplished by increasing multiplicity of infection (moi). Use of lentiviral vectors appeared to give rise to only a low genotoxic potential in this system. Naldini's group has recently published a promoter-trap strategy to evaluate differences in integration site selection between MLV and HIV in hematopoietic cells [9De Palma M. et al.Promoter Trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells.Blood. 2005; 105: 2307-2315Crossref PubMed Scopus (151) Google Scholar]. Despite the apparent reduced level of risk for genotoxic events with lentiviral vectors in this system, recent results have demonstrated the development of liver tumors in mice treated intravenously with a lentiviral vector either neonatally or in utero [10Themis, M., et al. (2005). Oncogenesis following delivery of a non-primate lentiviral gene therapy vector to fetal and neonatal mice. Mol. Ther. 12: In press.Google Scholar], suggesting that caution is warranted before making general conclusions about the relative safety of lentiviral vectors. The latter study provides an additional model system that may prove useful to analyze the genotoxic potential of particular vector systems and applications. The second session was chaired by George Stamatoyannopoulos (University of Washington) and covered the area of insertional mutagenesis and clonal expansion of genetically modified cells in clinical hematopoietic stem cell transfer and in studies in large animal models. An important concept that arose during this session was the notion that clonal expansion of genetically modified cells could actually be beneficial for engraftment. Indeed, much work over the past decade has been directed to just this issue, with the aim of providing a selective advantage to modified cells so as to promote their selective repopulation after transfer into models of myeloablation. The first talk was presented by Christof von Kalle who presented an update on the analysis of integration sites in the French X-SCID trial patients. Factors being examined include the tendency to insert at common sites or genes, common changes in gene expression in the clonally expanded lines, whether insertions activate or inactivate genes, and clonal expansion over time. Von Kalle further presented preliminary data on two patients from the German trial led by Manuel Grez (see below) treating Chronic Granulomatous Disease (CGD)—another monogenic immunodeficiency disorder being treated with gene-modified cells. Retroviral vector integration sites (RIS) were examined in repopulating stem and early progenitor cells. Large-scale mapping revealed that RIS distribution turned nonrandom five months after reinfusion of gene-corrected CD34+ cells. Activating insertions in the zinc finger transcription factor homolog MDS1/EVI1 and two other genes have subsequently expanded gene-corrected long-term myelopoiesis three- to five-fold in the two treated CGD patients without detrimental side effects thus far. These data provide direct evidence in humans that these genes influence regulation of normal long-term hematopoiesis. The data also support the notion that insertional side effects reinforced the therapeutic success observed in this trial, suggesting novel approaches for manipulating human stem and progenitor cells. Von Kalle concluded that a risk/benefit analysis of the clinical application of insertional vector systems in warranted just as with any other pharmaceutical agents. The next speaker, Christopher Baum, discussed his recent studies in mice [11Kustikova O. et al.Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (303) Google Scholar] that support the clinical findings reported above. Baum's group has shown that insertional upregulation of cellular proto-oncogenes or related signaling genes may trigger clonal dominance in both normal and malignant hematopoiesis. Furthermore, combinatorial activation of these genes in single cells and/or collaborating side effects of transgene expression have been shown to trigger leukemia [12Modlich U. et al.Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.Blood. 2005; 105: 4235-4246Crossref PubMed Scopus (155) Google Scholar]. Subsequent dose-escalation studies have demonstrated that self-inactivating retroviral vectors reduce the risk of these insertional side effects, leading Baum to reiterate the need for predictive murine models that could be used for dose-escalation safety studies. Such models could be useful to test potentially simple modifications that could enhance the safety of vectors currently in clinical use. Hans-Peter Kiem (Fred Hutchison Cancer Research Center, Seattle) moved the discussion to larger animal models. Kiem discussed studies demonstrating efficient gene transfer to hematopoietic repopulating cells in large animal models using gamma retroviral, lentiviral and foamy viral vectors in myeloablative transplant conditions. He stressed that safety and not efficacy had become the prime concern in the development of protocols for stem cell gene transfer in these animal models. Integration site analyses in these studies demonstrated polyclonal repopulation with no evidence of monoclonality or leukemia. Cynthia Dunbar described results of long-term follow-up studies from her lab investigating the outcomes of 46 rhesus macaques transplanted with retrovirally-marked CD34+ bone marrow (BM) or peripheral blood (PB) cells [13Kiem H.-P. et al.Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: No progression to clonal hematopoiesis or leukemia.Mol. Ther. 2004; 9: 389-395Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Overall, the results showed no evidence for progression to monoclonality or abnormal hematopoiesis in the reconstituted cells from these animals. However, one animal has now died following the development of leukemia that has been linked to the mouse leukemia virus-based vector. Evidence has now been found for retroviral integration into the BCL2A1 anti-apoptosis gene locus on chromosome 15. In addition, an evaluation of integration events in the other animals has revealed an overrepresentation of insertion at the MDS1-EVI1 locus—which has also been noted in the two CGD trial patients as discussed by von Kalle. While there is no evidence to date for progression towards oligo- or monoclonality in these animals, it is hypothesized that integration at this site may lead to immortalization of a transduced myeloid progenitor in recipient animals, thereby enhancing engraftment. Although not discussed in her presentation, Dunbar and colleagues have also recently published an analysis of differences in the genomic integration patterns of MLV and SIV vectors in primate hematopoietic stem and progenitor cells [14Hematti P. et al.Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.PLoS Biol. 2004; 2: e423Crossref PubMed Scopus (226) Google Scholar]. Fulvio Mavilio (University of Modena, Milan, Italy) wrapped up the session with a presentation characterizing transduction of T lymphocytes with retroviral vectors as a safe procedure, based on his group's analysis of four patients between four months and nine years after transplantation with transduced cells. Although retroviral vectors inserted into potentially dangerous genomic locations and activated target genes in about 15% of the cases, they observed no consequences in terms of selection, expansion or transformation of the transduced T cells. In fact, clonal analysis suggested that deleterious integrations were counterselected in patients in vivo. The Lunch Sesion, chaired by meeting co-organizer David A. Williams, was devoted to a discussion of the response of regulatory agencies to the adverse events associated with the French X-SCID trial. The session was kicked off by Katherine High (University of Pennsylvania) who presented her perspectives as a principal investigator on the AAV-Factor IX clinical gene therapy trial. She noted the significant challenges associated with the multilayered nature of the regulatory process, and stressed the need for FDA reviewers who are both scientifically active and sensitive to the issues faced by clinical researchers. High was followed by Klaus Cituchek (Paul-Ehrlich-Institute, Langen, Germany), who outlined the European regulatory process for gene therapy products. He further discussed the successful European trials for treatment of X-SCID, adenosine deaminase-deficient (ADA)-SCID and CGD patients with retrovirally-transduced hematopoietic cells. Cituchek noted that differences in the French and London X-SCID trials with regard to the incidence of adverse events might have been due to variations in the protocols and vectors used in each trial. Cituchek further noted that future regulatory decisions need to balance the inherent risk associated with integrating vectors against the risks associated with alternative therapies, as suggested recently in an editorial in these pages [3Baum C. et al.A Balanced Decision? Regulatory Reaction to the “Third Case”.Mol. Ther. 2005; 11: 819-820Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar]. Cituchek finished with a brief discussion of the recent finding that fetal and in utero delivery of lentiviral vectors gave rise to liver tumors in mice [10Themis, M., et al. (2005). Oncogenesis following delivery of a non-primate lentiviral gene therapy vector to fetal and neonatal mice. Mol. Ther. 12: In press.Google Scholar]. The SIN vectors used in the study lacked intact LTRs but did include a WPRE element and a CMV promoter. Several vector insertions occurred near known proto-oncogenes, signal transduction and cell-cycle regulation genes, although a causative link between tumorigenesis and vector insertion has not been established to date. Carolyn Wilson (FDA) presented a brief historical review of the early assumption that tumorigenesis associated with the clinical use of retroviral vectors would only be a risk only when a replication-defective retroviral vector was contaminated with a replication-competent retrovirus (RCR). The observation that leukemias developed in children with X-SCID after integration of a replication-defective vector in the absence of any evidence of contaminating RCR have forced a reevaluation of regulatory issues regarding the use of retroviral vectors. Informed consent documents must now clearly explain the newly appreciated risk. Sponsors need to justify the use of retroviral vectors in the context of the clinical indication by providing a risk/benefit analysis, and there is now a request to perform additional subject monitoring for clonality of vector integration sites. Wilson also presented a review of the recommendations from the most recent FDA Advisory Committee on this topic, as well as results from a statistical model to assess the likelihood of vector integration into LMO2 and plans for a large-scale animal study that will be performed through the National Toxicology Program to assess a preclinical model for predicting leukemogenic potential of retroviral vectors. The third session, chaired by Punam Malik (Children's Hospital, Los Angeles) covered new data on the safety, efficacy and evaluation of insertion in ongoing gene therapy trials. The first speaker, Maria Cavazzano-Calvo, provided an update on the reconstitution of the immune systems in the children treated in the French X-SCID trial, and further characterization of the leukemias that developed in three of the children [15Schmidt M. et al.Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial.Blood. 2005; 105: 2699-2706Crossref PubMed Scopus (68) Google Scholar]. Leukemic cells from all three affected patients contained insertion of the vector into the LMO2 gene. There were additional genetic mutations in the affected cells, but these were different in each patient. The developmental stage of the clonal leukemic cells also differed. The results overall suggest activation of ILγR and LMO2 in combination with additional genetic changes as causative factors. An additional contributing factor may include the young age of the affected children at the time of vector transduction, due to the predicted presence of a greater number of immature progenitor cells in the transduced cells from these patients. It has been hypothesized that the strong dysregulated expression of ILγR during early stages of T-cell development of these transduced progenitors may have predisposed the cells to transformation by LMO2 expression and/or effects due to the additional genetic changes. Cavazzano-Calvo also outlined simple measures currently under investigation by her group that might be taken to reduce risk of genotoxic events, such as the inclusion in the vector of a mobilizable suicide gene, and the use of vectors with self-inactivatable LTRs. Adrian Thrasher next provided an update on the treatment in the London trial of seven children with X-SCID [16Gaspar H.B. 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 (562) Google Scholar]. As with the children in the French trial, these children were treated by gene transfer of the missing growth factor receptor to bone marrow CD34+ cells using a retroviral vector. All patients developed largely normal immune function, although recovery was partial in one patient. Despite the similarity with the French X-SCID trial, leukemia has not developed in any of these patients. Interestingly, the kinetics of immunological reconstitution were qualitatively different than those observed for patients treated in the French X-SCID study. The significance of these observations in terms of efficacy or toxicity is not known. Finally, one patient with ADA-SCID has been treated with good correction of immunity at one year after gene therapy. Thrasher observed in summary that insertional mutagenesis was a finite risk and that better assays are needed to assess this risk, as with any other experimental pharmaceutical agent. As pointed out by the previous speaker, he suggested that relatively simple modifications to vector design may have significant impact on safety. Manuel Grez (Georg-Speyer-Haus, Frankfurt, Germany) provided an overview of results of treatment of two CGD patients with gene-modified cells. A gammaretroviral vector expressing the missing gene, encoding gp91phox, was used to transduce CD34+ cells isolated from these patients. Within three to four months of infusion of gene-transduced cells, the group observed an unexpected expansion of gene-modified cells with similar kinetics in both patients. Grez believes that the cell expansion resulted from the activation of growth promoting genes as a result of vector integration. However, myeloid cell proliferation was restricted and did not result in granulocyte numbers exceeding those before gene therapy. The results again suggest that insertional mutagenesis might have provided proliferative advantages to the transduced cells, which could be useful for those diseases where there is no intrinsic advantage imparted by the transgene. Importantly, therapeutic levels of the transgene product have been observed in both patients, who are well and have been free of severe infections since transplantation. While there is now evidence of clonal expansion of certain transduced cells, there is no evidence to date of leukemogenesis in these patients. Clearly, long-term follow-up will be necessary to closely monitor any risks associated with this treatment. Fanconi anemia is a genetic disease characterized by progressive bone marrow failure and aplastic anemia. Patrick Kelly (Cincinnati Children's Hospital) discussed recent efforts to treat Fanconi anemia patients with hematopoietic cell transfer. There are significant challenges to the treatment of this disease due to depletion of the HSC target cell and their sensitivity to standard ex vivo manipulation. Patients are at increased risk for spontaneous leukemia and are hypersensitive to agents used in nonmyeloablative preparatory regimens. Kelly and coworkers modified their methods in order to minimize cell loss and to monitor the patients closely post reinfusion for leukemic/clonal evolution, irrespective of the presence of proviral insertion sequences. Low numbers of HSC were collected from the FA patients, even early in the course of their bone-marrow failure (10-20 fold less CD34+ cells compared to X-SCID/ADA patients). To date, two patients have been infused with genetically modified cells. While gene transfer and functional correction of the FA phenotype was efficient in vitro (40-60%), detection of gene-corrected