期刊
EUROPEAN JOURNAL OF HUMAN GENETICS
卷 22, 期 5, 页码 667-674出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/ejhg.2013.198
关键词
allele-specific Col1a1 silencing; siRNA; shRNA; osteogenesis imperfecta; lentiviral vector
资金
- Region des Pays de la Loire [JG/ND/RECH N 660]
- Agence Nationale de la Recherche [N R07196NS]
- AFM TELETHON [N.16017]
- [PRIN20094C2H2M]
- [Cariplo 2011-0270]
- Fondazione Telethon Funding Source: Custom
Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation. published online 11 September 2013
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