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


This article has been updated


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.

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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.


  1. 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)

    CAS  Article  Google Scholar 

  2. 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)

    CAS  Article  Google Scholar 

  3. 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. 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)

    CAS  Article  Google Scholar 

  5. 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)

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 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)

    CAS  Article  Google Scholar 

  8. 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)

    CAS  Article  Google Scholar 

  9. 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)

    CAS  Article  Google Scholar 

  10. 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)

    CAS  Article  Google Scholar 

  11. 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)

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 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)

    CAS  Article  Google Scholar 

  15. 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. 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)

    CAS  Article  Google Scholar 

  17. 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)

    CAS  Article  Google Scholar 

  18. 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)

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 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)

    CAS  Article  Google Scholar 

  21. 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)

    ADS  CAS  Article  Google Scholar 

  22. 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)

    CAS  Article  Google Scholar 

  23. 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. 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)

    CAS  Article  Google Scholar 

  25. 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. 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)

    MathSciNet  CAS  Article  Google Scholar 

  27. 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)

    CAS  Article  Google Scholar 

  28. 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. 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)

    CAS  Article  Google Scholar 

  30. 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)

    CAS  Article  Google Scholar 

  31. 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)

    CAS  Article  Google Scholar 

  32. 32

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

  33. 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)

    CAS  Article  Google Scholar 

  34. 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)

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

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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




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
Extended Data Table 2 AAV cap gene sequence comparison
Extended Data Table 3 Tabular representation of vector comparison data from different experiments presented in the study

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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).

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