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Production and characterization of adeno-associated viral vectors

Nature Protocols volume 1, pages 1412–1428 (2006)Cite this article

Abstract

The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These methods have paved the way to a better understanding of the vector and, ultimately, its use in clinical applications. This review provides an up-to-date, detailed description of essential methods such as production, purification and titering and their application to characterize current AAV vectors for preclinical and clinical use.

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Figure 1: Schematic diagram summarizing the production and purification of rAAV.
Figure 2: Illustration of iodixanol gradients before and after centrifugation.
Figure 3
Figure 4: Dot blot of five rAAV preparations.
Figure 5: Graphical representation of the suggested portion of the transgene plasmid to be used as the template of the probe for dot-blot analysis.
Figure 6: An example of standard curve generated using Microsoft Excel.
Figure 7: Graphical representation of the difference between conventional ssAAV and scAAV vectors.
Figure 8: Transmission electron microscopy of rAAV.
Figure 9: Southern blot of CsCl gradient fractions of a scAAV preparation.
Figure 10: Southern blot of vector DNA isolated from HEK-293 cells infected with rAAV containing genomes of sizes 4,675 nucleotides (nt), 5,302 nt and 6,019 nt in the presence of transfected adenovirus helper (XX680) and AAV2 helper (pHelper).
Figure 11: Picture of the infectious center assay apparatus.
Figure 12: Infectious center assay of a purified rAAV preparation.
Figure 13: HeLa cells were infected by serially diluted scAAV (0.1 IU/cell–20 IU/cell).

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Acknowledgements

We thank A. Asokan, M. Hirsh, C. Li and Z. Wu for helpful discussions and critical reading of the manuscript. This work was supported in part by US National Institutes of Health grants GM059299, HL051818 and HL066973 (awarded to R.J.S.).

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Author notes

  1. Joshua C Grieger

    Present address: AlphaVax, Inc., 2 Triangle Drive, Research Triangle Park, North Carolina, 27709, USA

  2. Vivian W Choi

    Present address: CBR Institute for Biomedical Research, Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA

  3. Joshua C Grieger and Vivian W Choi: These authors contributed equally to this work.

Authors and Affiliations

  1. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Joshua C Grieger & R Jude Samulski

  2. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Joshua C Grieger, Vivian W Choi & R Jude Samulski

  3. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Vivian W Choi & R Jude Samulski

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  1. Joshua C Grieger
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Correspondence to R Jude Samulski.

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Grieger, J., Choi, V. & Samulski, R. Production and characterization of adeno-associated viral vectors. Nat Protoc 1, 1412–1428 (2006). https://doi.org/10.1038/nprot.2006.207

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