Morpholino antisense oligonucleotides (AONs) mediate ush2a exon 13 skipping, usherinΔexon 13 protein expression, and restoration of electroretinogram (ERG) in a mutant zebrafish model (A) Amino acid alignment of the sequences encoded by human and zebrafish USH2A exon 13. The (partial) EGF-lam domains are indicated. The cysteine residues required for 3D topology of the EGF-lam domains (green) are completely conserved between zebrafish and human. (B) Phosphorodiamidate morpholino oligonucleotide (PMO)-induced skipping of ush2a exon 13 in zebrafish larvae. ush2armc1 mutant embryos were injected with a combination of PMO1 and PMO2 (1 ng of each). Investigation of ush2a pre-mRNA splicing at 3 days post-fertilization (dpf) revealed the skipping of ush2a exon 13 upon injection of PMOs targeting ush2a exon 13. Uninjected ush2armc1 mutant zebrafish larvae and WT larvae were used as controls. (C) Subcellular localization of usherin in horizontal cryosections of larval (5 dpf) zebrafish retinae. Usherin was visualized with anti-usherin antibodies directed against the intracellular C-terminal tail of zebrafish usherin (green signal). Nuclei were stained with DAPI (blue signal), and the connecting cilium is labeled using anti-centrin antibodies (red). In WT larvae, usherin is present at the photoreceptor periciliary membrane, adjacent to the connecting cilium. In homozygous ush2armc1 larvae, no specific usherin signal could be detected. PMO-induced ush2a exon 13 skipping in ush2armc1 mutant larvae resulted in partial restoration of usherinΔexon 13 expression with the correct subcellular localization in the retina. OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer; wt: WT; ush2armc1, zebrafish with exon 13 mutation. (D) Scatterplot of the relative fluorescence intensity of anti-usherin staining in the periciliary region of all photoreceptors in the middle section of the larval zebrafish eye. The signal intensity is decreased in the ush2armc1 retina compared to WTs. Relative fluorescent signal intensity of anti-usherin staining is significantly increased in PMO-injected ush2armc1 mutants as compared to uninjected mutants (∗∗∗∗p < 0.0001, Kruskal-Wallis test followed by Dunn’s nonparametric post-test). (E) Average ERG b-wave traces from uninjected, control PMO-injected, exon 13 PMO-injected ush2armc1 larvae and WT controls at 5−6 dpf. PMO-induced skipping of ush2a exon 13 completely restored b-wave amplitudes in ush2armc1 larvae as compared to uninjected or control PMO-injected mutants. (F) Maximum b-wave amplitudes recorded in uninjected or control PMO-injected ush2armc1 larvae are significantly reduced as compared to ERG traces from age- and strain-matched WT controls. Maximum b-wave amplitudes recorded in PMO-injected ush2armc1 mutants are significantly improved as compared to ERG traces from uninjected or control PMO-injected ush2armc1 mutants and do not significantly differ from WTs (p > 0.99). Data are shown as mean ± SD, ∗p < 0.05, ∗∗p < 0.01, Kruskal-Wallis test followed by Dunn’s nonparametric post-test. (G) Quantification of ush2a Δexon 13 transcripts in uninjected and PMO-injected zebrafish larvae at 3 dpf.
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