Figure 6.

Synthetic regulatory sequences enhance blood-brain barrier endothelial cell specificity in vivo. (a) Schematic representation of the investigated AAV-BR1 constructs indicating the inverted terminal repeats at both ends as well as the position of the short Tie2 intron and the CMV enhancer relative to the promoter sequence (CAG, Cdh5, Ocln, Slc2a1, Slco1c1, blue) controlling the expression of the reporter gene eGFP. The CAG promoter served as control. (b) Representative immunostainings of the vector-expressed eGFP (green) and CD31 (red) in the cortex, hippocampus, and cerebellum, 14 to 15 days after injecting the indicated vectors. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. (c) The ratio of the length of the vessels expressing eGFP to the total vessel length was determined. Each vector was administered to 2 to 5 animals (AAV-BR1-CAG-eGFP (n = 3), AAV-BR1-S-C-Cdh5-eGFP-W (n = 2), AAV-BR1-C-Ocln-eGFP-W (n = 2), AAV-BR1-C-Slc2a1-S-eGFP-W (n = 5), AAV-BR1-C-Slco1c1-eGFP-W (n = 3)). Data are presented as mean ± SD analyzed by one-way ANOVA (vector, F(4/20) = 29.86, p < 0.0001), *p < 0.0332, ****p < 0.0001 (Tukey’s multiple comparison test, n = 5 images per vector). (d to f) The vector-encoded expression of the reporter gene eGFP in non-targeted cell types of the brain was reduced by short regulatory sequences. Representative immunostainings (d) of the expressed eGFP (green) and NeuN, GFAP, Iba1, Olig2 (red) in the cortex 14 to 15 days after injecting the vector AAV-BR1-CAG-eGFP. Arrows indicate neurons or astrocytes expressing eGFP. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. Quantification of eGFP-expressing neurons (e) and astrocytes (f) per mm³ in the cortex 14 to 15 days after injecting the vectors from (a). Data are presented as mean ± SD analyzed by one-way ANOVA. For neurons (e), F(4/15) = 29.22, p < 0.0001; for astrocytes (f), F(4/15) = 14.22, p < 0.0001. **p < 0.01, ***p < 0.001, ****p < 0.0001 (Tukey’s multiple comparison test, n = 3-5 animals per vector).