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

Abstract

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. 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 outcome. 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 studies, 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.

Figure 1

In vivo comparison between rAAV2 and rAAV8. (a) Representative histological images from the humanized FRG mouse livers transduced with 5×10¹⁰vg single-stranded (ss) and self-complementary (sc) rAAV2 and rAAV8. hAlb – human Albumin. Scale bar = 100µm. The percentage of transduced human hepatocytes was determined by individually analyzing and comparing cell counts from images of eGFP fluorescence and FAH immunostaining (see methods for details). (b) Quantification of data shown in (a). For ssAAV2 and ssAAV8 groups n=7/each; for scAAV2 n=4, for scAAV8 n=3, up to 10 sections per mouse were scored. (c) Ratio of vector genomes in human vs. mouse hepatocytes in vivo. Laser capture microdissection (LCM) followed by QPCR analysis was use to obtain relative vector copy (VC) numbers in each population (see Methods), error bars=SD, n=3.

Figure 2

In vivo AAV shuffled library screen. (a) Graphic representation of the screen. N=2 passage 1&2, n=1 passage 3&4. Percentage of human hepatocyte repopulation is listed. Selection was performed once (single biological repeat). (b) PCR analysis of liver lysates after each selection step. NTC–no template control. Marker–1kb+ DNA ladder. (c) Dot blot titer comparison between parental AAVs, AAV-DJ, and novel isolates for two transduction experiments, error bars=SD, n=3. (d) Genealogical relationship of isolates at protein (left panel) and DNA (right panel) levels.

Figure 3

Functional analysis of selected isolates. (a) Contribution comparison for the top 3 isolates in graphic form. Residues with unknown parental origin are indicated as “?”. (b) Transgene expression in primary human hepatocytes in culture (see Methods). Error bars represent SD, n=4 from a single biological repeat. (c) Intravenous immunoglobulin (IVIG) neutralization assay, n=4 (<15% variation). (d) Human hepatocyte growth factor competition assay. Data represents averages from two biological repeats with n=3 per repeat. (e) Comparison of hFIX expression in C57BL6 mice. N=5 animals/group up to day 54, n=2 at day 80. Single biological repeat, also see Extended Data Fig. 2c.

Figure 4

In vivo vector specificity analysis. (a) Luc expression in human hepatocellular carcinoma xenograft model 6 days after i.v. vector injection (n=5/group). Controls = naïve animals. The same results were observed in two independent biological repeats. (b) Luc expression in humanized FRG animals (hFRG) or naïve controls (Ctr). Serum human albumin (hAlb) levels and estimated percentages of human hepatocyte repopulation are given. All animals used in the study are shown – n=1+2 (rAAV3), n=1 (rAAV8), n=2+2 (rAAV-LK03) (c) In vivo comparisons between hFRG animals transduced with 5×10¹¹vg rAAV8 or rAAV-LK03. Representative histological images are shown. Cell counting was as in Fig. 1a and detailed in the methods. hFAH–human fumarylacetoacetate hydrolase. Scale bar = 100µm. The graph represents quantification of in vivo data from 3 animals.