AON-Mediated Exon Skipping to Bypass Protein Truncation in Retinal Dystrophies Due to the Recurrent CEP290 c.4723A > T Mutation. Fact or Fiction?

Abstract

Mutations in CEP290 encoding a centrosomal protein important to cilia formation cause a spectrum of diseases, from isolated retinal dystrophies to multivisceral and sometimes embryo–lethal ciliopathies. In recent years, endogenous and/or selective non-canonical exon skipping of mutant exons have been documented in attenuated retinal disease cases. This observation led us to consider targeted exon skipping to bypass protein truncation resulting from a recurrent mutation in exon 36 (c.4723A > T, p.Lys1575*) causing isolated retinal ciliopathy. Here, we report two unrelated individuals (P1 and P2), carrying the mutation in homozygosity but affected with early-onset severe retinal dystrophy and congenital blindness, respectively. Studying skin-derived fibroblasts, we observed basal skipping and nonsense associated–altered splicing of exon 36, producing low (P1) and very low (P2) levels of CEP290 products. Consistent with a more severe disease, fibroblasts from P2 exhibited reduced ciliation compared to P1 cells displaying normally abundant cilia; both lines presented however significantly elongated cilia, suggesting altered axonemal trafficking. Antisense oligonucleotides (AONs)-mediated skipping of exon 36 increased the abundance of the premature termination codon (PTC)-free mRNA and protein, reduced axonemal length and improved cilia formation in P2 but not in P1 expressing higher levels of skipped mRNA, questioning AON-mediated exon skipping to treat patients carrying the recurrent c.4723A > T mutation.

Suppression of Nonsense Mutations by New Emerging Technologies

“…Antisense oligos with exon-skipping ability were suggested two decades ago as a means to splice out PTC-harboring exons in frame [ 63 , 64 , 65 ]. However, this approach has a potential disadvantage, in that the alternatively spliced (exon-skipping) transcript may encode a non-functional protein, which needs careful assessment on a case-by-case scenario [ 66 , 67 ]. That said, it could be a valuable alternative, especially for diseases where gene replacement therapies prove very difficult, possibly because of the large size of the gene (difficult to be delivered to patients).…”

Spectrum of Disease Severity in Nonsyndromic Patients With Mutations in the CEP290 Gene: A Multicentric Longitudinal Study

“…16 Finally, evidence to support the hypomorphic character of truncating mutations is only available for a very small proportion of variants under investigation (hence could not be systematically considered for the entire cohort). [15][16][17]55 This study presents some limitations mainly related to its retrospective design. First, the differences in follow-up time and visit frequency.…”

“… 16 Finally, evidence to support the hypomorphic character of truncating mutations is only available for a very small proportion of variants under investigation (hence could not be systematically considered for the entire cohort). 15 – 17 , 55 …”

Molecular Therapies for Inherited Retinal Diseases—Current Standing, Opportunities and Challenges

“…For this mutation, the potential of AON-based therapy was demonstrated first in in vitro and in vivo models [96,100,101,102,103,104] and later on, in a phase I/II clinical trial [105]. Promising proof-of-concept studies employing AONs have also been developed for other mutations in CEP290 [106], and for other genes mutated in IRD such as OPA1 [107], CHM [108], USH2A [109] and ABCA4 [110,111,112,113].…”