Episomal Transgene Expression in Pluripotent Stem Cells

  • Michele M. P. Lufino
  • Anna R. Popplestone
  • Sally A. Cowley
  • Pauline A. H. Edser
  • William S. James
  • Richard Wade-Martins
Part of the Methods in Molecular Biology book series (MIMB, volume 767)


Herpes simplex type 1 (HSV-1) amplicon vectors possess a number of features that make them excellent vectors for the delivery of transgenes into stem cells. HSV-1 amplicon vectors are capable of efficiently transducing both dividing and nondividing cells and since the virus is quite large, 152 kb, it is of sufficient size to allow for incorporation of entire genomic DNA loci with native promoters. HSV-1 amplicon vectors can also be used to incorporate and deliver to cells a variety of sequences that allow extrachromosomal retention. These elements offer advantages over integrating vectors as they avoid transgene silencing and insertional mutagenesis. The construction of amplicon vectors carrying extrachromosomal retention elements, their packaging into HSV-1 viral particles, and the use of HSV-1 amplicons for stem cell transduction will be described.

Key words

HSV-1 amplicon iBAC Extrachromosomal vector Stem cells Gene expression vector 



This work was supported by the Parkinson’s UK Monument Trust Discovery Award; the Friedreich’s Ataxia Research Alliance, Ataxia UK and the National Ataxia Foundation; the Medical Research Council and the Biotechnology and Biological Sciences Research Council. M.M.P.L. is an Ataxia UK Research Fellow, A.R.P. is a Medical Research Council student and S.A.C. is a Wellcome Trust Research Fellow.


  1. 1.
    Spaete, R. R., and Frenkel, N. (1982) The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector Cell 30, 295–304.PubMedCrossRefGoogle Scholar
  2. 2.
    Fraefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, A. I. (1996) Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells J Virol 70, 7190–7.PubMedGoogle Scholar
  3. 3.
    Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X. O., and Chiocca, E. A. (2001) Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome Mol Ther 3, 591–601.PubMedCrossRefGoogle Scholar
  4. 4.
    Wade-Martins, R., Smith, E. R., Tyminski, E., Chiocca, E. A., and Saeki, Y. (2001) An infectious transfer and expression system for genomic DNA loci in human and mouse cells Nat Biotechnol 19, 1067–70.PubMedCrossRefGoogle Scholar
  5. 5.
    Wang, S., and Vos, J. M. (1996) A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo J Virol 70, 8422–30.PubMedGoogle Scholar
  6. 6.
    Piechaczek, C., Fetzer, C., Baiker, A., Bode, J., and Lipps, H. J. (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells Nucleic Acids Res 27, 426–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Lufino, M. M., Manservigi, R., and Wade-Martins, R. (2007) An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency Nucleic Acids Res 35, e98.PubMedCrossRefGoogle Scholar
  8. 8.
    Moralli, D., Simpson, K. M., Wade-Martins, R., and Monaco, Z. L. (2006) A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors EMBO Rep 7, 911–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat, N., McIntyre, E., Radford, I., Villeval, J. L., Fraser, C. C., Cavazzana-Calvo, M., and Fischer, A. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency N Engl J Med 348, 255–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M. P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C. S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J. I., de Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias, A., Stoppa-Lyonnet, D., Romana, S., Radford-Weiss, I., Gross, F., Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva, L. E., Wissler, M., Prinz, C., Rabbitts, T. H., Le Deist, F., Fischer, A., and Cavazzana-Calvo, M. (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Science 302, 415–9.Google Scholar
  11. 11.
    Hibbitt, O. C., Harbottle, R. P., Waddington, S. N., Bursill, C. A., Coutelle, C., Channon, K. M., and Wade-Martins, R. (2007) Delivery and long-term expression of a 135 kb LDLR genomic DNA locus in vivo by hydrodynamic tail vein injection J Gene Med 9, 488–97.PubMedCrossRefGoogle Scholar
  12. 12.
    Wade-Martins, R., Saeki, Y., and Chiocca, E. A. (2003) Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells Mol Ther 7, 604–12.PubMedCrossRefGoogle Scholar
  13. 13.
    Bowers, W. J., and Federoff, H. J. (2006) Herpes simplex virus type 1-derived amplicon vectors, Gene Transfer: Delivery and Expression of DNA and RNA, A Laboratory Manual, pg. 227–254, Cold Spring Harbor Press, Cold Spring Harbor, NY.Google Scholar
  14. 14.
    Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Nishikawa, S., Muguruma, K., and Sasai, Y. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells Nat Biotechnol 25, 681–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Ludwig, T. E., Bergendahl, V., Levenstein, M. E., Yu, J., Probasco, M. D., and Thomson, J. A. (2006) Feeder-independent culture of human embryonic stem cells Nat Methods 3, 637–46.PubMedCrossRefGoogle Scholar
  16. 16.
    Lufino, M. M., Edser, P. A., and Wade-Martins, R. (2008) Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression Mol Ther 16, 1525–38.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Michele M. P. Lufino
    • 1
  • Anna R. Popplestone
    • 1
  • Sally A. Cowley
    • 2
  • Pauline A. H. Edser
    • 1
  • William S. James
    • 2
  • Richard Wade-Martins
    • 1
  1. 1.Molecular Neurodegeneration and Gene Therapy Research Group, Department of Physiology, Anatomy and GeneticsUniversity of OxfordOxfordUK
  2. 2.Sir William Dunn School of PathologyUniversity of OxfordOxfordUK

Personalised recommendations