Abstract

Retinal dystrophies are a large and extremely variegate group of diseases causing visual impairments and eventually blindness. As of today, no cures are available for such conditions, and the most advanced clinical approaches are based on classical gene transfer techniques. The recent development of genetic tools designed to manipulate splicing offers a unique opportunity to target several of these diseases, potentially reaching a highly specific and controlled effect in contrast to the more investigated gene supplementation therapies. These molecular tools are mainly part of two classes: antisense oligonucleotides (AONs) and chimeric or adapted small nuclear RNAs (snRNAs). They function by altering how the splicing machinery recognize a target sequence, usually an exon, allowing to increase its presence (exon inclusion) or decrease it (exon skipping) in mature mRNA. The first aim of this work has been the identification of genes causing retinal dystrophies having mutations potentially targetable by splicing-correction approaches. Following screening of mutation databases we identified three genes, each of them having mutations with characteristic interesting for a possible application of such approaches: CACNA2D4, RPGR, and USH2A. The CACNA2D4 gene encodes for an accessory subunit of high-voltage-activated (HVA) calcium channels and, when mutated, causes retinal cone dystrophy 4 (RCD4). It was selected because of the presence of a mouse model of retinal dystrophy (Wycisk et al., 2006a), allowing an in vivo test of the designed strategy. Since the approach consisted in inducing skipping of the exon hosting the mutation, we analysed the splicing pattern of the target exon in vivo and in vitro; the functionality of the rescued protein resulting from therapeutic exon skipping. The analysis revealed on one side that the rescued protein was not functional, therefore showing how the approach was not feasible for CACNA2D4. Interestingly, we found evidence of two newly identified splicing isoforms of the gene, one of which mimics the effect of the mutation. A mutation in RPGR intron 9 has been shown to cause retinitis pigmentosa by increasing the inclusion in mature mRNA of an alternatively spliced exon (E9a) (Neidhardt et al., 2007). Using splicing reporter minigene assays, we were able to design efficient chimeric U1snRNAs able to mediate E9a skipping thus correcting the genetic defect. In the USH2A gene we instead identified an interesting region hosting 5% of known mutation which could be approachable with exon skipping. The implementation of assays able to assess functionality of USH2A and RPGR will be fundamental for the development of new splicing-correction approaches for these genes.