GFP Adeno-associated virus(AAV Serotype 9)

GM1 gangliosidosis is an autosomal recessive neurodegenerative disorder caused by a deficiency of lysosomal β-galactosidase (β-gal), leading to accumulation of GM1 gangliosides. Here, to develop a human GM1 gangliosidosis model in the central nervous system, researchers used CRISPR/Cas9 genome editing technology to target GLB1 exons 2 and 6, sites of common mutations in patients, to create isogenic induced pluripotent stem (iPS) cell lines with lysosomal β-gal deficiency. The researchers screened clones with <5% β-gal enzymatic activity of the parental cell line and confirmed GLB1 knockout clones using DNA sequencing. GLB1 knockout cerebral organoids were then generated from one of the GLB1 knockout iPS cell clones. Analysis of cultured GLB1 knockout organoids revealed progressive accumulation of GM1 ganglioside. Compared with organoids injected with AAV9-GFP, GLB1 knockout organoids microinjected with AAV9-GLB1 vector had significantly increased β-galactosidase activity and significantly reduced GM1 ganglioside content, demonstrating the efficacy of an AAV9 gene therapy-based approach in GM1 gangliosidosis.

AAV9-GLB1 was injected into the center of 10-week-old GLB1 knockout brain organoids, and β-galactosidase activity and GM1 ganglioside content were analyzed 5 weeks after injection. Controls included uninjected GLB1 knockout organoids, GLB1 knockout organoids injected with an equivalent titer of AAV9-green fluorescent protein (AAV9-GFP), and uninjected syngeneic control organoids. All GLB1 knockout organoids used were from the same culture to minimize variability. The relative β-galactosidase activity of AAV9-GLB1-injected organoids was significantly increased compared with uninjected and AAV9-GFP-treated organoids (Figure 1A and B). Both immunohistochemistry (Figure 1C and D) and HPTLC analysis (Figure 1E and F) showed that GM1 ganglioside content was significantly reduced in AAV9-GLB1-treated organoids compared with AAV9-GFP-treated organoids.

Figure 1. Treatment of GLB1 Knockout Cerebral Organoids with AAV9-GLB1 Gene Therapy. (Latour Y L, et al., 2019)