[go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Srivastava, A., Lusby, E.W. & Berns, K.I. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J. Virol. 45, 555–564 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Chiorini, J.A., Kim, F., Yang, L. & Kotin, R.M. Cloning and characterization of adeno-associated virus type 5. J. Virol. 73, 1309–1319 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Davidson, B.L. et al. Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc. Natl. Acad. Sci. USA 97, 3428–3432 (2000).

    Article  CAS  Google Scholar 

  4. Gao, G.P. et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99, 11854–11859 (2002).

    Article  CAS  Google Scholar 

  5. Gao, G. et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc. Natl. Acad. Sci. USA 100, 6081–6086 (2003).

    Article  CAS  Google Scholar 

  6. Kay, M.A. et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24, 257–261 (2000).

    Article  CAS  Google Scholar 

  7. Manno, C.S. et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101, 2963–2972 (2003).

    Article  CAS  Google Scholar 

  8. Moss, R.B. et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 125, 509–521 (2004).

    Article  Google Scholar 

  9. Samulski, R.J., Chang, L.S. & Shenk, T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 63, 3822–3828 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Berns, K.I. & Linden, R.M. The cryptic life style of adeno-associated virus. Bioessays 17, 237–245 (1995).

    Article  CAS  Google Scholar 

  11. Xiao, X., Li, J. & Samulski, R.J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol. 70, 8098–8108 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Halbert, C.L., Rutledge, E.A., Allen, J.M., Russell, D.W. & Miller, A.D. Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J. Virol. 74, 1524–1532 (2000).

    Article  CAS  Google Scholar 

  13. Koeberl, D.D., Alexander, I.E., Halbert, C.L., Russell, D.W. & Miller, A.D. Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors. Proc. Natl. Acad. Sci. USA 94, 1426–1431 (1997).

    Article  CAS  Google Scholar 

  14. Snyder, R.O. et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet. 16, 270–276 (1997).

    Article  CAS  Google Scholar 

  15. McCown, T.J., Xiao, X., Li, J., Breese, G.R. & Samulski, R.J. Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res. 713, 99–107 (1996).

    Article  CAS  Google Scholar 

  16. Lai, K., Kaspar, B.K., Gage, F.H. & Schaffer, D.V. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 6, 21–27 (2003).

    Article  CAS  Google Scholar 

  17. Erles, K., Sebokova, P. & Schlehofer, J.R. Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV). J. Med. Virol. 59, 406–411 (1999).

    Article  CAS  Google Scholar 

  18. Moskalenko, M. et al. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure. J. Virol. 74, 1761–1766 (2000).

    Article  CAS  Google Scholar 

  19. Wobus, C.E. et al. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J. Virol. 74, 9281–9293 (2000).

    Article  CAS  Google Scholar 

  20. Sun, J.Y., Anand-Jawa, V., Chatterjee, S. & Wong, K.K. Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther. 10, 964–976 (2003).

    Article  CAS  Google Scholar 

  21. Peden, C.S., Burger, C., Muzyczka, N. & Mandel, R.J. Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain. J. Virol. 78, 6344–6359 (2004).

    Article  CAS  Google Scholar 

  22. Nguyen, J.B., Sanchez-Pernaute, R., Cunningham, J. & Bankiewicz, K.S. Convection-enhanced delivery of AAV-2 combined with heparin increases TK gene transfer in the rat brain. Neuroreport 12, 1961–1964 (2001).

    Article  CAS  Google Scholar 

  23. Smith-Arica, J.R. et al. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells 5, 51–62 (2003).

    Article  CAS  Google Scholar 

  24. Hughes, S.M., Moussavi-Harami, F., Sauter, S.L. & Davidson, B.L. Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol. Ther. 5, 16–24 (2002).

    Article  CAS  Google Scholar 

  25. Dong, J.Y., Fan, P.D. & Frizzell, R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther. 7, 2101–2112 (1996).

    Article  CAS  Google Scholar 

  26. Hermonat, P.L., Labow, M.A., Wright, R., Berns, K.I. & Muzyczka, N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J. Virol. 51, 329–339 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Rabinowitz, J.E., Xiao, W. & Samulski, R.J. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus. Virology 265, 274–285 (1999).

