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.

  • Short Communication
  • Published:

Lack of genotoxicity due to foamy virus vector integration in human iPSCs

Gene Therapy volume 20, pages 868–873 (2013)Cite this article

Subjects

Abstract

Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes the retroviral and lentiviral vectors commonly used for deriving induced pluripotent stem cells (iPSCs). We previously used integrating foamy virus (FV) vectors expressing OCT4, SOX2, MYC and KLF4 to reprogram osteogenesis imperfecta mesenchymal stem cells (MSCs). Here, we have studied the effects of 10 FV vector proviruses on neighboring gene expression in four iPSC lines and their corresponding iPSC-derived MSC (iMSCs). Gene expression profiles in these iPSC lines showed that none of the 38 genes within 300 kb up- or downstream of integrated proviruses had a significant difference in mRNA levels, including five genes with proviruses in their transcription units. In the iMSCs derived from these iPSCs, the same type of analysis showed a single dysregulated transcript out of 46 genes found near proviruses. This frequency of dysregulation was similar to that of genes lacking nearby proviruses, so it may have been due to interclonal variation and/or measurement inaccuracies. While the number of integration sites examined in this paper is limited, our results suggest that integrated FV proviruses do not impact the expression of chromosomal genes in pluripotent human stem cells or their differentiated derivatives. This interpretation is consistent with previous reports that FV vectors have minimal genotoxicity, even when integrating near or within genes.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.

    Article  CAS  PubMed  Google Scholar 

  2. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest 2008; 118: 3143–3150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–409.

    Article  CAS  PubMed  Google Scholar 

  4. Krause D . Gene therapy for Wiskott–Aldrich syndrome: benefits and risks. The Hematologist 2011; 8: 10.

    Google Scholar 

  5. Modlich U, Bohne J, Schmidt M, von Kalle C, Knoss S, Schambach A et al. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood 2006; 108: 2545–2553.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotech 2006; 24: 687–696.

    Article  CAS  Google Scholar 

  7. Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E et al. Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther 2008; 16: 718–725.

    Article  CAS  PubMed  Google Scholar 

  8. Hargrove PW, Kepes S, Hanawa H, Obenauer JC, Pei D, Cheng C et al. Globin lentiviral vector insertions can perturb the expression of endogenous genes in [beta]-thalassemic hematopoietic cells. Mol Ther 2008; 16: 525–533.

    Article  CAS  PubMed  Google Scholar 

  9. Winkler T, Cantilena A, Métais J-Y, Xu X, Nguyen A-D, Borate B et al. No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells. Stem Cells 2010; 28: 687–694.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ronen K, Negre O, Roth S, Colomb C, Malani N, Denaro M et al. Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat [beta]-thalassemia. Mol Ther 2011; 19: 1273–1286.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Biasco L, Ambrosi A, Pellin D, Bartholomae C, Brigida I, Roncarolo MG et al. Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell. EMBO Mol Med 2011; 3: 89–101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Modlich U, Navarro S, Zychlinski D, Maetzig T, Knoess S, Brugman MH et al. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther 2009; 17: 1919–1928.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 2010; 467: 318–322.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Maruggi G, Porcellini S, Facchini G, Perna SK, Cattoglio C, Sartori D et al. Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design. Mol Ther 2009; 17: 851–856.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kane NM, Nowrouzi A, Mukherjee S, Blundell MP, Greig JA, Lee WK et al. Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. Mol Ther 2010; 18: 2139–2145.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Trobridge G, Josephson N, Vassilopoulos G, Mac J, Russell DW . Improved foamy virus vectors with minimal viral sequences. Mol Ther 2002; 6: 321–328.

    Article  CAS  PubMed  Google Scholar 

  17. Russell DW, Miller AD . Foamy virus vectors. J Virol 1996; 70: 217–222.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Moebes A, Enssle J, Bieniasz PD, Heinkelein M, Lindemann D, Bock M et al. Human foamy virus reverse transcription that occurs late in the viral replication cycle. J Virol 1997; 71: 7305–7311.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Yu SF, Sullivan MD, Linial ML . Evidence that the human foamy virus genome is DNA. J Virol 1999; 73: 1565–1572.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Trobridge GD, Miller DG, Jacobs MA, Allen JM, Kiem HP, Kaul R et al. Foamy virus vector integration sites in normal human cells. Proc Natl Acad Sci USA 2006; 103: 1498–1503.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hendrie PC, Huo Y, Stolitenko RB, Russell DW . A rapid and quantitative assay for measuring neighboring gene activation by vector proviruses. Mol Ther 2008; 16: 534–540.

