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.

  • Article
  • Published:

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Nature Biotechnology volume 21, pages 1040–1046 (2003)Cite this article

This article has been updated

Abstract

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type–specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Design and characteristics of the random peptide AAV display library construct.
Figure 2: Two-step system for production of a random AAV display peptide library from library plasmids.
Figure 3: PCR amplification of virus DNA comprising the modified cap gene section isolated at different stages of selection.
Figure 4: Replication and transduction efficiencies of selected AAV clones displaying a targeting motif.

Similar content being viewed by others

Change history

  • 17 August 2003

    HTML updated

References

  1. Somia, N. & Verma, I.M. Gene therapy: trials and tribulations. Nat. Rev. Genet. 1, 91–99 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. McCormick, F. Cancer gene therapy: fringe or cutting edge? Nat. Rev. Cancer 1, 130–141 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Marshall, E. Gene therapy a subject in leukemia-like disease. Science 298, 34–35 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Wickham, T.J. Targeting adenovirus. Gene Ther. 7, 110–114 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Trepel, M., Arap, W. & Pasqualini, R. In vivo phage display and vascular heterogeneity: implications for targeted medicine. Curr. Opin. Chem. Biol. 6, 399–404 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Larochelle, A., Peng, K.W. & Russell, S.J. Lentiviral vector targeting. Curr. Top. Microbiol. 261, 143–163 (2002).

    CAS  Google Scholar 

  7. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366 (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Arap, W. et al. Steps toward mapping the human vasculature by phage display. Nat. Med. 8, 121–127 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Barry, M.A., Dower, W.J. & Johnston, S.A. Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat. Med. 2, 299–305 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Laakkonen, P., Porkka, K., Hoffman, J.A. & Ruoslahti, E. A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat. Med. 8, 751–755 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Trepel, M., Arap, W. & Pasqualini, R. Modulation of the immune response by systemic targeting of antigens to lymph nodes. Cancer Res. 61, 8110–8112 (2001).

    CAS  PubMed  Google Scholar 

  12. Ellerby, H.M. et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5, 1032–1038 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Curnis, F. et al. Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nat. Biotechnol. 18, 1185–1190 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Bartlett, J.S., Kleinschmidt, J., Boucher, R.C. & Samulski, R.J. Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab 'gamma)(2) antibody. Nat. Biotechnol. 17, 181–186 (1999).

    Article  CAS  PubMed  Google Scholar 

  15. Reynolds, P.N. et al. A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo. Mol. Ther. 2, 562–578 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Nettelbeck, D.M. et al. Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105). Mol. Ther. 3, 882–891 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Ponnazhagan, S., Mahendra, G., Kumar, S., Thompson, J. & Castillas, J.M.J. Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands. J. Virol. 76, 12900–12907 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Trepel, M., Grifman, M., Weitzman, M.D. & Pasqualini, R. Molecular adaptors for vascular-targeted adenoviral gene delivery. Hum. Gene Ther. 11, 1971–1981 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Nicklin, S.A. et al. Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells. Mol. Ther. 4, 174–181 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Grifman, M. et al. Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids. Mol. Ther. 3, 964–975 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Monahan, P.E. & Samulski, R.J. AAV vectors: is clinical success on the horizon? Gene Ther. 7, 24–30 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Summerford, C. & Samulski, R.J. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72, 1438–1445 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Summerford, C., Bartlett, J.S. & Samulski, R.J. alpha V beta 5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat. Med. 5, 78–82 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Qing, K. et al. Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat. Med. 5, 71–77 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Samulski, R.J., Chang, L.S. & Shenk, T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J. Virol. 61, 3096–3101 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Becerra, S.P., Koczot, F., Fabisch, P. & Rose, J.A. Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. J. Virol. 62, 2745–2754 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Opie, S.R., Warrington, K.H., Agbandje-McKenna, M., Zolotukhin, S. & Muzyczka, N. Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J. Virol. 77, 6995–7006 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kern, A. et al. Identification of a heparin binding motif on AAV-2 capsids. J. Virol. (in the press).

