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Background Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. Methods We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. Results We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. Conclusion Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies., Highlights • Ex vivo corrected autologous HSCs might cure Autosomal Recessive Osteopetrosis (ARO). • There is no animal model to prove in vivo functional rescue of corrected human cells. • NSG oc/oc mice display osteoclast-rich cell-autonomous ARO and immunodeficiency. • Human CD34+ cell-transplanted NSG oc/oc mice show human cell chimerism in the BM. • Further improvements will allow in vivo evaluating corrected patient-derived cells.

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC 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 hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC.