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


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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.


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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

Authors and Affiliations



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

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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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