Figure 7.

Silencing of Endogenous C9orf72 by AAV5-miC

(A) Transduction efficiency by AAV5 in HEK293T cells. Cells were transduced with 1e12 genomic copies (gc) AAV5-GFP and visualized 3 days post-transduction. (B) Levels of mature miC32_101 and miC46_101 guide strands in transduced cells. Cells were transduced with 2e10, 2e11, and 2e12 gc AAV5-miC32_101 and AAV5-miC46_101. RNA was isolated 3 days post-transduction to determine expression of the mature miC32_101 and miC46_101 by TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to PBS-treated cells. (C) Silencing of C9orf72 in transduced HEK293T cells, performed as described in (B). Total C9orf72 was determined by qRT-PCR. mRNA input was normalized to GAPDH and set relative to PBS-treated cells. (D) Correlation of miC32_101 expression levels and C9orf72 knockdown in cells upon transduction with AAV5-miC32_101. Pearson correlation (r) = −0,925. (E) Correlation of miC46_101 expression levels and C9orf72 knockdown in cells transduced with AAV5-miC46_101 (r = −0,808). (F) Levels of mature miC32_101 and miC46_101 guide strands in transduced iPSC-derived frontal brain-like neurons from an FTD patient. Cells were transduced with 2e11 and 2e12 gc AAV5-miC32_101 and AAV5-miC46_101. RNA was isolated 7 days post-transduction to determine expression of the mature miC32_101 and miC46_101 by TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV5-GFP-treated cells. (G) Reduction of C9orf72 in iPSC-derived frontal brain-like neurons from an FTD patient, performed as described in (F). Total C9orf72 levels were determined by qRT-PCR. mRNA input was normalized to GAPDH and set relative to cells treated with AAV5-GFP. Data were evaluated using two-way ANOVA, multiple-comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Each graph represents the mean values with SD of 2 independent experiments.