    Article  CAS  PubMed  Google Scholar 

  22. Bauer TR, Allen JM, Hai M, Tuschong LM, Khan IF, Olson EM et al. Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors. Nat Med 2008; 14: 93–97.

    Article  CAS  PubMed  Google Scholar 

  23. Ohmine K, Li Y, Bauer TR, Hickstein DD, Russell DW . Tracking of specific integrant clones in dogs treated with foamy virus vectors. Hum Gene Ther 2011; 22: 217–224.

    Article  CAS  PubMed  Google Scholar 

  24. Deyle DR, Khan IF, Ren G, Wang PR, Kho J, Schwarze U et al. Normal collagen and bone production by gene-targeted human osteogenesis imperfecta iPSCs. Mol Ther 2012; 20: 204–213.

    Article  CAS  PubMed  Google Scholar 

  25. Tsukiyama T, Niwa O, Yokoro K . Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells. Mol Cell Biol 1989; 9: 4670–4676.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hwang NS, Varghese S, Lee HJ, Zhang Z, Ye Z, Bae J et al. In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proc Natl Acad Sci USA 2008; 105: 20641–20646.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G et al. Patterns of somatic mutation in human cancer genomes. Nature 2007; 446: 153–158.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Prickett TD, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR et al. Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 2009; 41: 1127–1132.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008; 455: 1069–1075.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Soung YH, Lee JW, Kim SY, Wang YP, Jo KH, Moon SW et al. Somatic mutations of the ERBB4 kinase domain in human cancers. Int J Cancer 2006; 118: 1426–1429.

    Article  CAS  PubMed  Google Scholar 

  31. Recchia A, Bonini C, Magnani Z, Urbinati F, Sartori D, Muraro S et al. Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells. Proc Natl Acad Sci USA 2006; 103: 1457–1462.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li CL, Xiong D, Stamatoyannopoulos G, Emery DW . Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator. Mol Ther 2009; 17: 716–724.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Arumugam PI, Higashimoto T, Urbinati F, Modlich U, Nestheide S, Xia P et al. Genotoxic potential of lineage-specific lentivirus vectors carrying the beta-globin locus control region. Mol Ther 2009; 17: 1929–1937.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Almarza D, Bussadori G, Navarro M, Mavilio F, Larcher F, Murillas R . Risk assessment in skin gene therapy: viral-cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors. Gene Ther 2011; 18: 674–681.

    Article  CAS  PubMed  Google Scholar 

  35. Du Y, Jenkins NA, Copeland NG . Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells. Blood 2005; 106: 3932–3939.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Moiani A, Paleari Y, Sartori D, Mezzadra R, Miccio A, Cattoglio C et al. Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts. J Clin Invest 2012; 122: 1653–1666.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Knight S, Zhang F, Mueller-Kuller U, Bokhoven M, Gupta A, Broughton T et al. Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing. J Virol 2012; 86: 9088–9095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chamberlain JR, Deyle DR, Schwarze U, Wang P, Hirata RK, Li Y et al. Gene targeting of mutant COL1A2 alleles in mesenchymal stem cells from individuals with osteogenesis imperfecta. Mol Ther 2008; 16: 187–193.

    Article  CAS  PubMed  Google Scholar 

  39. Tucker KL, Wang Y, Dausman J, Jaenisch R . A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res 1997; 25: 3745–3746.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 2009; 324: 797–801.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was funded by NIH grants AR48328, AR48328-09S1, DK55759, GM86497, HL53750 and AR53917.

Author information

Author notes

  1. D R Deyle

    Present address: 3Current address: Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA.,

Authors and Affiliations

  1. Departments of Medicine, University of Washington, Seattle, WA, USA

    D R Deyle, I F Khan, G Ren & D W Russell

  2. Departments of Biochemistry, University of Washington, Seattle, WA, USA

    D W Russell

Authors
  1. D R Deyle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I F Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D W Russell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D W Russell.

Ethics declarations

Competing interests

The authors declare no conflict of interests.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deyle, D., Khan, I., Ren, G. et al. Lack of genotoxicity due to foamy virus vector integration in human iPSCs. Gene Ther 20, 868–873 (2013). https://doi.org/10.1038/gt.2013.6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2013.6

Keywords

Search

Advanced search

Quick links