cells post-infusion in one patient is very low and transient (less the 6 weeks post infusion). While still early in the process, the most significant barrier to overcome may be the low number of HSC collected for genetic correction. The final session was chaired by Brian Sorrentino (St. Jude Children's Research Hospital) and covered the development of three newer vector systems that are in early stages of preclinical analysis. David Russell (University of Washington) described an analysis of the integration preferences of Foamy Virus (FV) vectors, which are very efficient at transducing hematopoietic stem cells. Russell's lab has mapped nearly 3000 FV vector integration sites in human CD34+ cells and fibroblasts. Compared to other retroviral vectors or even random chromosomal insertions, FV vectors were less likely to integrate in genes, although they did have a modest preference for integration near transcription start sites and CpG islands. Michele Calos (Stanford University) described work from her lab on the use of ϕC31 integrase, a site-specific integrase isolated from a bacteriophage that mediates recombination between its recognition site placed on an incoming vector and a set of partially matching sequences present in the host cell genome [17Groth A.C. Calos M.P. Phage integrases: Biology and applications.J. Mol. Biol. 2004; 335: 667-678Crossref PubMed Scopus (332) Google Scholar]. Because the enzyme requires a significant sequence match in order to cause integration, the number of integration sites is orders of magnitude smaller than the number utilized by retroviral integrases or transposases. By studying integration events in several human cell lines, Calos’ lab identified approximately 135 integration sites in the human genome, with nearly half of the integration events occurring at about 10-20 “hotspot” sites. The sites identified to date are not in well-characterized proto-oncogenes such as LMO2 or the IRγR. Calos suggests that the ϕC31 integrase system may be safer for gene therapy than more randomly integrating vectors, and work is underway to enhance further the specificity of the integrase. Finally, Wolfgang Hammerschmidt completed the session with a description of his efforts to create Epstein-Barr Virus-based replicons for gene transfer applications. The meeting provided a great deal of new data that taken together is providing a clearer picture of the risk associated with the use of integrating vectors in HSC transduction for gene therapy. While the only clinical cases of insertional mutagenesis leading to leukemogenesis to date are associated with retroviral vectors with intact LTRs, there is new animal data suggesting that lentiviral vectors may be associated with some degree of risk in other experimental settings [10Themis, M., et al. (2005). Oncogenesis following delivery of a non-primate lentiviral gene therapy vector to fetal and neonatal mice. Mol. Ther. 12: In press.Google Scholar]. Various vector systems have been shown to integrate, thereby creating some level of at least theoretical risk for each. These risks can be attenuated by strategies to inactivate strong promoters and enhancers and to modify other genetic elements that might affect the target site of integration or the expression of genes in the vicinity of an integrated vector. The genotoxic potential of each new vector and vector system will need to be assessed rigorously in appropriately designed animal models, which should also include consideration of the contribution of the transgene to potential toxicity. The ability to control where and with what frequency a vector integrates would obviate the main culprit in insertional mutagenesis, which is the activation of potentially dangerous oncogenes. A better understanding of the mechanism and sequence preferences of retroviral integrases should allow a more targeted approach to retroviral transduction. Eventually, newer integration strategies based on engineered transposase or integrase systems might afford even greater control over integration of therapeutic transgenes, although the transfer of these systems to clinical study is yet some time away. The mechanism that is emerging in the case of the adverse events in the French X-SCID trial suggests potential collaboration between the IL2γR transgene, insertional activation of LMO2 and additional genetic defects within the affected clonal lines—and thus appears generally consistent with the multi-hit model of tumorigenesis. However, another potential contributing factor is the young age at treatment of the children who developed leukemia. They thus likely received a greater number of transduced immature progenitor cells in the subsequent reinfusion of gene-modified cells. Indeed, these three patients received the highest overall number of transduced CD34+ cells/kg relative to the other patients in the trial, and certainly far more than the animals in the preclinical studies. This factor alone may have contributed significantly to the increased risk leading to these adverse events. In general, appropriately designed experimental studies in animal models should allow us to evaluate, individually and in combination, each of the putative factors underlying these adverse events and should allow us to decipher the relative contribution of each of these components. Such studies should go a long way towards an eventual full understanding of the etiology of leukemogenesis secondary to insertional mutagenesis by retroviral vectors.