title: Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy.
abstract: 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 ATRwtcontext), 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 U1ATRpartially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSSmice. 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.
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Scalet
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Balestra
authors.affiliation: Center for Genomic Science, Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, Milano 20139, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Rohban
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Bovolenta
authors.affiliation: Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari 46, Ferrara 44121, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Perrone
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: Center for Genomic Science, Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, Milano 20139, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Campaner
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy. Electronic address: pnm@unife.it.
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004392:Dwarfism / Q000235:genetics / Q000628:therapy*; D005091:Exons; D019066:Facies; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use*; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008831:Microcephaly / Q000235:genetics / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000627:therapeutic use*; D017354:Point Mutation; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics / Q000627:therapeutic use*
chemical_list: D016376:Oligonucleotides, Antisense; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
keywords: (up to 6) Seckel syndrome-1; Antisense oligonucleotide; Exonic splicing silencer; correction approaches; modified U1snRNA