The siRNA had modified 3’dAdT overhangs to enhance the conjugation with the in vivo-jetPEI transfection reagent used as vehicle. The formulation was done in a 5% glucose solution to increase stability. Mice were injected with the solution within 15– 20 min of its preparation. The injected volumes were 100 μl for young mice and 200 μl for adult and old mice. The treatment was performed i.p. or s.c., with 4 mg/Kg of control scrambled siRNA or Clcn7- G213R-siRNA, three times a week for 2 to 12 weeks.

Autosomal Dominant Osteopetrosis type 2 (ADO2) is a rare bone disease characterized by dense and brittle bones due to impairment of osteoclast bone resorption. Dominant negative mutations of the CLCN7 gene affect about 70% of ADO2 patients. ADO2 has no cure and our recent work established that it is suitable for gene silencing by a specific small interfering RNA that does not affect the normal mRNA, thus inducing a condition of pseudo-haplosufficiency and rescuing the bone phenotype. We performed a systematic study to test the likelihood that the therapy could progress towards clinical trials, treating Clcn7(G213R/WT) ADO2 mice with Clcn7(G213R)-specific siRNA and investigating the bone phenotype by muCT and histomorphometry, and safety, by histopathology and serology. We demonstrated that our Clcn7(G213R) siRNA is not only effective in pre-pubertal ADO2 male mice as we showed in our previous study, but also in adult and ageing mice, in males and females, by intraperitoneal and subcutaneous administration. Furthermore, the study also showed safety following prolonged chronic administration and allowed us to identify specific end-points to be potentially used in clinical trials. These results may pave the way towards regulatory toxicity studies, through which the therapy, that is patent-protected, can obtain approval from public health authorities for the transition to the Phase I/II clinical trials. The study also suggests that similar strategies could be applied to other autosomal dominant bone diseases, opening an avenue for a wider use of the RNA interference therapy in rare genetic disorders.