Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy

• Published in: Biochimica et biophysica acta. Molecular basis of disease 2017-01, Vol.1863 (1), p.15-20
• Main Authors: Scalet, Daniela, Balestra, Dario, Rohban, Sara, Bovolenta, Matteo, Perrone, Daniela, Bernardi, Francesco, Campaner, Stefano, Pinotti, Mirko
• Format: Article
• Language: English
• Subjects: (up to 6) Seckel syndrome-1; Animals; Antisense oligonucleotide; Ataxia Telangiectasia Mutated Proteins - genetics; Base Sequence; Cell Line; correction approaches; Dwarfism - genetics; Dwarfism - therapy; Exonic splicing silencer; Exons; Facies; Genetic Therapy; Genetic Vectors - genetics; Genetic Vectors - therapeutic use; HEK293 Cells; Humans; Introns; Lentivirus - genetics; Mice; Microcephaly - genetics; Microcephaly - therapy; modified U1snRNA; Oligonucleotides, Antisense - genetics; Oligonucleotides, Antisense - therapeutic use; Point Mutation; RNA Splicing; RNA, Small Nuclear - genetics; RNA, Small Nuclear - therapeutic use
• ISSN: 0925-4439; 1879-260X
• DOI: 10.1016/j.bbadis.2016.09.011

The c.2101A>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 ATR 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 ATRwt 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.2101A 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.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5’ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome. [Display omitted] •The deleterious ATR c.2101A>G mutation depends on the unfavorable splicing context•The mutation promotes exon-skipping by strengthening an Exonic Splicing Silencer•An antisense oligonucleotide targeting the c.2101G position recovers exon inclusion•A compensatory U1snRNA delivered by Lentivirus rescues ATR expression in MEFSS-1