Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ex vivo transduction and transplantation of Cdkn2a−/− HPCs.
Figure 2: Representative examples of hematopoietic tumors developed in mice transplanted with Cdkn2a−/− HPCs, treated with the indicated vectors (lentiviral or retroviral) or mock-transduced (MT).
Figure 3: Survival of mice transplanted with Cdkn2a−/− HPCs treated with the indicated vector (lentiviral or retroviral, 1 to 3 indicates the rounds of infection), or mock-transduced (MT).
Figure 4: Molecular analysis of tumors developed in transplanted mice.
Figure 5: Vector integration site analysis in cells before transplant and in tumors and retrospective mouse survival analysis.

References

  1. Schroder, A. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).

    Article  CAS  Google Scholar 

  2. Wu, X., Li, Y., Crise, B. & Burgess, S.M. Transcription start regions in the human genome are favored targets for MLV integration. Science 300, 1749–1751 (2003).

    Article  CAS  Google Scholar 

  3. Mitchell, R.S. et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol. 2, E234 (2004).

    Article  Google Scholar 

  4. Hematti, P. et al. Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells. PLoS Biol. 2, e423 (2004).

    Article  Google Scholar 

  5. De Palma, M. et al. Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 105, 2307–2315 (2005).

    Article  CAS  Google Scholar 

  6. Nakai, H. et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat. Genet. 34, 297–302 (2003).

    Article  CAS  Google Scholar 

  7. Kohn, D.B. et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates. Nat. Med. 4, 775–780 (1998).

    Article  CAS  Google Scholar 

  8. Malech, H.L. et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc. Natl. Acad. Sci. USA 94, 12133–12138 (1997).

    Article  CAS  Google Scholar 

  9. Aiuti, A. et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–2413 (2002).

    Article  CAS  Google Scholar 

  10. Cavazzana-Calvo, M., Lagresle, C., Hacein-Bey-Abina, S. & Fischer, A. Gene therapy for severe combined immunodeficiency. Annu. Rev. Med. 56, 585–602 (2005).

    Article  CAS  Google Scholar 

  11. Gaspar, H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 (2004).

    Article  CAS  Google Scholar 

  12. Bordignon, C. & Roncarolo, M.G. Therapeutic applications for hematopoietic stem cell gene transfer. Nat. Immunol. 3, 318–321 (2002).

    Article  CAS  Google Scholar 

  13. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    Article  CAS  Google Scholar 

  14. Coffin, J., Hughes, S.H. & Varmus, H.E. Retroviruses (Cold Spring Harbor Laboratory Press, Plainview, 2000).

    Google Scholar 

  15. Lund, A.H. et al. Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice. Nat. Genet. 32, 160–165 (2002).

    Article  CAS  Google Scholar 

  16. Suzuki, T. et al. New genes involved in cancer identified by retroviral tagging. Nat. Genet. 32, 166–174 (2002).

    Article  CAS  Google Scholar 

  17. Mikkers, H. et al. High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat. Genet. 32, 153–159 (2002).

    Article  CAS  Google Scholar 

  18. Mikkers, H. & Berns, A. Retroviral insertional mutagenesis: tagging cancer pathways. Adv. Cancer Res. 88, 53–99 (2003).

    CAS  PubMed  Google Scholar 

  19. Akagi, K., Suzuki, T., Stephens, R.M., Jenkins, N.A. & Copeland, N.G. RTCGD: retroviral tagged cancer gene database. Nucleic Acids Res. 32 Database issue, D523–527 (2004).

    Article  CAS  Google Scholar 

  20. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G. & Jenkins, N.A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221–226 (2005).

    Article  CAS  Google Scholar 

  21. Du, Y., Spence, S.E., Jenkins, N.A. & Copeland, N.G. Cooperating cancer gene identification via oncogenic retrovirus-induced insertional mutagenesis. Blood 106, 2498–2505 (2005).

    Article  CAS  Google Scholar 

  22. Collier, L.S., Carlson, C.M., Ravimohan, S., Dupuy, A.J. & Largaespada, D.A. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276 (2005).

