Nature Medicine
9, 367 - 369 (2003)
doi:10.1038/nm0403-367
Safety of retroviral gene marking with a truncated NGF receptorC. Bonini1, M. Grez2, C. Traversari3, F. Ciceri1, S. Marktel1, 4, G. Ferrari5, M. Dinauer6, M. Sadat6, A. Aiuti5, S. Deola5, M. Radrizzani3, A. Hagenbeek7, J. Apperley4, S. Ebeling7, A. Martens7, H.J. Kolb8, M. Weber8, F. Lotti5, A. Grande9, E. Weissinger10, J.A. Bueren11, M. Lamana11, J.H.F. Falkenburg12, M.H.M. Heemskerk12, T. Austin13, S. Kornblau14, F. Marini14, C. Benati3, Z. Magnani1, S. Cazzaniga1, S. Toma3, C. Gallo-Stampino3, M. Introna15, S. Slavin16, P.D. Greenberg17, M. Bregni1, F. Mavilio9
& C. Bordignon1, 31 Cancer Immunotherapy and Gene Therapy Program and Bone Marrow Transplantation Unit, Istituto Scientifico H.S. Raffaele and University Vita e Salute, Milan, Italy 2 Georg-Speyer-Haus, Frankfurt, Germany 3 Molmed S.p.A., Milan, Italy 4 Department of Haematology, Faculty of Medicine, Imperial College Hammersmith Hospital, London, UK 5 H.S. Raffaele-Telethon Institute for Gene Therapy, Milan, Italy 6 Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA 7 Department of Haematology, University Medical Center, Utrecht, The Netherlands 8 GSF-National Research Center for Environment and Health, Munich, Germany 9 Department of Biomedical Sciences, University of Modena School of Medicine, Modena, Italy 10 Hematology and Oncology, Medical School, Hannover, Germany 11 Hematopoietic Gene Therapy Program, CIEMAT/Fundación Marcelino Botín, Madrid, Spain 12 Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands 13 Liver Stem Cell Program, Stem Cells Inc., Palo Alto, California, USA 14 Section of Molecular Hematology and Therapy, Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA 15 Molecular Immunohematology Laboratory, Department of Immunology and Cell Biology, Istituto di Ricerche Farmacologiche 'Mario Negri', Milan, Italy 16 Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital, Jerusalem, Israel 17 Program of Immunology, Fred Hutchinson Cancer Research Center and Department of Immunology and Medicine, University of Washington, Seattle, Washington, USA
Correspondence should be addressed to C. Bonini bonini.chiara@hsr.itTo the editor
Random integration into the host cell genome and inappropriate transgene expression are major safety concerns for the clinical use of retroviral vectors. Li et al. recently reported a leukemic transformation of mouse bone marrow cells caused by integration of a transgene-carrying retroviral vector into the Evi1 proto-oncogene. They suggested that expression of the transgene, a truncated form of the p75 low-affinity nerve growth factor receptor ( LNGFR) with most of the intracytoplasmic tail deleted (from residue 248), contributed to the leukemic progression1. Because LNGFR is used as a surface marker in gene therapy clinical trials aimed at controlling graft-versus-host disease (GVHD) after bone marrow transplantation (BMT)2,
3, a critical assessment of the potential risks associated with the use of such a molecule is essential.
