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.

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model

Nature volume 506, pages 382–386 (2014)Cite this article

Subjects

This article has been updated

Abstract

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials1,2,3. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome4,5,6,7,8. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human–murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies4,5,8, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly—approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: In vivo comparison between rAAV2 and rAAV8.
Figure 2: In vivo AAV-shuffled library screen.
Figure 3: Functional analysis of selected isolates.
Figure 4: In vivo vector specificity analysis.

Change history

  • 10 January 2014

    Changes were made to the keys of Fig. 3c, e.

References

  1. Bainbridge, J. W. et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 358, 2231–2239 (2008)

    Article  CAS  Google Scholar 

  2. Gaudet, D. et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther. 20, 361–369 (2013)

    Article  CAS  Google Scholar 

  3. Bennett, J. et al. AAV2 gene therapy readministration in three adults with congenital blindness. Sci. Trans. Med. 4, 120ra115 (2012)

    Article  Google Scholar 

  4. Nietupski, J. B. et al. Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression. Mol. Ther. 19, 1999–2011 (2011)

    Article  CAS  Google Scholar 

  5. Jiang, H. et al. Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood 108, 3321–3328 (2006)

    Article  CAS  Google Scholar 

  6. Nathwani, A. C. et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med. 365, 2357–2365 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nature Med. 12, 342–347 (2006)

    Article  CAS  Google Scholar 

  9. Nathwani, A. C. et al. Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood 109, 1414–1421 (2007)

    Article  CAS  Google Scholar 

  10. Nathwani, A. C. et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 107, 2653–2661 (2006)

    Article  CAS  Google Scholar 

  11. Davidoff, A. M. et al. Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models. Mol. Ther. 11, 875–888 (2005)

    Article  CAS  Google Scholar 

  12. Nonnenmacher, M. & Weber, T. Intracellular transport of recombinant adeno-associated virus vectors. Gene Ther. 19, 649–658 (2012)

    Article  CAS  Google Scholar 

  13. Azuma, H. et al. Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice. Nature Biotechnol. 25, 903–910 (2007)

    Article  CAS  Google Scholar 

  14. Grimm, D. et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J. Virol. 82, 5887–5911 (2008)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  16. 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 

  17. Maheshri, N., Koerber, J. T., Kaspar, B. K. & Schaffer, D. V. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nature Biotechnol. 24, 198–204 (2006)

    Article  CAS  Google Scholar 

  18. Li, W. et al. Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Mol. Ther. 16, 1252–1260 (2008)

    Article  CAS  Google Scholar 

  19. Pulicherla, N. et al. Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol. Ther. 19, 1070–1078 (2011)

    Article  CAS  Google Scholar 

  20. Asuri, P. et al. Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells. Mol. Ther. 20, 329–338 (2012)

    Article  CAS  Google Scholar 

  21. Yang, L. et al. A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc. Natl Acad. Sci. USA 106, 3946–3951 (2009)

    Article  ADS  CAS  Google Scholar 

  22. Choi, V. W., McCarty, D. M. & Samulski, R. J. AAV hybrid serotypes: improved vectors for gene delivery. Curr. Gene Ther. 5, 299–310 (2005)

    Article  CAS  Google Scholar 

  23. Rutledge, E. A., Halbert, C. L. & Russell, D. W. Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2. J. Virol. 72, 309–319 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Petek, L. M., Fleckman, P. & Miller, D. G. Efficient KRT14 targeting and functional characterization of transplanted human keratinocytes for the treatment of epidermolysis bullosa simplex. Mol. Ther. 18, 1624–1632 (2010)

    Article  CAS  Google Scholar 

  25. Calcedo, R., Vandenberghe, L. H., Gao, G., Lin, J. & Wilson, J. M. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J. Infect. Dis. 199, 381–390 (2009)

    Article  Google Scholar 

  26. Boutin, S. et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum. Gene Ther. 21, 704–712 (2010)

    Article  MathSciNet  CAS  Google Scholar 

  27. Ling, C. et al. Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3. Hum. Gene Ther. 21, 1741–1747 (2010)

    Article  CAS  Google Scholar 

  28. Hazari, S. et al. Hepatocellular carcinoma xenograft supports HCV replication: a mouse model for evaluating antivirals. World J. Gastroenterol. 17, 300–312 (2011)

    Article  Google Scholar 

  29. Thomas, C. E., Storm, T. A., Huang, Z. & Kay, M. A. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J. Virol. 78, 3110–3122 (2004)

    Article  CAS  Google Scholar 

  30. Cunningham, S. C., Dane, A. P., Spinoulas, A., Logan, G. J. & Alexander, I. E. Gene delivery to the juvenile mouse liver using AAV2/8 vectors. Mol. Ther. 16, 1081–1088 (2008)

    Article  CAS  Google Scholar 

  31. Nakai, H. et al. A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction. J. Virol. 76, 11343–11349 (2002)

    Article  CAS  Google Scholar 

  32. Grompe, M. & Strom, S. Mice with human livers. Gastroenterology (2013)

  33. Lieber, A., Peeters, M. J., Gown, A., Perkins, J. & Kay, M. A. A modified urokinase plasminogen activator induces liver regeneration without bleeding. Hum. Gene Ther. 6, 1029–1037 (1995)

