Exon-skipping as a therapeutic approach for treatment of retina degeneration in patients carrying the intronic USH2A c.7595-2144A>G mutation

Abstract

Purpose : Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by hearing aids or cochlear implants, but for the loss of vision currently no treatment is available. Recently we reported the identification of the first deep intronic mutation in the USH2A gene (c.7595-2144A>G) leading to inclusion of a pseudoexon (PE40), thereby predictably giving rise to a truncated protein. In this study we explore the therapeutic potential of AONs to restore the native USH2A transcript in minigene splice assays and in patient-derived fibroblast cells.

Methods : The PE40-containing region with 500 bp of flanking sequence was cloned into a minigene splice assay in order to model PE40 splicing in HEK293T cells. Next we used antisense oligonucleotides (AONs) directed against intron-exon boundaries and exonic splice enhancer (ESE) sites to mask USH2A PE40 recognition during splicing. AON efficacy in the natural context of the USH2A gene was confirmed using fibroblast cells from patients carrying the c.7595-2144A>G mutation in compound heterozygosity.

Results : The minigene splice assay showed incorporation of PE40 into the transcript after splicing, thereby mimicking the aberrant splicing as seen in cells of patients carrying the USH2A c.7595-2144A>G mutation. We designed and evaluated the therapeutic potential of two AONs (2'OMe-PT backbone) in both the minigene splice assay and in patient-derived fibroblast cells. We could show that both AONs were capable of redirecting splicing towards the exclusion of PE40, albeit with different efficacies.

Conclusions : By using AONs we were able to specifically restore normal USH2A splicing in both a minigene splice assay and in patient-derived fibroblast cells. Exclusion of PE40 from the mature transcript restores the reading frame and would thus lead to wildtype USH2A protein after translation. Following this approach we expect to be able to stop the progression of this devastating blinding aspect of Usher syndrome and provide USH2A patients carrying the c.7595-2144A>G mutation a prospect on vision in the future.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.