  29. Wobus, C.E. et al. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: Epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J. Virol. 74, 9281–9293 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Moskalenko, M. et al. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure. J. Virol. 74, 1761–1766 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Girod, A. et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat. Med. 5, 1052–1056 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Rabinowitz, J.E., Xiao, W.D. & Samulski, R.J. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus. Virology 265, 274–285 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Wu, P. et al. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism. J. Virol. 74, 8635–8647 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shi, W.F., Arnold, G.S. & Bartlett, J.S. Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors. Hum. Gene Ther. 12, 1697–1711 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Xie, Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc. Nat. Acad. Sci. USA 99, 10405–10410 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li, J., Samulski, R.J. & Xiao, X. Role for highly regulated rep gene expression in adeno-associated virus vector production. J. Virol. 71, 5236–5243 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Pajusola, K. et al. Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells. J. Virol. 76, 11530–11540 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. St. Croix, B. et al. Genes expressed in human tumor endothelium. Science 289, 1197–1202 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Wu, Q. & Maniatis, T. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97, 779–790 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Hauswirth, W.W., Lewin, A.S., Zolotukhin, S. & Muzyczka, N. Production and purification of recombinant adeno-associated virus. Meth. Enzymol. 316, 743–761 (2000).

    Article  CAS  Google Scholar 

  41. Heilbronn, R., Burkle, A., Stephan, S. & zur Hausen, H. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification. J. Virol. 64, 3012–3018 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Xiao, X., Li, J. & Samulski, R.J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72, 2224–2232 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Dubielzig, R., King, J.A., Weger, S., Kern, A. & Kleinschmidt, J.A. Adeno-associated virus type 2 protein interactions: formation of pre-encapsidation complexes. J. Virol. 73, 8989–8998 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Grimm, D. et al. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther. 6, 1322–1330 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Weger, S., Wistuba, A., Grimm, D. & Kleinschmidt, J.A. Control of adeno-associated virus type 2 cap gene expression: relative influence of helper virus, terminal repeats, and Rep proteins. J. Virol. 71, 8437–8447 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Zolotukhin, S., Potter, M., Hauswirth, W.W., Guy, J. & Muzyczka, N. A “humanized” green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70, 4646–4654 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Hugo A. Katus, Florian Otto, Roland Mertelsmann, Christoph Peters, Christoph Leder and Mirta Grifman for helpful discussions and critical reading of the manuscript, Marie Follo and Andreas Hunziker for DNA sequencing, Lothar Pilz for statistical analyses, Elisete de Lima-Hahn, Katrin Schlenker, Kristin Schmidt and Petra Poberschin for technical assistance. This work was supported by the US Department of Defense Grant DAMD 17-01-1-0003 to M.T. M.T. was also supported in part by the Susan G. Komen Breast Cancer Foundation, O.M. is supported by the Deutsche Forschungsgemeinschaft (MU 1654/2-1) and R.P. is supported by the National Cancer Institute (CA 99106). The 293T cells were used with kind permission of David Baltimore, and the Kasumi cells were a gift from Michael Lübbert. We thank the Laboratoire de Thérapie Génique, Nantes, France, for providing wild-type Ad5, and Jude Samulski for the pSub201, pXX2 and pXX6 plasmids.

Author information

Author notes

  1. Oliver J Müller, Felix Kaul, Jürgen A Kleinschmidt and Martin Trepel: These authors contributed equally to this work.

Authors and Affiliations

  1. Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, Heidelberg, D-69120, Germany

    Oliver J Müller & Jürgen A Kleinschmidt

  2. Department of Hematology and Oncology, University of Freiburg Medical Center, Hugstetter Strasse 55, Freiburg, D-79106, Germany

    Felix Kaul & Martin Trepel

  3. University of Freiburg Medical Center, Institute for Molecular Medicine and Cell Research, Hugstetter Strasse 55, Freiburg, D-79106, Germany

    Martin Trepel

  4. The Salk Institute, Laboratory of Genetics, P.O. Box 85800, La Jolla, 92183-5800, California, USA

    Matthew D Weitzman

  5. The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 427, Houston, 77030-4095, Texas, USA

    Renata Pasqualini & Wadih Arap

Authors
  1. Oliver J Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Felix Kaul
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew D Weitzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Renata Pasqualini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wadih Arap
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jürgen A Kleinschmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Martin Trepel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jürgen A Kleinschmidt.

Ethics declarations

Competing interests

The University of Texas and its researchers W.A. and R.P. have equity in NTTX Biotechnology, which is subject to certain restrictions under university policy. The University of Texas manages the terms of these arrangements in accordance with conflict-of-interest policies.

Supplementary information

Supplementary Table 1 (PDF 16 kb)

Supplementary Table 2 (PDF 18 kb)

Supplementary Figure 1

Note: The version of Supplementary Figure 1 originally published online contained an error in the figure legend. The last sentence of the legend should read: …the statistical analysis was performed using a χ2-test showing that the hypothesis of independence can be refused (α=0.05; p=00032). (PDF 188 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Müller, O., Kaul, F., Weitzman, M. et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol 21, 1040–1046 (2003). https://doi.org/10.1038/nbt856

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt856

This article is cited by

Search

Advanced 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