Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy

The c.2101 A > G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through AIR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6 +/- 1% of correct transcripts only) depends on the poor exon 9 definition (47 +/- 4% in the ATR(wt) context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101 A nucleotide is conserved across species, and the SS causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101 A > C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28 +/- 7% of correct transcripts). The observation that an antisense oligonucleotide (AON(ATR)) targeting the c.2101 A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1(ATR)) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63 +/- 3%) in the ATR(SS)-allele. Most importantly, upon lentivirus-mediated delivery, the U1(ATR) partially rescued ATR mRNA splicing (from similar to 19% to similar to 54%) and protein (from negligible to similar to 6%) in embryonic fibroblasts derived from humanized ATR(SS) mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101 A > G mutation and identify two potential complementary RNA-based therapies for Secicel syndrome. (C) 2016 Elsevier B.V. All rights reserved.