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Pseudoexon exclusion by antisense therapy in 6‐pyruvoyl‐tetrahydropterin synthase deficiency
Author(s) -
Brasil Sandra,
Viecelli Hiu Man,
Meili David,
Rassi Anahita,
Desviat Lourdes R.,
Pérez Belen,
Ugarte Magdalena,
Thöny Beat
Publication year - 2011
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21529
Subject(s) - biology , minigene , rna splicing , antisense therapy , exon , tetrahydrobiopterin , morpholino , exon skipping , microbiology and biotechnology , antisense rna , genetics , alternative splicing , gene , oligonucleotide , gene knockdown , rna , nitric oxide synthase , biochemistry , enzyme , locked nucleic acid
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. Hum Mutat 32:1–9, 2011. © 2011 Wiley‐Liss, Inc.