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.

  • Research Article
  • Published:

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation

Gene Therapy volume 11, pages 711–721 (2004)Cite this article

Abstract

Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six- to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay – referred to as repeat-anchored integration capture (RAIC) PCR – that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

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
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Herweijer H, Wolff JA . Progress and prospects: naked DNA gene transfer and therapy. Gene Therapy 2003; 10: 453–458.

    Article  CAS  Google Scholar 

  2. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA . DNA vaccines. Annu Rev Immunol 1997; 15: 617–648.

    Article  CAS  Google Scholar 

  3. Nishikawa M, Huang L . Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 2001; 12: 861–870.

    Article  CAS  Google Scholar 

  4. Bigey P, Bureau MF, Scherman D . In vivo plasmid DNA electrotransfer. Curr Opin Biotechnol 2002; 13: 443–447.

    Article  CAS  Google Scholar 

  5. Gehl J . Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 2003; 177: 437–447.

    Article  CAS  Google Scholar 

  6. Trezise AEO . In vivo DNA electrotransfer. DNA Cell Biol 2002; 21: 869–877.

    Article  CAS  Google Scholar 

  7. Fattori E, La-Monica N, Ciliberto G, Toniatti C . Electro-gene-transfer: a new approach for muscle gene delivery. Somat Cell Mol Genet 2002; 27: 75–83.

    Article  CAS  Google Scholar 

  8. Nichols WW, Ledwith BJ, Manam SV, Troilo PJ . Potential DNA vaccine integration into host cell genome. Ann NY Acad Sci 1995; 772: 30–39.

    Article  CAS  Google Scholar 

  9. Ledwith BJ et al. Plasmid DNA vaccines: assay for integration into host genomic DNA. Dev Biol (Basel) 2000; 104: 33–43.

    CAS  Google Scholar 

  10. Ledwith BJ et al. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology 2000; 43: 258–272.

    Article  CAS  Google Scholar 

  11. Manam S et al. Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirology 2000; 43: 273–281.

    Article  CAS  Google Scholar 

  12. Martin T et al. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther 1999; 10: 759–768.

    Article  CAS  Google Scholar 

  13. Rizzuto G et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 1999; 96: 6417–6422.

    Article  CAS  Google Scholar 

  14. Minami M, Poussin K, Brechot C, Paterlini P . A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome. Genomics 1995; 29: 403–408.

    Article  CAS  Google Scholar 

  15. Sorensen AB, Duch M, Jorgensen P, Pedersen FS . Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method. J Virol 1993; 67: 7118–7124.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Longo MC, Berninger MS, Hartley JL . Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990; 93: 125–128.

    Article  CAS  Google Scholar 

  17. Krayev AS et al. The nucleotide sequence of the ubiquitous repetitive DNA sequence B1 complementary to the most abundant class of mouse fold-back RNA. Nucleic Acids Res 1980; 8: 1201–1215.

    Article  CAS  Google Scholar 

  18. Waterston RH, et al. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature 2002; 420: 520–562.

    Article  CAS  Google Scholar 

  19. Zhang G et al. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001; 12: 427–438.

    Article  CAS  Google Scholar 

  20. Hui EK-W, Wang P-C, Lu SJ . Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants. Cell Mol Life Sci 1998; 54: 1403–1411.

    Article  CAS  Google Scholar 

  21. Jin YF, Ishibashi T, Nomoto A, Masuda M . Isolation and analysis of retroviral integration targets by solo long terminal repeat inverse PCR. J Virol 2002; 76: 5540–5547.

    Article  CAS  Google Scholar 

  22. Würtele H, Little KCE, Chartrand P . Illegitimate DNA integration in mammalian cells. Gene Therapy 2003; 10: 1791–1799.

    Article  Google Scholar 

  23. Merrihew RV et al. High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome. Mol Cell Biol 1996; 16: 10–18.

    Article  CAS  Google Scholar 

  24. Bode J et al. Fatal connections: when DNA ends meet on the nuclear matrix. J Cell Biochem 2000; Suppl 35: 3–22.

    Article  Google Scholar 

  25. Wu X, Li Y, Crise B, Burgess SM . Transcription start regions in the human genome are favored targets for MLV integration. Science 2003; 300: 1749–1751.

    Article  CAS  Google Scholar 

  26. Nakai H et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet 2003; 34: 297–302.

    Article  CAS  Google Scholar 

  27. Zhang L, Nolan E, Kreitschitz S, Rabussay DP . Enhanced delivery of naked DNA to the skin by non-invasive in vivo electroporation. Biochim Biophy Acta 2002; 1572: 1–9.

    Article  CAS  Google Scholar 

  28. Gilbert RA, Jaroszeski MJ, Heller R . Novel electrode designs for electrochemotherapy. Biochim Biophys Acta 1997; 1334: 9–14.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Carolann Beare, Steven Hall, Donna Lynch, and Brenda Givler for assistance with the antemortem portion of the study, and Yuan Liu for assistance with analysis of the integration sites using Bioinformatics. We also thank Warren Nichols for his continued interest and ever insightful discussions.

Author information

Authors and Affiliations

  1. Department of Biologics Safety Assessment, Merck Research Laboratories, West Point, PA, USA

    Z Wang, P J Troilo, X Wang, T G Griffiths II, S J Pacchione, A B Barnum, L B Harper, C J Pauley, Z Niu, L Denisova, T T Follmer & B J Ledwith

  2. Istituto Di Ricerche Di Biologia Molecolare, 00040 Pomezie, Rome, Italy

    G Rizzuto, G Ciliberto, E Fattori & N L Monica

  3. Department of Regulatory Affairs, Merck Research Laboratories, Blue Bell, PA, USA

    S Manam

Authors
  1. Z Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P J Troilo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. X Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T G Griffiths II
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S J Pacchione
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A B Barnum
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. L B Harper
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. C J Pauley
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Z Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. L Denisova
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. T T Follmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. G Rizzuto
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. G Ciliberto
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. E Fattori
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. N L Monica
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. S Manam
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. B J Ledwith
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Z., Troilo, P., Wang, X. et al. Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther 11, 711–721 (2004). https://doi.org/10.1038/sj.gt.3302213

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302213

Keywords

This article is cited by

Search

Advanced search

Quick links