Bone Abstracts

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare genetic disease due to reduced osteoclast function. Clinical manifestations are variable, and in some cases the symptoms, including frequent fractures, osteomyelitis, hematologic and neural failures, are already evident during childhood and worsen with age. In 70% of cases, ADO2 is caused by heterozygous dominant negative mutations of the CLCN7 gene, encoding the Cl−/H+ antiporter type 7. We hypothesized that silencing the mutant CLCN7 transcript by small interfering (si)RNA could rescue the normal phenotype, and tested this hypothesis in the first animal model for this disease: the mouse harbouring the heterozygous Clcn7G213R mutation (ADO2 mice), recently developed by our group. We used a mutation-driven strategy to design and test in vitro various siRNAs against this mutation, finding a siRNA that specifically silenced the mutant transcript in the Clcn7G213R-transfected HEK293 cells (−85%, P=0.02), without affecting the WT allele. Of note, treatment with the Clcn7G213R-siRNA, rescued the in vitro bone resorption in ADO2 mice osteoclasts (+2.6-fold, P=0.003). This siRNA was conjugated with the delivery reagent ‘in-vivo-JetPEI’ and injected i.p. in ADO2 mice. Pharmacodynamic experiments evidenced 4 mg/kg every 48 h to be the most effective treatment regimen, strongly decreasing the mutant mRNA in tibias of treated mice (−80%, P=0.01). Consistently, 2-weeks treatment down-regulated Clcn7G213R mRNA 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 normalized (+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 normalization 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. The invention is protected by the patent application RM2014A000272.

Disclosure: The authors declared no competing interests.