In a collaborative effort between 17 independent groups of investigators, we have accumulated both pre-clinical and clinical evidence supporting the safety of LNGFR as a cell-marking molecule. Cumulative data obtained from >300 mice transplanted with bone marrow cells transduced with LNGFR-expressing retroviral vectors showed normal engraftment, persistence and differentiation of LNGFR-expressing hematopoietic stem-progenitor cells (HSCs) in primary, secondary and tertiary BMT recipients, with no adverse events (Table 1 and Supplementary Information online). Over 100 of these mice were monitored for >20 weeks after BMT; more than 70 animals, including 16 recipients of secondary or tertiary BMT, were monitored for >28 weeks. Considering that a total of >1  109 transduced cells were transplanted, and assuming an average of one retroviral integration per cell, we estimate the risk of oncogenic transformation after transduction with a LNGFR-encoding retroviral vector to be <1 in 109 integration events. Therefore, expression of LNGFR could not have increased the expected frequency of an insertional oncogenesis event, which has been previously estimated at 10-8 to 10-7 per insertion event4. Expression of LNGFR did not alter the function or survival of T lymphocytes derived from peripheral blood mononuclear cells transduced with a variety of vectors and studied in different animal models. In pre-clinical models of post-BMT GVHD, no difference in the ability to induce donor chimerism or to mediate GVHD was observed for LNGFR-expressing T cells, as compared with control T cells, in 356 mice, 200 rats and 3 dogs (Table 1 and Supplementary Information online), again with no adverse events.
 | Table 1. Studies supporting the safety of retroviral gene marking with  LNGFR: cumulative results from animal models | | |  |
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Analysis of 102 independent transductions of human peripheral lymphocytes with two different vectors (SFCMM-3 and SFCM) encoding the same LNGFR detected no change in the expression of markers of lineage, activation or adhesion, or in the proliferative capacity of T cells, as assayed by limiting dilution after polyclonal in vitro stimulation. All cells remained strictly dependent on interleukin-2 for growth and survival, and the addition of potentially stimulatory doses of 50−100 ng/ml of nerve growth factor did not induce cell proliferation, expression of the CD25 activation marker or secretion of tumor necrosis factor (see Supplementary Information online), thus providing direct evidence that LNGFR is a safe and inert gene marker for T lymphocytes even in the presence of high concentrations of the ligand. Most importantly, no toxicity or other adverse effects have been associated with the use of LNGFR as a surface marker for genetically modified lymphocytes in phase 1 clinical studies aimed at treating or preventing post-BMT GVHD2,
3. Treatment of 31 patients with donor lymphocytes transduced with two different vectors (SFCMM-2 or SFCMM-3) encoding the same LNGFR resulted in engraftment (up to 40% of circulating mononuclear cells) and long-term persistence (>80 months) of transduced cells. No acute or chronic adverse or toxic events related to the gene transfer procedure or to transgene expression were observed during these trials, which involved infusion of >1011 cells generated by >50 independent transductions (Table 2).
| | Table 2. Studies supporting the safety of retroviral gene marking with LNGFR: results from clinical trials | | | |
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These pre-clinical and clinical studies provide evidence that the use of LNGFR as a cell-surface marker is safe, non toxic and non-tumorigenic in both mice and men. The data presented by Li et al. correlated for the first time a leukemic transformation with the integration of a replication-defective retroviral vector. Recently, occurrence of T-cell lymphoproliferative disorders in patients treated with gene therapy for X-linked severe combined immunodeficiency has been correlated with the integration of the therapeutic vector into a T-cell proto-oncogene5, indicating that in a clinical setting, insertional oncogenesis is more than just a theoretical possibility. In light of these safety concerns, it is essential that the potential risks associated with any gene therapy approach are assessed on the basis of safety studies of the appropriate size and significance. The increased risk of leukemic transformation proposed by Li et al. as a consequence of the expression of a truncated neurotrophin receptor is not supported by the experimental and clinical data presented in this cooperative study.
Note: Supplementary information is available at the Nature Medicine website.
REFERENCES
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- Bonini, C. et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276, 1719–1724 (1997). | Article | PubMed | ISI | ChemPort |
- Rosenberg, N. & Jolicoeur, P. Retroviral pathogenesis. in Retroviruses (eds. Coffin, J.M., Hughes, S.H. & Varmus, H.) 413–416 (Cold Spring Harbor Laboratory Press, New York, 1997).
- Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003). | Article | PubMed | ISI |
Competing interests statement:
The authors declare competing financial interests. |
