Good News for Gene Therapy
http://www.100md.com
《新英格兰医药杂志》
Gene therapy has endured several setbacks in recent years. One setback involved infants born with X-linked severe combined immunodeficiency (SCID) resulting from a mutation of the c gene who were successfully treated with an infusion of autologous hematopoietic stem cells transduced with a defective retroviral vector.1 The enthusiasm generated by the apparent cure of 9 of the 10 infants turned to alarm when, nearly three years after treatment, T-cell leukemia developed in 2 of the boys.2 A recent study by Davé and colleagues,3 however, suggests that this setback may have been due to a coincidence and that it is possible to reduce the likelihood that such an event will recur.
Before their transfusion into the patients with SCID, the autologous stem cells were transduced with a retrovirus containing the c gene — the rationale being that the genetically tweaked cells, replete with an intact and functional c gene, would populate the patients' marrow and thereby cure them of the disease. In both of the patients in whom leukemia developed, the retrovirus carrying the c gene had inserted itself near LMO2, an oncogene that is activated as a result of translocations in acute lymphoblastic leukemia.4 The insertion caused overexpression of the LMO2 protein, implicating LMO2 in the cause of the leukemia (Figure 1).
Figure 1. A Potential Mechanism of Leukemogenesis.
T-cell leukemia developed in two children with SCID who were treated with CD34+ stem cells transduced with a viral vector containing IL2RG, which encodes the common subunit of the interleukin-2 receptor. A recent study by Davé et al.3 suggests that the c transgene was inserted close to the LMO2 gene, activating the T-cell oncogene. Overexpression of the LMO2 protein in T cells blocks the differentiation of the cells and thus increases their susceptibility to leukemia. This finding has potential implications for the design of gene-therapy protocols.
This event was unexpected. Although replication-competent retroviruses are routinely used to induce cancer and identify oncogenes in experimental systems, experts in the field believed that the risk of inducing cancer with replication-defective viruses, such as those used in gene therapy, was very slim.4 This is because the genesis of cancer requires multiple synergizing mutations in the same cell. Although a defective retrovirus can undoubtedly cause sporadic activation of a single oncogene, a single round of infection would be extremely unlikely to activate multiple oncogenes in the same cell. This view has been bolstered by the findings that replication-defective retroviral vectors that are not carrying oncogenes do not cause cancer in animal models and have been harmless in a large number of gene-therapy trials.
The leukemias occurring in the two patients with SCID challenge this view. Could it be that in patients with SCID, the therapeutic c transgene itself acts as an oncogene, and therefore, insertion of the transgene near LMO2 constitutes a "double hit" in promoting tumor development? Although an examination of the T-cell clones from the patients provided no data to support this hypothesis,2 Davé et al. have recently obtained supporting data from another source. These authors screened the Mouse Retroviral Cancer Gene data base, which contains the sequences of more than 3000 insertions from nearly 1000 retrovirally induced hematopoietic tumors. They found two insertions near Lmo2 and two near the endogenous c gene. Remarkably, one tumor contained two clonal insertions: one near the c gene and the other near Lmo2 — statistically, an extremely unlikely event. This leads to two important conclusions: the c gene can act as an oncogene when under control of the retroviral promoter (a promoter is a stretch of DNA that activates gene transcription), and the c gene and Lmo2 are collaborating oncogenes.
The data of Davé et al.3 explain why leukemia developed after gene therapy in the two patients with SCID: the insertion of the c vector near LMO2 represents a double hit, meaning that the cells might then be only one mutation away from overt tumor development. The vast expansions in the clonal populations of cells in such patients, although critical to the success of the therapy, increase the chance that additional genetic glitches will occur.
Although these findings are doubtless discomforting to the patients with SCID who were treated with the c vector and their families, it is good news for the field of gene therapy. The findings mean that in most gene-therapy applications, the therapeutic gene does not have oncogenic potential. They also offer a rational basis for improving gene-therapy protocols. For example, in the case of patients with SCID, one might seek to mitigate — or otherwise compensate for — signaling by the c receptor or to modify the vector so that it is not as likely to activate juxtaposed genes. Of course, the latter approach would increase the safety of any gene-therapy protocol.
If the therapeutic gene that is inserted has no oncogenic potential, the risk of cancer should be low — exactly how low is hard to predict. If retroviruses show a strong preference for integration in loci that happen to harbor proto-oncogenes or tumor-suppressor genes, the risk might be higher than presumed. And if the disruption of such loci facilitates clonal expansion over time, the risk of the occurrence of mutations that have a synergistic effect will increase. Our ability to prevent such adverse events will depend on the availability of improved vectors, the findings of additional studies involving appropriate models, and a thorough assessment of the putative oncogenic capacity of genes, including marker genes,5 that are incorporated in the various gene-therapy vectors. In the meantime, we should carefully assess on a case-by-case basis the risk factors associated with a particular gene-therapy protocol.
Editor's note: Dr. Berns is a member of the Central Committee on Research Involving Human Subjects in the Netherlands.
Source Information
From the Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam.
References
Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002;346:1185-1193.
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415-419.
