Fig. 3
AAV-BR1 vector distribution and its effect on organ weight and cholesterol accumulation in peripheral tissue. A Biodistribution of AAV-BR1-NPC2 [viral genomes (vg)] in brain, lung, liver, and spleen, analyzed by quantitative qPCR at 12 weeks of age. B Relative Npc2 gene expression in the cerebrum and lung tissue was analyzed by RT-qPCR. The Npc2 gene expression was significantly lower in the cerebrum of untreated Npc2−/− compared to Npc2+/+, whereas no significant difference was seen between AAV-BR1-NPC2-treated Npc2−/− mice and Npc2+/+. The Npc2 gene expression in the lungs of treated and untreated Npc2−/− mice was significantly lower compared to wild-type littermates. Data are analyzed with a one-way ANOVA (FCerebrum[2,6] = 9.64, p = 0.013, FLung[2,6] = 74.52, p < 0.0001) with Tukey’s multiple comparisons test (*p = 0.011, ***p ≤ 0.0001). A, B Data are presented as mean ± SD (n = 3 mice/group). High responders (n = 2) are indicated with green/black triangles, while low responders (n = 1) are indicated with green triangles. C Injection with the AAV-BR1-NPC2 vector had no significant effect on organ size, and both Npc2−/− (n = 9 mice) and AAV-BR1 treated Npc2−/− mice (n = 10 mice) had significantly lower brain weight and a significantly larger lung and spleen compared to wild-type (WT) mice (Npc2+/+) (n = 10 mice) analyzed with a one-way ANOVA (FBrain[2,26] = 20.00, p < 0.0001, FLung[2,26] = 19.49, p < 0.0001), FSpleen[2,26] = 24.34, p < 0.0001) with Tukey’s multiple comparisons tests (***p < 0.001, ****p < 0.0001). There was no significant difference in the size of the liver between the three groups (FLiver[2,26] = 20.1866, p = 0.8309). Data for the brain, liver, and spleen are presented as mean ± SD, whereas data for the lungs are presented as geometric mean with a 95% confidence interval. D, E Cholesterol concentrations were evaluated in the liver, lung, spleen, and serum in the three groups (n = 7 mice/group). The cholesterol levels in the investigated organs were significantly higher in Npc2−/− and AAV-BR1-NPC2-treated Npc2−/− mice compared to Npc2+/+ age-matched controls (****p < 0.0001). However, the cholesterol concentration in the spleen of treated mice was significantly lower compared to untreated Npc2−/− mice (**p = 0.0089). No significant differences were observed in serum. All data were analyzed with one-way ANOVA (FLiver[2,18] = 36.37, p < 0.0001), FLung[2,18] = 23.93, p < 0.0001, FSpleen[2,18] = 83.17, p < 0.0001, FSerum[2,18] = 0.31, p = 0.7351) with Tukey’s multiple comparisons test. Data are presented as geometric mean with a 95% confidence interval for D and as mean ± SD for E. High responders (n = 4) are indicated with green/black triangles, while low responders (n = 3) are indicated with green triangles. F Visceral pathology was analyzed with hematoxylin and eosin staining. Images are representative of n = 5–7 mice/group. Arrows point to lipid-laden macrophages in the liver (top panel), lung (mid panel), and spleen (bottom panel). Asterisks indicate the accumulation of eosinophilic granular material in the alveolar lumen, shown in higher magnification in the black boxes. Scale bars are 100 µm and 20 µm (black box)