Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta.

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.

• Authors: Julie Rousseau, Roberta Gioia, Pierre Layrolle, Blandine Lieubeau, Dominique Heymann, Antonio Rossi, Joan C Marini, Valerie Trichet, Antonella Forlino
• Journal: European journal of human genetics : EJHG (Eur J Hum Genet) Vol. 22 Issue 5 Pg. 667-74 (May 2014) ISSN: 1476-5438 [Electronic] England
• PMID: 24022296 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Collagen Type I
• Collagen Type I, alpha 1 Chain

Topics

• Alleles
• Animals
• Cell Line
• Collagen Type I (genetics, metabolism)
• Collagen Type I, alpha 1 Chain
• Disease Models, Animal
• Exons
• Fibroblasts (metabolism)
• Gene Expression Regulation
• Gene Silencing
• Humans
• Mice
• Mice, Knockout
• Mutation
• Osteogenesis Imperfecta (genetics)
• Protein Biosynthesis
• RNA Interference