Figure 4.

Effect of AMOs Targeting the 147–286 Region on Exon 2 Inclusion in Patient Fibroblasts

(A) RT-PCR analysis of patient fibroblasts carrying the c-32-13T > G transfected with AMOs 1, 2, and 3 delivered as single units or combined (1+2+3) to a final concentration of 15 μM. Cells transfected with Endo-Porter and not transfected (NT) were used as controls. The agarose gel picture shows a representative result of three independent experiments. (B) qRT-PCR analysis of GAA exon 2 inclusion (N) after AMO transfection in patient fibroblasts. The relative abundance of the N form is expressed as the fold of increase over fibroblasts treated with Endo-Porter reagent. qRT-PCR data are represented as mean ± SEM of three independent experiments (NS, not significant; ***p < 0.001). (C) Western blot analysis of endogenous mature GAA protein in patient fibroblasts after AMO treatment. Total protein extract was collected 72 hr after transfection with AMOs and analyzed with anti-GAA antibody. The western blot picture shows a representative result of three independent experiments. Beta actin protein was chosen as internal control. (D) GAA enzymatic activity in patient fibroblasts treated with AMOs was analyzed after 48 hr from transfection. Treatment with the Endo-Porter reagent was used as control. The GAA enzymatic activity data are represented as mean ± SEM of at least three independent experiments (*p < 0.05, ***p < 0.001).