    Article  CAS  Google Scholar 

  34. Arbetman, A. E. et al. Novel caprine adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties. J. Virol. 79, 15238–15245 (2005)

    Article  CAS  Google Scholar 

  35. Lisowski, L. et al. Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression. Mol. Ther. 20, 1912–1923 (2012)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institutes of Health grants HL092096 and HL064274 to M.A.K. and DK048252 to M.G.; L.L. was supported in part by the Berry Fellowship Foundation; I.E.A. by Australian National Health and Medical Research Council (NHMRC) grant 1008021.

Author information

Author notes

  1. Leszek Lisowski & Allison P. Dane

    Present address: Present addresses: Gene Transfer, Targeting and Therapeutics Core, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, San Diego, California 92037, USA (L.L.); Department of Haematology, University College London Cancer Institute, London WC1E 6BT, UK (A.P.D.).,

Authors and Affiliations

  1. Departments of Pediatrics and Genetics, Stanford University, School of Medicine, 269 Campus Drive, Stanford, 94305, California, USA

    Leszek Lisowski, Kirk Chu, Yue Zhang & Mark A. Kay

  2. Gene Therapy Research Unit, The Children's Hospital at Westmead and Children’s Medical Research Institute, Locked Bag 4001, Westmead, 2145 New South Wales, Australia,

    Allison P. Dane, Sharon C. Cunningham & Ian E. Alexander

  3. Yecuris Corporation, Portland, 97062, Oregon, USA

    Elizabeth M. Wilson

  4. Oregon Stem Cell Center, Oregon Health and Science University, Portland, 97239, Oregon, USA

    Sean Nygaard & Markus Grompe

  5. Discipline of Paediatrics and Child Health, The University of Sydney, 2145 New South Wales, Australia,

    Ian E. Alexander

Authors
  1. Leszek Lisowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Allison P. Dane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kirk Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sharon C. Cunningham
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elizabeth M. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sean Nygaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Markus Grompe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ian E. Alexander
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mark A. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.L. helped with study design, performed experiments and data analysis, prepared figures and the manuscript. A.D. performed some of the experiments and data analysis, and assisted in figure preparation and manuscript editing. K.C. helped in performing some of the experiments. Y.Z. performed some of the vector sequence analysis. S.C.C. performed some of the animal studies and assisted in manuscript editing. E.M.W. generated the human transplanted FRG mice in Fig. 4c. S.N. injected the animals and prepared tissues for the experiment in Fig. 4c. M.G. helped with establishing the FRG colony and provided advice on in vivo human hepatocyte repopulation. I.E.A. helped with study design and manuscript editing. M.A.K. helped with study coordination, manuscript writing and editing. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Mark A. Kay.

Ethics declarations

Competing interests

Oregon Health and Science University (OHSU) and M.G. have a significant financial interest in Yecuris Corp., a company that has some commercial interests in the FRG mouse. M.A.K. has a minor equity stake with stock options value >US$5,000 and has no role in the company. E.M.W. is an employee of Yecuris and has no equity. M.G. and Yecuris have no ownership or intellectual property rights to any of the new AAV vectors described herein including AAV-LK03.

Extended data figures and tables

Extended Data Figure 1 IVIG neutralization assay optimization on Huh-7 cells using rAAV2-RSV-Luc2.

Gamunex and Gammagard IVIGs were compared at two different temperatures. See Methods for experimental details.

Extended Data Figure 2 In vivo vectors comparison in C57/BL6 animals.

a, b, In vivo average VCN analysis in tissues harvested on day 54 (a) from the first in vivo rAAV-hFIX experiment and on day 7 (b) from the second in vivo rAAV-hFIX experiment. c, In vivo hFIX expression levels. hFIX levels obtained from the first in vivo rAAV-hFIX comparison (solid colour lines, from day 5 until day 80) are presented on the same graph with data obtained during the second in vivo experiment (dotted lines, days 2, 4 and 7).

Extended Data Figure 3 Time course of Luc signal in animals shown in Fig. 4a.

a, b, Data for days 2, 4 and 6 were collected and are shown. In a, all animals are shown with the same pseudo-scale, whereas in b, auto-scale was selected for each group.

Extended Data Figure 4 Detailed analysis of bioluminescence for animals shown in Fig. 4b.

The table represents detailed information on signal for each animal/ROI.

Extended Data Table 1 Relative transduction efficiency of in vivo AAV isolates and wild-type AAV serotypes in tissue culture cell lines
Full size table
Extended Data Table 2 AAV cap gene sequence comparison
Full size table
Extended Data Table 3 Tabular representation of vector comparison data from different experiments presented in the study
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures 1-4. (PDF 5149 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lisowski, L., Dane, A., Chu, K. et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506, 382–386 (2014). https://doi.org/10.1038/nature12875

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12875

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research