Abstract
Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a−/− mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.
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Acknowledgements
We are grateful to F.R. Santoni de Sio for help and suggestions and A. Aiuti for critical reading of this manuscript. This work was supported by grants from Telethon (TIGET grant), EU (Project LSHB-CT-2004-005242, CONSERT), National Institutes of Health (2 P01 HL053750-11), AIRC (1192) and the Italian Ministry of Scientific Research to L.N. E.M. would like to thank B. Franco for the useful discussion about the 'Brute Force Approach'.
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Supplementary information
Supplementary Fig. 1
FACS analysis of tumor infiltrated BMs from 14 primary recipients (1st) and the relative FACS analyses of secondary recipients (2nd). (DOC 962 kb)
Supplementary Fig. 2
Survival of mice transplanted with different doses of Cdkn2a-HPC. (DOC 417 kb)
Supplementary Table 1
Tumor Phenotypes. (DOC 1136 kb)
Supplementary Table 2
Tumor Integrations. (DOC 445 kb)
Supplementary Table 3
Pre-transplant integrations. (DOC 1269 kb)
Supplementary Table 4
Raw data used for overrepresentation statistical analysis of GO classes in the four different datasets. (DOC 767 kb)
Supplementary Data
Histopathological description of the hematopoietic tumors shown in Figure 2>. (DOC 29 kb)
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Montini, E., Cesana, D., Schmidt, M. et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 24, 687–696 (2006). https://doi.org/10.1038/nbt1216
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DOI: https://doi.org/10.1038/nbt1216
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