    Article  CAS  Google Scholar 

  23. Wu, X., Luke, B.T. & Burgess, S.M. Redefining the common insertion site. Virology 344, 292–295 (2006).

    Article  CAS  Google Scholar 

  24. Baum, C. et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol. Ther. 9, 5–13 (2004).

    Article  CAS  Google Scholar 

  25. Modlich, U. et al. Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 105, 4235–4246 (2005).

    Article  CAS  Google Scholar 

  26. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    Article  CAS  Google Scholar 

  27. Kay, M.A., Glorioso, J.C. & Naldini, L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat. Med. 7, 33–40 (2001).

    Article  CAS  Google Scholar 

  28. Verma, I.M. & Weitzman, M.D. Gene therapy: twenty-first century medicine. Annu. Rev. Biochem. 74, 711–738 (2005).

    Article  CAS  Google Scholar 

  29. Pawliuk, R. et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294, 2368–2371 (2001).

    Article  CAS  Google Scholar 

  30. May, C., Rivella, S., Chadburn, A. & Sadelain, M. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood 99, 1902–1908 (2002).

    Article  CAS  Google Scholar 

  31. Imren, S. et al. High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells. J. Clin. Invest. 114, 953–962 (2004).

    Article  CAS  Google Scholar 

  32. Biffi, A. et al. Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells. J. Clin. Invest. 113, 1118–1129 (2004).

    Article  CAS  Google Scholar 

  33. Serrano, M. et al. Role of the INK4a locus in tumor suppression and cell mortality. Cell 85, 27–37 (1996).

    Article  CAS  Google Scholar 

  34. Sherr, C.J. Principles of tumor suppression. Cell 116, 235–246 (2004).

    Article  CAS  Google Scholar 

  35. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).

    Article  CAS  Google Scholar 

  36. Roberts, M.R. et al. Antigen-specific cytolysis by neutrophils and NK cells expressing chimeric immune receptors bearing zeta or gamma signaling domains. J. Immunol. 161, 375–384 (1998).

    CAS  PubMed  Google Scholar 

  37. Schmidt, M. et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 100, 2737–2743 (2002).

    Article  CAS  Google Scholar 

  38. Ailles, L. et al. Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells. Mol. Ther. 6, 615–626 (2002).

    Article  CAS  Google Scholar 

  39. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  Google Scholar 

  40. Hosack, D.A., Dennis, G., Jr., Sherman, B.T., Lane, H.C. & Lempicki, R.A. Identifying biological themes within lists of genes with EASE. Genome Biol. 4, R70 (2003).

    Article  Google Scholar 

  41. Klug, C.A., Cheshier, S. & Weissman, I.L. Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny. Blood 96, 894–901 (2000).

    CAS  PubMed  Google Scholar 

  42. Zentilin, L. et al. Variegation of retroviral vector gene expression in myeloid cells. Gene Ther. 7, 153–166 (2000).

    Article  CAS  Google Scholar 

  43. Hwang, H.C. et al. Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis. Proc. Natl. Acad. Sci. USA 99, 11293–11298 (2002).

    Article  CAS  Google Scholar 

  44. Johnson, C., Lobelle-Rich, P.A., Puetter, A. & Levy, L.S. Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites. J. Virol. 79, 57–66 (2005).

    Article  CAS  Google Scholar 

  45. Nielsen, A.A., Sorensen, A.B., Schmidt, J. & Pedersen, F.S. Analysis of wild-type and mutant SL3–3 murine leukemia virus insertions in the c-myc promoter during lymphomagenesis reveals target site hot spots, virus-dependent patterns, and frequent error-prone gap repair. J. Virol. 79, 67–78 (2005).

    Article  CAS  Google Scholar 

  46. Futreal, P.A. et al. A census of human cancer genes. Nat. Rev. Cancer 4, 177–183 (2004).

    Article  CAS  Google Scholar 

  47. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57–70 (2000).

    Article  CAS  Google Scholar 

  48. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79–90 (1998).

    Article  CAS  Google Scholar 

  49. Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

    Article  CAS  Google Scholar 

Download references

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'.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luigi Naldini.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Supplementary Methods (DOC 148 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1216

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing