Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterised by an increase in bone density due to the failure of bone resorption by impaired osteoclast development or function. The only therapy is haematopoietic stem cell transplantation, which, however, is not effective in osteoclast-poor RANKL-dependent ARO, since in bone RANKL is produced mainly by stromal cells. On the other hand, mesenchymal stem cells (MSCs) transplantation (MSCT) could represent a possible effective therapy. To verify this hypothesis, we established bone marrow derived MSCs (BM-MSCs) lines from the Rankl-/- mouse model, which recapitulates the human disease, and we developed a third generation lentiviral vector expressing human soluble RANKL (hsRANKL) for their correction. This vector carries hsRANKL under the CMV promoter and GFP under the hPGK promoter. The lentivirus production was performed by calcium phosphate transfection in HEK293T cells with the pMDL-g/pRRE, pMD2-VSVg, pRSV-Rev plasmids. In order to evaluate transduction efficiency, the produced vector was tested by transducing HEK293T cells at different multiplicity of infection (MOI). Fluorescence microscopy and FACS analysis showed about 100% GFP+ cells, while hsRANKL production, assessed by western blot and ELISA on the culture supernatant, increased proportionally to the MOI (ranging from 1 to 100) and was stable over time. However, the higher the MOI (50 and 100 MOI), the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRANKL transduction in Rankl-/- BM-MSCs at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl-/- control cells the cytokine was not detected, in corrected cells RANKL production and secretion was measurable and comparable with sRANKL levels in wild type (WT) mouse and human BM-MSCs. We are currently testing the transduced MSCs by in vitro functional assays; then we will use them in Rankl-/- mice.
Disclosure: This work was supported by FP7 [HEALTH.2013.2.1.1-1] -SYBIL-System biology for the functional validation of genetic determinants of skeletal diseases.