Davé UP, Jenkins NA, Copeland NG. Gene therapy insertional mutagenesis insights. Science 2004;303:333-333.
McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2004;350:913-922.
Li Z, Dullmann J, Schiedlmeier B, et al. Murine leukemia induced by retroviral gene marking. Science 2002;296:497-497.(Anton Berns, Ph.D.)
Before their transfusion into the patients with SCID, the autologous stem cells were transduced with a retrovirus containing the c gene — the rationale being that the genetically tweaked cells, replete with an intact and functional c gene, would populate the patients' marrow and thereby cure them of the disease. In both of the patients in whom leukemia developed, the retrovirus carrying the c gene had inserted itself near LMO2, an oncogene that is activated as a result of translocations in acute lymphoblastic leukemia.4 The insertion caused overexpression of the LMO2 protein, implicating LMO2 in the cause of the leukemia (Figure 1).
Figure 1. A Potential Mechanism of Leukemogenesis.
T-cell leukemia developed in two children with SCID who were treated with CD34+ stem cells transduced with a viral vector containing IL2RG, which encodes the common subunit of the interleukin-2 receptor. A recent study by Davé et al.3 suggests that the c transgene was inserted close to the LMO2 gene, activating the T-cell oncogene. Overexpression of the LMO2 protein in T cells blocks the differentiation of the cells and thus increases their susceptibility to leukemia. This finding has potential implications for the design of gene-therapy protocols.
This event was unexpected. Although replication-competent retroviruses are routinely used to induce cancer and identify oncogenes in experimental systems, experts in the field believed that the risk of inducing cancer with replication-defective viruses, such as those used in gene therapy, was very slim.4 This is because the genesis of cancer requires multiple synergizing mutations in the same cell. Although a defective retrovirus can undoubtedly cause sporadic activation of a single oncogene, a single round of infection would be extremely unlikely to activate multiple oncogenes in the same cell. This view has been bolstered by the findings that replication-defective retroviral vectors that are not carrying oncogenes do not cause cancer in animal models and have been harmless in a large number of gene-therapy trials.
The leukemias occurring in the two patients with SCID challenge this view. Could it be that in patients with SCID, the therapeutic c transgene itself acts as an oncogene, and therefore, insertion of the transgene near LMO2 constitutes a "double hit" in promoting tumor development? Although an examination of the T-cell clones from the patients provided no data to support this hypothesis,2 Davé et al. have recently obtained supporting data from another source. These authors screened the Mouse Retroviral Cancer Gene data base, which contains the sequences of more than 3000 insertions from nearly 1000 retrovirally induced hematopoietic tumors. They found two insertions near Lmo2 and two near the endogenous c gene. Remarkably, one tumor contained two clonal insertions: one near the c gene and the other near Lmo2 — statistically, an extremely unlikely event. This leads to two important conclusions: the c gene can act as an oncogene when under control of the retroviral promoter (a promoter is a stretch of DNA that activates gene transcription), and the c gene and Lmo2 are collaborating oncogenes.
The data of Davé et al.3 explain why leukemia developed after gene therapy in the two patients with SCID: the insertion of the c vector near LMO2 represents a double hit, meaning that the cells might then be only one mutation away from overt tumor development. The vast expansions in the clonal populations of cells in such patients, although critical to the success of the therapy, increase the chance that additional genetic glitches will occur.
Although these findings are doubtless discomforting to the patients with SCID who were treated with the c vector and their families, it is good news for the field of gene therapy. The findings mean that in most gene-therapy applications, the therapeutic gene does not have oncogenic potential. They also offer a rational basis for improving gene-therapy protocols. For example, in the case of patients with SCID, one might seek to mitigate — or otherwise compensate for — signaling by the c receptor or to modify the vector so that it is not as likely to activate juxtaposed genes. Of course, the latter approach would increase the safety of any gene-therapy protocol.
If the therapeutic gene that is inserted has no oncogenic potential, the risk of cancer should be low — exactly how low is hard to predict. If retroviruses show a strong preference for integration in loci that happen to harbor proto-oncogenes or tumor-suppressor genes, the risk might be higher than presumed. And if the disruption of such loci facilitates clonal expansion over time, the risk of the occurrence of mutations that have a synergistic effect will increase. Our ability to prevent such adverse events will depend on the availability of improved vectors, the findings of additional studies involving appropriate models, and a thorough assessment of the putative oncogenic capacity of genes, including marker genes,5 that are incorporated in the various gene-therapy vectors. In the meantime, we should carefully assess on a case-by-case basis the risk factors associated with a particular gene-therapy protocol.
Editor's note: Dr. Berns is a member of the Central Committee on Research Involving Human Subjects in the Netherlands.
Source Information
From the Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam.
References
Hacein-Bey-Abina S, Le Deist F, Carlier F, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002;346:1185-1193.
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415-419.
Davé UP, Jenkins NA, Copeland NG. Gene therapy insertional mutagenesis insights. Science 2004;303:333-333.
McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2004;350:913-922.
Li Z, Dullmann J, Schiedlmeier B, et al. Murine leukemia induced by retroviral gene marking. Science 2002;296:497-497.(Anton Berns, Ph.D.)