A STEP-BY-STEP PROCESS TO GENERATE FUNCTIONAL OSTEOCLASTS FROM SITE SPECIFIC GENE-CORRECTED INDUCED PLURIPOTENT STEM CELLS: AN AUTOLOGOUS CELL THERAPY APPROACH TO TREAT AUTOSOMAL RECESSIVE OSTEOPETROSIS.

Autosomal recessive osteopetrosis (ARO) is a human severe inherited disorder leading to increased bone mass, decreased bone strength with risk of multiple fractures and progressive narrowing of the medullary cavity. Among the heterogeneous group of osteopetrosis, ARO shows the most severe phenotype, leading to death early in life if not treated. The most frequently mutated gene is the T-cell immune regulator 1 (TCIRG1), encoding the a3 subunit of the vacuolar-type proton transporting ATPase pump, which mediates the acidification of the bone/osteoclast interface, causing defects in the resorbing activity of osteoclasts. The spontaneous mouse model oc/oc well recapitulates the disease features. Hematopoietic stem cell (HSC) transplantation is the only treatment so far available to cure the disease, due to the hematopoietic origin of osteoclasts, however its success is limited to the restricted number of available matched donors. Thus, the transplant of autologous corrected hematopoietic cells represents a valid alternative therapeutic option. In particular, due to the limitations in performing gene targeting of primary cells, induced pluripotent stem cells (iPSc) obtained from patients could represent an unlimited source of autologous cells in which it is possible to perform target gene correction. They can be subsequently differentiated towards hematopoietic progenitors to perform an autologous transplantation. In the present thesis, I have exploited murine iPSc as a potential source of cells to be differentiated into functional osteoclasts able to resorb bone. To this end, I have pursued the following strategy: (a) generation of vector-free iPSc from wild type (wt) and oc/oc mice employing a third generation Cre-excisable lentiviral vector carrying the reprogramming genes Oct4, Sox2, Klf4; (b) correction of oc/oc iPSc replacing the entire mutated Tcirg1 gene through homologous recombination, by using a BAC engineered vector; (c) set up a protocol for the differentiation of wt, uncorrected and corrected oc/oc iPSc into the hematopoietic lineage, generating both mature cells and high proliferative potential colony-forming progenitors; (d) further differentiation of obtained myeloid precursors into osteoclasts with rescued bone resorbing activity upon gene-correction. The process is aimed at generating transplantable hematopoietic cells, including osteoclast precursors, with the final goal of transplanting them in the ARO mouse model oc/oc to revert the phenotype, thus providing a proof of principle for an autologous cell therapy approach to treat ARO.