Directed Evolution of Adeno-Associated Virus (AAV) as Vector for Muscle Gene Therapy

  • Lin Yang
  • Juan Li
  • Xiao Xiao
Part of the Methods in Molecular Biology book series (MIMB, volume 709)


Adeno-associated virus (AAV) is emerging as a vector of choice for muscle gene therapy because of its effective and stable transduction in striated muscles. AAV naturally evolve into multiple serotypes with diverse capsid gene sequences that are apparently the determinants of their tissue tropism and infectivity. Certain AAV serotypes show robust gene transfer upon direct intramuscular injection, while others are effective in crossing the endothelial barrier to reach muscle when delivered intravenously. Muscular dystrophy gene therapy requires efficient body-wide muscle gene transfer. However, preferential liver transduction by nearly all natural AAV serotypes could be an undesirable feature for muscle-directed applications, especially by means of systemic gene delivery. Here we describe a method of in vitro evolution and in vivo selection of AAV capsids that target striated muscles and detarget the liver. Using DNA shuffling technology, we have generated a capsid gene library by in vitro scrambling and shuffling the capsid genes of natural AAV1 to AAV9. To minimize the bias and limitation of in vitro screening on culture cells, we performed direct in vivo panning in adult mice after intravenous injection of the shuffled capsid library that packaged their own coding sequences. The AAV variants enriched in the heart and muscle are retrieved by capsid gene PCR and subsequently characterized for their tissue tropisms. This directed evolution and in vivo selection method should be useful in generating novel gene therapy vectors for muscle and heart and other tissues.

Key words

AAV Transduction DNA shuffling In vivo selection Tissue tropism 



This work was supported by National Institutes of Health grants AR 45967 and AR 50595.


  1. 1.
    Xiao, X., Li, J., and Samulski, R. J. (1996) Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70, 8098–8108.PubMedGoogle Scholar
  2. 2.
    Duan, D., Sharma, P., Yang, J., Yue, Y., Dudus, L., Zhang, Y., et al. (1998) Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol 72, 8568–8577.PubMedGoogle Scholar
  3. 3.
    Kay, M. A., Manno, C. S., Ragni, M. V., Larson, P. J., Couto, L. B., McClelland, A., et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24, 257–261.PubMedCrossRefGoogle Scholar
  4. 4.
    Wang, Z., Zhu, T., Qiao, C. P., Zhou, L. Q., Wang, B., Zhang, J., et al. (2005) Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotech 23, 321–328.CrossRefGoogle Scholar
  5. 5.
    Inagaki, K., Fuess, S., Storm, T. A., Gibson, G. A., Mctiernan, C. F., Kay, M. A., et al. (2006) Robust systemic transduction with AAV9 vectors in mice: Efficient global cardiac gene transfer superior to that of AAV8. Mol Ther 14, 45–53.PubMedCrossRefGoogle Scholar
  6. 6.
    Stemmer, W. P. (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91, 10747–10751.PubMedCrossRefGoogle Scholar
  7. 7.
    Yang, L., Jiang, J., Drouin, L. M., Agbandje-McKenna, M., Chen, C., Qiao, C., Pu, D., Hu, X., Wang, D. Z., Li, J., Xiao, X. (2009) A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl Acad Sci USA 106, 3946–3951.PubMedCrossRefGoogle Scholar
  8. 8.
    Gao, G., Alvira, M. R., Somanathan, S., Lu, Y., Vandenberghe, L. H., Rux, J. J., Calcedo, R., et al. (2003) Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc Natl Acad Sci USA 100, 6081–6086.PubMedCrossRefGoogle Scholar
  9. 9.
    Soong, N. W., Nomura, L., Pekrun, K., Reed, M., Sheppard, L., Dawes, G., et al. (2000) Molecular breeding of viruses. Nat Genet 25, 436–439.PubMedCrossRefGoogle Scholar
  10. 10.
    Powell, S. K., Kaloss, M. A., Pinkstaff, A., McKee, R., Burimski, I., Pensiero, M., et al. (2000) Breeding of retroviruses by DNA shuffling for improved stability and processing yields. Nat Biotech 18, 1279–1282.CrossRefGoogle Scholar
  11. 11.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Third Edition New York.Google Scholar
  12. 12.
    Müller, O. J., Kaul, F., Weitzman, M.D., Pasqualini, R., Arap, W., Kleinschmidt, J.A., et al. (2003) Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotech 21, 1040–1046.CrossRefGoogle Scholar
  13. 13.
    Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Lin Yang
    • 1
  • Juan Li
    • 1
  • Xiao Xiao
    • 1
  1. 1.Division of Molecular Pharmaceutics, Eshelman School of PharmacyUniversity of North Carolina at Chapel HillChapel HillUSA

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