Fig. 4.

Treatment of GLB1 Knockout Cerebral Organoids with AAV9-GLB1 Gene Therapy.

GLB1 knockout cerebral organoids were injected with AAV9-GFP or AAV9-GLB1 at 10 weeks and harvested for analysis 5 weeks later. (A) Representative X-gal staining of AAV9-GFP–injected and AAV9-GLB1–injected organoids. Scale bars, 500 μm. (B) β-gal enzyme activity of whole cerebral organoid lysates (compared to uninjected isogenic controls whose value was set at 100%). Data represent the mean relative β-gal activity ± SD (n = 5 organoids for each treatment); ^⁎p < .05, t-test analysis). (C) Representative images of AAV9-GFP– and AAV9-GLB1–injected organoids stained with anti-GM1 (green) and anti-β3 tubulin (red) antibodies. Nuclei were visualized with DAPI staining (blue). Arrows indicate areas of GM1 storage. Scale bars: white, 500 μm; yellow, 65 μm. (D) Quantification of GM1 fluorescent signal normalized to DAPI of AAV9-GLB1–injected organoids compared with AAV9-GFP–injected organoids. Data represent mean GM1 content normalized with DAPI ± SD, n = 3 injected organoids per treatment and 16 immunostained fields per analysis (^⁎⁎⁎p < .001, t-test analysis between AAV9-GFP-injected and AAV9-GLB1-injected organoids). (E) Representative HPTLC plate of gangliosides extracted from AAV9-GFP–injected and AAV9-GLB1–injected organoids. Gangliosides extracted from 3 organoids were applied per lane for AAV9-GFP- and AAV9-GLB1-injected organoids. Std, monosialogangliosides used as standards. (F) Quantification of GM1 content of AAV9-GFP–injected and AAV9-GLB1–injected organoids by HPTLC. GM1 content was determined by densitometry and normalized with the corresponding phospholipid concentrations. Data represent mean GM1 content of AAV9-GLB1–injected organoids relative to AAV9-GFP–injected organoids ± SD, n = 3 lanes (^⁎⁎p < .01, t-test analysis between AAV9-GFP-injected and AAV9-GBL1-injected values). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)