A comprehensive study of AAV tropism across C57BL/6 mice, BALB/c mice, and crab-eating macaques

Figures

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  Figure 1. AAV transduction differences between C57BL/6 and BALB/c mice (A) Schematic of the AAV constructs containing two sets of 7nt-barcodes. (B) Schematic of the AAV tropism analysis procedure in C57BL/6 and BALB/c mice. We injected 5E12 vg/kg AAV pool intravenously into mice (N = 3). The infection and transduction efficiency of AAV library was assessed at the DNA, RNA, and protein levels. (C) Quantitative analysis of vector DNA abundance of overall AAV pools in C57BL/6 and BALB/c mice using qPCR. Vector copy numbers were normalized to per diploid genome using the mouse Gapdh gene as a control. N = 3; means ± SD; ns, not significant; unpaired t tests. (D) Quantitative analysis of the AAV RNA abundance of overall AAV pools between C57BL/6 and BALB/c mice. N = 3; means ± SD; ns, not significant; ∗∗∗p < 0.001; unpaired t tests. (E–H) Representative images of tdTomato expression in tissues of the brain (E), spinal cord (F), lung (G), and bone marrow (H). The right graph shows the quantification of tdTomato+ cells among all cells. N = 3; means ± SD; ∗∗∗p < 0.001; unpaired t tests. (I) Pearson correlation elucidating the variation of the transduction pattern of each capsid between C57BL/6 and…

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  Figure 2. Overall AAV transduction differences between C57BL/6 mice and crab-eating macaques (A) Schematic of the AAV tropism analysis procedure in crab-eating macaques. We injected 5E12 vg/kg AAV pool intravenously to macaques (N = 3, male). Rituximab and rapamycin were applied to suppress the potential immune response. The infection and transduction efficiency of AAV pool was assessed at the DNA, RNA, and protein levels. (B) Quantitative analysis of the vector DNA abundance of overall pooled AAVs in C57BL/6 mouse and crab-eating macaques using qPCR. Vector copy numbers were normalized to per diploid genome using mouse or macaque Gapdh gene as an internal control. N = 3; means ± SD; ns, not significant; ∗∗p < 0.01; unpaired t tests. (C) Quantitative analysis of AAV RNA abundance of overall AAV library between C57BL/6 mice and crab-eating macaques. N = 3; means ± SD; ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; unpaired t tests. (D–G) Representative images of tdTomato expression in tissues including liver (D), heart (E), muscle (F), and brain (G). The right graph shows the quantification of tdTomato+ cells among all cells. N = 3; means ± SD; ∗∗∗p < 0.001; unpaired t tests. The…

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  Figure 3. AAV transduction differences of each capsid between C57BL/6 mice and crab-eating macaques (A) Heatmap of AAV DNA abundance of each serotype in each crab-eating macaque (N = 3). The DNA abundance of each capsid was calculated from the total DNA abundance of the pooled AAVs and the proportion of each capsid. Every three cells in the heatmap represents independent data from three individual macaques to show variation between animals. (B) Heatmap of AAV RNA abundance of each serotype in each crab-eating macaque (N = 3). (C) Pearson correlation elucidating the variation of transduction pattern in each capsid between C57BL/6 mice and crab-eating macaques. (D and E) Quantitative analysis of AAV RNA abundance of AAV4 (D) or AAV.CAP-B10 (E) in C57BL/c mice and crab-eating macaques. N = 3; means ± SD; ns, not significant; ∗∗p < 0.01; ∗∗∗p < 0.001; unpaired t tests.

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  Figure 4. AAV capsid variants with the top transduction efficiencies across species Heatmap of the RNA abundance of each AAV capsid in the liver (A), muscle (B), brain (C), lung (D), colon (E), kidney (F), pancreas (G), and testes (H). The RNA abundance of each capsid was calculated from the total RNA abundance of pooled AAVs. The RNA abundance of the macaque brain was averaged by the RNA abundance of 10 regions of the macaque brain. Numbers 1–5 represent the transduction rank of the AAV capsids in each tissue. The red arrow highlights the capsids that performed well across strains and species. Representative images of tdTomato expression in kidney (F), pancreas (G), and testes (H) are shown in the right panels. White dashed circles indicate the glomerulus in the kidneys (F) and the pancreatic islets in the pancreas (G), respectively.

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  Figure 5. AAV4 exhibits different transduction patterns from AAV9 in C57BL/6 mice (A) Schematic of the tropism analysis procedure for AAV4 and AAV9 in C57BL/6 mice. 3E12 vg/kg AAV4 or AAV9 was intravenously injected into mice (N = 3). The infection and transduction efficiency of the AAV pool was assessed at the DNA, RNA, and protein level. (B) Quantitative analysis of the vector DNA abundance of AAV4 and AAV9. N = 3; means ± SD; ns, not significant; ∗∗∗p < 0.001; unpaired t tests. (C) Quantitative analysis of the vector RNA abundance of AAV4 and AAV9. N = 3; means ± SD; ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; unpaired t tests. (D–I) Representative images of tdTomato expression delivered by AAV4 or AAV9 in tissues, including liver (D), brown adipose (E), testes (F), lung (G), pancreas (H), and kidney (I). The right graph shows the quantification of the tdTomato+ cell proportion. N = 3; means ± SD; ∗p < 0.05; ∗∗∗p < 0.001; unpaired t tests. White dashed circles indicate the glomerulus in the kidneys and the pancreatic islets in the pancreas respectively. (J) Quantitative analysis of the vector DNA abundance of AAV4 in crab-eating macaques. (K) Heatmap of the vector DNA…