title: Pseudoexon exclusion by antisense therapy in 6-pyruvoyl-tetrahydropterin synthase deficiency.
abstract: Antisense oligonucleotide therapy to modulate splicing mutations in inherited diseases is emerging as a treatment option also for metabolic defects. In this article, we report the effect of cellular antisense therapy to suppress pseudoexon activation in primary dermal fibroblasts from patients with mutations in the PTS gene encoding 6-pyruvoyltetrahydropterin synthase (PTPS), which leads to tetrahydrobiopterin and monoamine neurotransmitter deficiency. Pathogenic inclusion of SINE or LINE-derived cryptic exons in different PTPS patients due to the intronic mutations c.84-322A>T, c.163 + 695_163 + 751del57, or c.164-712A>T was demonstrated by transcript analysis in fibroblasts and minigene ex vivo assays. Antisense morpholino oligonucleotides (AMOs) directed to the pseudoexons 3' or 5' splice sites were designed with the aim of preventing the pathological pseudoexon inclusion. At the time of AMO transfection, we investigated patients' cells for correct PTS-mRNA splicing and functional recovery of the PTPS protein. Transcriptional profiling after 24 hr posttransfection revealed a dose- and sequence-specific recovery of normal splicing. Furthermore, PTPS enzyme activity in all three patients' fibroblasts and the pterin profile were close to normal values after antisense treatment. Our results demonstrate proof-of-concept for pseudoexon exclusion therapy using AMO in inherited metabolic disease.
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Brasil
authors.forename: Hiu Man
authors.initials: HM
authors.lastname: Viecelli
authors.forename: David
authors.initials: D
authors.lastname: Meili
authors.forename: Anahita
authors.initials: A
authors.lastname: Rassi
authors.forename: Lourdes R
authors.initials: LR
authors.lastname: Desviat
authors.forename: Belen
authors.initials: B
authors.lastname: Pérez
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.forename: Beat
authors.initials: B
authors.lastname: Thöny
mesh_terms: D020087:Alu Elements / Q000235:genetics; D001483:Base Sequence; D001708:Biopterins / Q000378:metabolism; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D019798:Neopterin / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D010661:Phenylketonurias / Q000188:drug therapy* / Q000201:enzymology; D019761:Phosphorus-Oxygen Lyases / Q000172:deficiency* / Q000235:genetics; D012326:RNA Splicing; D059467:Transcriptome / Q000235:genetics
chemical_list: D016376:Oligonucleotides, Antisense; D001708:Biopterins; D019798:Neopterin; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase