Rousseau, J., Gioia, R., Layrolle, P. et al. (6 more authors) (2014) Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta. European Journal of Human Genetics, 22 (5). pp. 667-674. ISSN 1018-4813
Abstract
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2014 Macmillan Publishers Limited |
Keywords: | allele-specific Col1a1 silencing; siRNA; shRNA; osteogenesis imperfecta; lentiviral vector |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > The Medical School (Sheffield) > Division of Genomic Medicine (Sheffield) > Department of Oncology and Metabolism (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 18 May 2016 14:59 |
Last Modified: | 03 Nov 2016 01:54 |
Published Version: | http://dx.doi.org/10.1038/ejhg.2013.198 |
Status: | Published |
Publisher: | Nature Publishing Group |
Refereed: | Yes |
Identification Number: | 10.1038/ejhg.2013.198 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:98159 |