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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

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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.

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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.

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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.

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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

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  1. Oliver J Müller
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Correspondence to Jürgen A Kleinschmidt.

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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)

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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

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