A protein domain-oriented approach to expand the opportunities of therapeutic exon skipping for -associated retinitis pigmentosa.

Loss-of-function mutations in are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). We previously presented skipping of exon 13 as a promising treatment paradigm for -associated RP. However, RP-associated mutations are often private, and evenly distributed along the gene. In order to broaden the group of patients that could benefit from therapeutic exon skipping strategies, we expanded our approach to other exons in which unique loss-of-function mutations have been reported by implementing a protein domain-oriented dual exon skipping strategy. We first generated zebrafish mutants carrying a genomic deletion of the orthologous exons of the frequently mutated human exons 30-31 or 39-40 using CRISPR-Cas9. Excision of these in-frame combinations of exons restored usherin expression in the zebrafish retina and rescued the photopigment mislocalization typically observed in mutants. To translate these findings into a future treatment in humans, we employed assays to identify and validate antisense oligonucleotides (ASOs) with a high potency for sequence-specific dual exon skipping. Together, the and data demonstrate protein domain-oriented ASO-induced dual exon skipping to be a highly promising treatment option for RP caused by mutations in .