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Directed evolution of adeno-associated virus yields enhanced gene delivery vectors

Nature Biotechnology volume 24, pages 198–204 (2006)Cite this article

Abstract

Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties.

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Figure 1: A forward genetics approach to creating 'designer' rAAV vectors.
Figure 2: Heparin-binding characteristics of wild-type AAV versus the viral library.
Figure 3: Selection of antibody-escape mutants from generated viral capsid libraries.
Figure 4: Neutralization profiles of antibody-evading mutants.
Figure 5: rAAV-Epo was produced with wild-type, r2.4 and r2.15 capsids.
Figure 6: Mutation profile of neutralizing antibody-evading mutants.

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Acknowledgements

We thank Wilson Mok, Diana Chai, Kirti Magudia, and Robert Teachnor for technical assistance. This work was funded by National Science Foundation Graduate Fellowships (to N.M. and J.K.), Project A.L.S. funding (to B.K.), and Whitaker Foundation and ALS Association funding (to D.S.).

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Authors and Affiliations

  1. The Department of Chemical Engineering and The Helen Wills Neuroscience Institute, The University of California, Berkeley, 94720-1462, California, USA

    Narendra Maheshri, James T Koerber & David V Schaffer

  2. Department of Gene Therapy and Division of Molecular Medicine Columbus Children's Research Institute and The Ohio State University, Columbus, 43205, Ohio, USA

    Brian K Kaspar

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  1. Narendra Maheshri
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Correspondence to David V Schaffer.

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Supplementary information

Supplementary Fig. 1

Heparin binding characterization of individual clones. (PDF 17 kb)

Supplementary Fig. 2

Heparin chromatograms of antibody evading mutants. (PDF 16 kb)

Supplementary Fig. 3

Neutralization profile of selected AbE mutants on a second rabbit serum source. (PDF 12 kb)

Supplementary Fig. 4

Neutralization profile of heat-inactivated serum. (PDF 15 kb)

Supplementary Table 1

Titers of antibody escape mutants. (PDF 7 kb)

Supplementary Note

The neutralization assay. (PDF 16 kb)

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Maheshri, N., Koerber, J., Kaspar, B. et al. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol 24, 198–204 (2006). https://doi.org/10.1038/nbt1182

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