Figure 4.

Treatment with Ex41 skip ASO induces the expression of a near full-length CEP290 protein. (a) CEP290 gene expression analysis of fibroblasts obtained from control heterozygote fibroblasts C2 and JBTS5 fibroblasts P1 and P2 treated with 5 μM Std ASO or 5 μM Ex41 skip ASO and serum starved for 48 h. In heterozygote control C2, ASO-induced skipping of exon 41 results in a 2-fold increase of CEP290 transcript levels. Similarly, treatment with Ex41 skip ASO leads to a 2-fold increase of CEP290 transcript levels in JBTS5 fibroblasts P2 (59% of CEP290 expression levels in heterozygote C2 cells) and to a 5-fold increase in JBTS5 fibroblasts P1 (133% of CEP290 expression levels in heterozygote C2 cells). Each bar represents mean value from three replicates. Values are normalised to heterozygote control C2. (b) Western blot demonstrates the presence of full-length CEP290 protein (290 kDa) in C2 protein lysates, whereas protein lysates from P2 cells treated with Std ASO show absence of full-length protein. Treatment with increasing concentrations (1 μM, 5 μM and 10 μM) of Ex41 skip ASO for 48 h results in a rescue of CEP290 protein expression in P2 cells. Vinculin (124 kDa) serves as loading control. (c) P1 protein lysates treated with Std ASO show a near absence of full-length protein. CEP290 exon 41 skipping induced by 5 μM Ex41 skip ASO in P1 cells demonstrates the rescue of CEP290 protein translation to levels higher than in heterozygote C2 cells. Vinculin serves as loading control.