Genetic autosomal dominant diseases are generally due to heterozygous missense mutations that could be eradicated by RNA interference. We hypothesised that this approach could cure ADO2 and tested this treatment in ADO2 mice carrying a G213R amino acid substitution in the CLC-7 protein, encoded by the Clcn7 gene. Using a systematic mutation-driven strategy, we designed and tested in-vitro various small interfering (si)RNAs against this mutation and found a Clcn7G213R-siRNA that silenced specifically the mutant transcript in transfected HEK293 cells (−85%, P=0.02), without affecting the WT gene and rescuing bone resorption in ADO2 osteoclasts (+2.6-fold, P=0.003). This siRNA was made sticky by 3′dAdT overhangs, conjugated with the delivery system in-vivo-JetPEI and injected i.p. in ADO2 mice. Time- and dose-dependent experiments evidenced 4 mg/kg every 48 h to be the most effective treatment, decreasing the mutant mRNA in tibias (−80%, P=0.01). Two-weeks treatment of ADO2 mice, down-regulated Clcn7G213R mRNA expression in bone and other organs, increased the serum bone resorption marker CTX over the osteoclast marker TRAcP 5b (+1.8-fold, P=0.002), and decreased trabecular BV/TV (−19%, P=0.04) and Tb.N (−16%, P=0.05) vs scrambled-siRNA treated ADO2 mice. After 4 weeks, trabecular BV/TV (−21%, P=0.03) and trabecular variables (Tb.N −19%, Tb.Sp +1.2-fold, P<0.03) returned to WT level, with a full rescue of the bone phenotype. In the rescued ADO2 mice, serum CTX/TRAcP, osteoclast number and erosion surface/bone surface were normalised (+1.2-fold, −32%,+2.1-fold, P=0.03, P=0.01, P=0.02, respectively, vs control ADO2 mice), while osteoblast and dynamic parameters were unremarkable. Treatment was well tolerated, with no adverse events, and with normalisation of liver aspartate aminotransferase. To the best of our knowledge, this is the first experimental curative treatment of ADO2, which rescued osteoclast function and returned the bone phenotype to normal by a systemic RNA interference strategy. The invention is protected by the patent application RM2014A000272, which could provide the means to develop this siRNA strategy for therapy in humans.
Disclosure: The authors declared no competing interests. Telethon (Grant numbers GGP06019, GGP09018 and GGP14014); The European Union funded project SYBIL FP7-HEALTH-2013-INNOVATION (Grant number 602300).