    Article  CAS  Google Scholar 

  28. Girod, A. et al. Genetic capsid modifications allow efficient re-targeting of adeno- associated virus type 2. Nat Med. [published erratum appears in Nat. Med. 1999 Dec;5(12):1438] 5, 1052–1056 (1999).

    Article  CAS  Google Scholar 

  29. Shi, W., Arnold, G.S. & Bartlett, J.S. Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors. Hum. Gene Ther. 12, 1697–1711 (2001).

    Article  CAS  Google Scholar 

  30. Müller, O.J. et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat. Biotechnol. 21, 1040–1046 (2003).

    Article  Google Scholar 

  31. Perabo, L. et al. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display. Mol. Ther. 8, 151–157 (2003).

    Article  CAS  Google Scholar 

  32. Opie, S.R., Warrington, Jr., K.H., Jr., Agbandje-McKenna, M, Zolotukhin, S. & Muzyczka, N. Identification of amino acid residues in the capsid proteins of adeno- associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J. Virol. 77, 6995–7006 (2003).

    Article  CAS  Google Scholar 

  33. Walters, R.W. et al. Structure of adeno-associated virus serotype 5. J. Virol. 78, 3361–3371 (2004).

    Article  CAS  Google Scholar 

  34. Xie, Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc. Natl. Acad. Sci. USA 99, 10405–10410 (2002).

    Article  CAS  Google Scholar 

  35. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    Article  CAS  Google Scholar 

  36. May, O., Nguyen, P.T. & Arnold, F.H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat. Biotechnol. 18, 317–320 (2000).

    Article  CAS  Google Scholar 

  37. Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    Article  CAS  Google Scholar 

  38. Daugherty, P.S., Chen, G., Iverson, B.L. & Georgiou, G. Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proc. Natl. Acad. Sci. USA 97, 2029–2034 (2000).

    Article  CAS  Google Scholar 

  39. Soong, N.W. et al. Molecular breeding of viruses. Nat. Genet. 25, 436–439 (2000).

    Article  CAS  Google Scholar 

  40. Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).

    Article  CAS  Google Scholar 

  41. Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999).

    Article  CAS  Google Scholar 

  42. Rabinowitz, J.E. et al. Cross-Packaging of a Single Adeno-Associated Virus (AAV) Type 2 Vector Genome into Multiple AAV Serotypes Enables Transduction with Broad Specificity. J. Virol. 76, 791–801 (2002).

    Article  CAS  Google Scholar 

  43. Summerford, C. & Samulski, R.J. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72, 1438–1445 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Huttner, N.A. et al. Genetic modification of the adeno-associated virus type 2 capsid reduce the affinity and the neutralizating effects of human serum antibodies. Gene Ther. 10, 2139–2147 (2003).

    Article  CAS  Google Scholar 

  45. Gao, G. et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J. Virol. 78, 6381–6388 (2004).

    Article  CAS  Google Scholar 

  46. Hewat, E. & Blaas, D. in Antibodies in Viral Infections, vol. 260 (ed. Burton, D.R.) 29–44, (Springer-Verlag, Berlin, 2001).

    Book  Google Scholar 

  47. Hansen, J., Qing, K., Kwon, H.J., Mah, C. & Srivastava, A. Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts. J. Virol. 74, 992–996 (2000).

    Article  CAS  Google Scholar 

  48. Walters, R.W. et al. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J. Biol. Chem. 276, 20610–20616 (2001).

    Article  CAS  Google Scholar 

  49. Batard, P., Jordan, M. & Wurm, F. Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270, 61–68 (2001).

    Article  CAS  Google Scholar 

  50. Kern, A. et al. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J. Virol. 77, 11072–11081 (2003).

    Article  CAS  Google Scholar 

Download references

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.).

Author information

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

Authors
  1. Narendra Maheshri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James T Koerber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brian K Kaspar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David V Schaffer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David V Schaffer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1182

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing