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. 2023 Jun 8;14:3345. doi: 10.1038/s41467-023-38582-7

Fig. 1. Engineered AAVs can specifically target the brain endothelial cells in mouse following systemic delivery.

Fig. 1

A An overview of the engineering and characterization of the novel capsids. (1): Evolution of AAV9 using Multiplexed-CREATE and (2): identification of a novel vector, AAV-X1, that transduces brain endothelial cells specifically and efficiently following systemic administration in mice. (3): Combinatorial peptide substitution and point mutation to further refine the novel vector’s tropism, yielding improved vectors. (4): Transferring the X1 peptide to the AAV1 backbone to enable serotype switching for sequential AAV administration. (5): Utilizing AAV-X1 to transform the brain endothelial cells into a biofactory, producing Hevin for the CNS. (6): Validation of the novel AAVs across rodent models (genetically diverse mice strains and rats), NHPs (marmosets and macaques), and ex vivo human brain slices. Created with BioRender.com. B (Left) Representative images of AAV (AAV9, AAV-X1) vector-mediated expression of eGFP (green) in the brain (scale bar: 2 mm). (Right) Zoomed-in images of AAV-X1-mediated expression of eGFP (green) across brain regions, including the cortex, hippocampus, thalamus, and midbrain (scale bar: 50 µm). (C57BL/6J, n = 3 per group, 3E11 vg IV dose per mouse, 3 weeks of expression). C (Left) Representative images of AAV (AAV9, PHP.V1, BR1, and AAV-X1) vector-mediated expression of eGFP (green) in the cortex. Tissues were co-stained with GLUT1 (magenta) (scale bar: 50 µm). (Middle) Percentage of AAV-mediated eGFP-expressing cells that overlap with the GLUT1+ marker across brain regions, representing the efficiency of the vectors’ targeting of GLUT1+ cells. A two-way ANOVA and Tukey’s multiple comparisons tests with adjusted P values are reported (****P < 0.0001. P = 1.8e-15 for AAV9 versus X1.1 in the cortex. P = 1.8e-15 for AAV9 versus X1.1 in the hippocampus. P = 1.8e-15 for AAV9 versus X1.1 in the thalamus). Each data point shows the mean of three slices per mouse. (Right) Percentage of GLUT1+, NeuN+, S100β+ and Iba1+ markers in AAV-mediated eGFP-expressing cells across brain regions, representing the specificity of the vectors’ targeting of GLUT1+ cells. (C57BL/6J, n = 3 per group, 3E11 vg IV dose per mouse, 3 weeks of expression). Source data are provided as a Source Data file.