A humanized mouse model for adeno-associated viral gene therapy

Abstract

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg^-/-/Rag2^-/-/Fah^-/-/Aavr^-/- (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Fig. 1. Generation of the TIRFA strain.

a AAVR gene was knocked out in TIRF zygotes using CRISPR/Cas9 gene editing tools. b AAVR gene scheme showing the sgRNAs used to target exons 4 and exons 5 and the primers binding sites used to screen the mice. On the right, DNA gel electrophoresis shows PCR bands of heterozygous and homozygous mice. c, d Human albumin levels of humanized TIRF control and TIRFA mice transplanted with two different hepatocyte donors (n = 18 for TIRF donor#1, n = 23 for TIRF donor#2, n = 7 for TIRFA donor#1 and n = 14 for TIRFA donor#2) c and its increase over time d (n = 3 for TIRF donor#1, n = 4 for TIRF donor#2, n = 3 for TIRFA donor#1 and n = 6 for TIRFA donor#2), e FAH immunostaining and H&E of TIRF and TIRFA humanized mice livers f tdTomato immunostaining of different organs from a TIRFA mouse injected with AAV8-tdTomato virus. On the right, amplification of the heart tissue. g Immunofluorescence staining of the livers from TIRF and TIRFA humanized mice injected with AAV8 virus expressing tdTomato (red). Human cells are stained with hLDH (green). Results observed in (b) and (e–g) were observed in at least 3 independent samples per condition. sgRNA single guide RNA, PCR polymerase chain reaction, H&E Hematoxylin and Eosin, FAH fumarylacetoacetate hydrolase, AAV adeno-associated virus, RFP red fluorescent protein, hLDH human lactate dehydrogenase. Data are presented as means ± SD. Significance was validated with a two-sided student t-test. Source data are provided as a Source Data file. Scale bars represent 50, 100, or 200 μm, respectively.

Fig. 2. Tropism of AAV viruses in humanized TIRFA mice.

Humanized TIRF and TIRFA mice were injected (1 × 10¹² gc/mouse) with AAV8 (a, b) or AAV9 (c, d) expressing tdTomato and harvested after a month. Immunohistochemistry showing co-staining of hLDH (magenta) and   tdTomato (brown) in livers of humanized TIRF and TIRFA mice (low and high repopulated). Quantification of human hepatocytes expressing tdTomato from mice injected with (b) AAV8 (n = 6 and n = 9 lobes for humanized TIRF and TIRFA, respectively) or (d) AAV9 (n = 5 lobes for both humanized TIRF and TIRFA). hLDH human lactate dehydrogenase. Data are presented as means ± SD. Significance was validated with a two-sided student t-test. ***P ≤ 0.001 and ****P ≤ 0.0001. Source data are provided as a Source Data file. Scale bars represent 100 μm.

Fig. 3. Detection of AAV DNA and RNA in human liver chimeric mice.

Humanized TIRF and TIRFA mice were intravenous injected with 1 × 10¹² gc/mouse AAV8-tdTomato or AAV9-tdTomato. Shown are representative in situ hybridization of AAV DNA (a, c, e) or RNA (b, d, f) from chimeric liver sections. Detection of DNA (a) and RNA (b) of AAV9-tdTomato one month after injection. Detection of DNA (c) and RNA (d) of AAV8-tdTomato one month (c, d) and 3 days (e, f) after injection. Murine with adjacent human areas is shown and semiquantitatively scored: “−” no nucleotide detection, “(+)” minimal, “+” little, “++” abundant DNA, respectively RNA. The boxed area is shown with higher magnification. Results observed in these figures were observed in at least 3 independent samples per condition. Scale bars represent 25 μm.

Fig. 4. Tropism of AAV viruses for different human tissues in humanized TIRFA mice.

H&E staining of a teratoma from a TIRFA humanized mice (a) injected with AAV9-GFP virus (a, b, c) teratoma co-stained with GFP (green) and different markers (red) (c): Sox17, Snail, Cytokeratin 14 (K14), lactate dehydrogenase (LDH), cytokeratin 19 (K19), FAS receptor and Glutamine synthetase (GS). (d, e) Human tumor of a TIRF (d) and TIRFA (e) humanized mouse injected with AAV9-tdTomato stained with hLDH (magenta) and tdTomato (brown). H&E Hematoxylin and Eosin. GFP green fluorescent protein, RFP red fluorescent protein, hLDH human lactate dehydrogenase. Representative results were observed in at least 3 independent samples per condition. Scale bars represent 100 or 200 μm, respectively.