Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing

Silvia Maruelli; Roberta Besio; Julie Rousseau; Nadia Garibaldi; Jérôme Amiaud; Bénédicte Brulin; Pierre Layrolle; Virginie Escriou; Antonio Rossi; Valerie Trichet; Antonella Forlino

doi:10.1016/j.mbplus.2020.100028

Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing

Matrix Biol Plus. 2020 Jan 31:6-7:100028. doi: 10.1016/j.mbplus.2020.100028. eCollection 2020 May.

Affiliations

¹ Department of Molecular Medicine, Biochemistry Unit, University of Pavia, Pavia, Italy.
² INSERM, Université de Nantes, UMR1238, Phy-Os, Bone sarcomas and remodeling of calcified tissues, Faculty of Medicine, University of Nantes, Nantes, France.
³ Université de Paris, UTCBS, CNRS, INSERM, F-75006 Paris, France.

Abstract

Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)₃ homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy.

Keywords: BCP, biphasic calcium phosphate; Collagen; D-MEM, Dulbecco-modified Eagle's medium; EDS, Ehlers Danlos syndrome; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; Gene therapy; MEF, murine embryonic fibroblast; MSC, mesenchymal stem cell; NMD, nonsense mediated RNA decay; OI, osteogenesis imperfecta; Osteogenesis imperfecta; PBS, phosphate buffered saline; RNAi, RNA interference; SDS, sodium dodecyl sulphate; Silencing; TRAP, tartrate-resistant acid phosphatase; shRNA, short hairpin RNA; siRNA; siRNA, small interfering RNA.