Objectives: The Brtl mouse, a unique model for the autosomal dominant forms of osteogenesis imperfecta was used to prove the feasibility of a lentiviral-shRNA-based strategy to improve collagen quality by targeting the mutant Col1a1 allele at the point mutation responsible for the causative substitution Gly349Cys. The ability to specifically suppress the mutant allele should convert the moderate Brtl outcome to the mild one caused by quantitative defect.
Methods: A model of human embryonic kidney cell lines which express the firefly luciferase gene combined with either wild-type or mutant Brtl Col1a1 exon 23 sequences enabled the identification of two siRNAs which were effective specifically against mutant sequence and not active against the wild-type allele\. The corresponding shRNA subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts at transcript and protein levels, respectively measured by allele specific qPCR and collagen quantification after fluorescent labeling and SDSPAGE in non reducing conditions.
Results: No effect was detected on cell proliferation, but a preferential reduction of the mutant allele transcript (up to 52%) associated to a decrease of the mutant protein (about 40%) was obtained. Interestingly a down regulation of Hsp47, a specific collagen chaperone known to be up regulated in some osteogenesis imperfecta case, was also detected. Brtl fibroblasts stably expressing the shRNA of interest and down-regulating the mutant Col1a1 were seeded in alginate to measure engraftment in mice and collagen synthesis in vivo.
Conclusion: Our data support the use of cautiously designed, lentivirus delivered-shRNAs as a strategy to specifically suppress the mutant allele making appealing the modification of mesenchymal stem cells of osteogenesis imperfecta patients for autologous transplantation.
22 Jun 2013 - 25 Jun 2013