Exon Skipping Through Chimeric Antisense U1 snRNAs to Correct Retinitis Pigmentosa GTPase-Regulator (RPGR) Splice Defect.

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator (RPGR) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming at restoring impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1_asRNAs) and tested their efficacy in vitro, upon transient cotransfection with RPGR minigene reporter systems in HEK-293T, 661W, and PC-12 cell lines. We thus identified three chimeric U1_asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5'antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T and 661W cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1_snRNA exon skipping-based approach to correct splicing defects in RPGR.