Adeno-associated virus vectors can be efficiently produced without helper virus (original) (raw)

Gene Therapy volume 5, pages 938–945 (1998)Cite this article

Abstract

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-Mr, E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-Mr, E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

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Authors and Affiliations

  1. Avigen, Inc., Alameda, CA, USA
    T Matsushita, S Elliger, C Elliger, G Podsakoff, GJ Kurtzman & P Colosi
  2. University of Southern California School of Medicine, Los Angeles, CA, USA
    T Matsushita
  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
    L Villarreal
  4. Department of Urology, University of Southern California School of Medicine, Los Angeles, CA, USA
    Y Iwaki
  5. Childrens Hospital Oakland Research Institute, 747 52nd Street, Oakland, 95609, CA
    S Elliger
  6. California Academy of Sciences, Golden Gate Park, San Francisco, 94118, CA, USA
    C Elliger

Authors

  1. T Matsushita
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  2. S Elliger
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  3. C Elliger
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  4. G Podsakoff
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  5. L Villarreal
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  6. GJ Kurtzman
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  7. Y Iwaki
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  8. P Colosi
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Matsushita, T., Elliger, S., Elliger, C. et al. Adeno-associated virus vectors can be efficiently produced without helper virus.Gene Ther 5, 938–945 (1998). https://doi.org/10.1038/sj.gt.3300680

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