title: 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.affiliation: 1] INSERM, UMR 957, Nantes, France [2] Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Rousseau
authors.affiliation: Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Gioia
authors.affiliation: 1] INSERM, UMR 957, Nantes, France [2] Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Layrolle
authors.affiliation: 1] INRA USC707, Nantes, France [2] LUNAM Université, Oniris University Nantes EA 4644 IECM, Nantes, France.
authors.forename: Blandine
authors.initials: B
authors.lastname: Lieubeau
authors.affiliation: 1] INSERM, UMR 957, Nantes, France [2] Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Heymann
authors.affiliation: Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Rossi
authors.affiliation: Bone and Extracellular Matrix Branch (BEMB), NICHD, NIH, Bethesda, MD, USA.
authors.forename: Joan C
authors.initials: JC
authors.lastname: Marini
authors.affiliation: 1] INSERM, UMR 957, Nantes, France [2] Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France.
authors.forename: Valerie
authors.initials: V
authors.lastname: Trichet
authors.affiliation: Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Forlino
mesh_terms: D000483:Alleles; D000818:Animals; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D004195:Disease Models, Animal; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D010013:Osteogenesis Imperfecta / Q000235:genetics*; D014176:Protein Biosynthesis; D034622:RNA Interference
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain
references.citation: PLoS One. 2012;7(11):e49757
references.pmid: 23152933
references.citation: J Bone Miner Res. 2013 Jan;28(1):73-80
references.pmid: 22836659
references.citation: J Med Genet. 1979 Apr;16(2):101-16
references.pmid: 458828
references.citation: Blood. 2011 May 19;117(20):5332-9
references.pmid: 21403130
references.citation: Aging Cell. 2003 Aug;2(4):209-17
references.pmid: 12934714
references.citation: Mol Ther. 2012 Mar;20(3):483-512
references.pmid: 22186795
references.citation: Stem Cells. 2007 Dec;25(12):3183-93
references.pmid: 17823236
references.citation: Blood. 2001 Mar 1;97(5):1227-31
references.pmid: 11222364
references.citation: Nature. 1998 Feb 19;391(6669):806-11
references.pmid: 9486653
references.citation: Hum Mutat. 2007 Mar;28(3):209-21
references.pmid: 17078022
references.citation: Am J Hum Genet. 2004 Apr;74(4):752-60
references.pmid: 15024692
references.citation: Nat Rev Endocrinol. 2011 Jun 14;7(9):540-57
references.pmid: 21670757
references.citation: Nucleic Acids Res. 2012 Apr;40(8):3741-52
references.pmid: 22169954
references.citation: Ther Clin Risk Manag. 2010 Sep 07;6:367-81
references.pmid: 20856683
references.citation: Nucleic Acids Res. 2000 Oct 15;28(20):4013-20
references.pmid: 11024182
references.citation: Nat Biotechnol. 2003 Jun;21(6):635-7
references.pmid: 12754523
references.citation: Nat Chem Biol. 2006 Dec;2(12):711-9
references.pmid: 17108989
references.citation: J Pathol. 1998 Feb;184(2):212-8
references.pmid: 9602714
references.citation: J Musculoskelet Neuronal Interact. 2012 Sep;12(3):183-8
references.pmid: 22947550
references.citation: Matrix Biol. 2007 Oct;26(8):604-14
references.pmid: 17662583
references.citation: Oncogene. 2007 Oct 11;26(46):6653-64
references.pmid: 17471233
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3451-8
references.pmid: 9254703
references.citation: Int J Med Sci. 2008;5(6):361-5
references.pmid: 19015742
references.citation: Science. 2009 Nov 6;326(5954):818-23
references.pmid: 19892975
references.citation: Anal Biochem. 2004 Nov 1;334(1):199-200
references.pmid: 15464970
references.citation: Blood. 2009 Jul 9;114(2):459-68
references.pmid: 19414862
references.citation: FASEB J. 2008 Nov;22(11):3846-52
references.pmid: 18653764
references.citation: J Biol Chem. 1999 Dec 31;274(53):37923-31
references.pmid: 10608859
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):183-8
references.pmid: 12518064
references.citation: Stem Cells. 2012 Jul;30(7):1465-76
references.pmid: 22511244
references.citation: Proteomics. 2007 Jun;7(11):1877-91
references.pmid: 17520686
references.citation: J Cell Sci. 2010 Jul 1;123(Pt 13):2145-54
references.pmid: 20554893
references.citation: J Clin Invest. 1996 Jan 15;97(2):448-54
references.pmid: 8567966
references.citation: Science. 1999 Oct 29;286(5441):950-2
references.pmid: 10542148
references.citation: Eur J Hum Genet. 2004 Oct;12(10):864-6
references.pmid: 15241481
references.citation: Blood. 2008 Feb 1;111(3):1717-25
references.pmid: 17967940
references.citation: Cell. 2003 Oct 17;115(2):199-208
references.pmid: 14567917
references.citation: Rev Endocr Metab Disord. 2008 Jun;9(2):153-60
references.pmid: 18404382
references.citation: PLoS One. 2008 May 21;3(5):e2248
references.pmid: 18493311
references.citation: Cancer Gene Ther. 2010 Jun;17(6):387-97
references.pmid: 20075983
references.citation: PLoS Genet. 2006 Sep 8;2(9):e140
references.pmid: 16965178
references.citation: Cytotherapy. 2010 Sep;12(5):576-8
references.pmid: 20735162
references.citation: Transplantation. 2005 Jun 15;79(11):1607-14
references.pmid: 15940052
references.citation: Science. 2002 Apr 19;296(5567):550-3
references.pmid: 11910072
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7195-200
references.pmid: 12782788
references.citation: Genes Dev. 2002 Apr 15;16(8):948-58
references.pmid: 11959843