Extended Data Fig. 5: Dose-dependent exon 5 skipping with human CLN3-directed ASOs.
From: Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease
a, RT-PCR of RNA isolated from heterozygous CLN3+/Δex7/8 human fibroblast cell line treated with increasing concentrations (3.125 to 200 nM) of ASO-20 or ASO-28, as labeled. The spliced products are labeled at left of gel. Size markers (bp) are shown at right of gel. Quantitation of exon 5 skipping is shown as [Δ578/(Δ578 + Δ78)] x 100. Results reproduce ASO activity observed in homozygous CLN3Δex7/8 human fibroblast cell line shown in Fig. 1i. b, Exon 5 skipped (%) in relationship to the log of the dose is plotted. The half-maximal effective concentration (EC50) was calculated after fitting the data using non-linear regression with a variable slope. c, RT-PCR analysis of exon 5 skipping in three different CLN3 Batten disease patient-derived cell lines homozygous for CLN3Δex7/8 transfected with the indicated ASOs. d, Quantification of the percentage of exon 5 skipped calculated as: [Δ578/(Δ578 + Δ78) x 100]. Bars are mean ± s.e.m. ASO-C (n = 3) vs. ASO-20 (n = 3), P < 0.0001; ASO-C vs. ASO-28 (n = 3), P < 0.0001; ASO-20 vs. ASO-26, P = 0.9216. One-way ANOVA with Tukey’s multiple comparisons test; ****P < 0.0001, non-significant (ns). The spliced products are labeled at left of gel. Size markers (bp) are shown at right of gel.
