期刊
HUMAN MUTATION
卷 32, 期 9, 页码 1019-1027出版社
WILEY-BLACKWELL
DOI: 10.1002/humu.21529
关键词
alternative splicing; antisense oligonucleotides; pseudoexons; splicing therapy; tetrahydrobiopterin deficiency
资金
- Fundacao para a Ciencia e Tecnologia from Portugal [SFRH/BD/45753/2008]
- Comision Interministerial de Ciencia y Tecnologia [SAF2007-61350]
- Fondo de Investigaciones Sanitarias [PI10/00455]
- Fundacion Ramon Areces to the Centro de Biologia Molecular Severo Ochoa
- Swiss National Science Foundation
- Fundação para a Ciência e a Tecnologia [SFRH/BD/45753/2008] Funding Source: FCT
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 32:1019-1027, 2011. (C) 2011 Wiley-Liss, Inc.
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