Figure 6.

In Vitro Analysis of Off-Target Gene Regulation

(A–F) Graphs showing hSOD1 mRNA level fold change as determined by qRT-PCR in HEK293T cells transduced with 50,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 3) (A); isogenic Tet-inducible Flp-FRT HEK293 cells expressing either WT or G93A hSOD1 transduced with 50,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 3) (B); iAstrocyte cells transduced with 250,000 vg/cell scAAV9 viral vectors expressing off-target constructs, harvested 120 hr post-transduction (n = 10) (C); PCA plot of virally transduced isogenic Tet-inducible Flp-FRT HEK293 cell microarray data colored by treatment (scAAV9_hSOD1si, blue; scAAV9_hSOD1si_2, yellow; scAAV9_misSOD1si, pink; and scAAV9_H1emp, white) (D); volcano plots showing differentially expressed genes in scAAV9_hSOD1si transduced isogenic Tet-inducible Flp-FRT HEK293 cells (E) and iAstrocytes (F) compared to scAAV9_H1emp transduced cells (Probeset color key: green, p < 0.05, fold change > 1.5; orange, p > 0.05, fold change > 1.5; red, p < 0.05, fold change < 1.5). SOD1 probeset is highlighted by red dashed circles. Data are presented as the mean ± SEM. Data were analyzed by one-way ANOVA (A and C) or two-way ANOVA (B) followed by post hoc Dunnett’s multiple comparisons test with respect to mock. *p < 0.05, **p < 0.01, and ****p < 0.0001.