PROJECT SUMMARY/ABSTRACT Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH), the most common form of hereditary rickets. In XLH, elevated fibroblast growth factor 23 (FGF23) causes renal phosphate (Pi) wasting, hypophosphatemia, reduced 1,25-dihydroxyvitamin D (1,25D), and secondary hyperparathyroidism, all contributing to mineralization disturbances in the skeleton and dentition. Yet current treatments lack efficacy and no treatments are available to specifically improve associated dentoalveolar defects that substantially affect oral health and quality of life in individuals with XLH. Limited efficacy of treatments to date is in part related to the complex etiology of mineralization defects in XLH, including local perturbations that have been overlooked and gone unaddressed. Conventional therapy for XLH, consisting of oral 1,25D and Pi, shows limited improvement of skeletal and dental defects. A recent FGF23-neutralizing antibody (FGF23Ab) treatment targeting excess FGF23 is poised to become standard-of-care. Neither preclinical nor clinical trials of FGF23Ab evaluated dentoalveolar effects. In a pilot study, we found FGF23Ab made limited improvements similar to 1,25D in the Hyp mouse model of XLH. The inability of FGF23Ab and 1,25D therapies to resolve XLH mineralization defects reflects gaps in knowledge about functions of PHEX and pathological mechanisms of XLH, preventing effective treatments. Two mineralization regulators disturbed in XLH are not addressed by current treatments and likely contribute to persistent defects by acting locally in bone and tooth extracellular matrices. PHEX cleaves and inactivates mineralization inhibitor, osteopontin (OPN). Increased OPN in bones and teeth in XLH inhibits mineralization. Additionally, increased production of inorganic pyrophosphate (PPi), a potent mineralization inhibitor, occurs in Hyp mice in association with increased ANK and ENPP1, and decreased tissue-nonspecific alkaline phosphatase (TNAP). Thus, disruptions at both systemic (high FGF23, low 1,25D and Pi) and local (increased OPN and PPi) levels contribute to XLH-associated mineralization disorders. Local factors have not been targeted by treatments to date. TNAP promotes mineralization in local ECM by both reducing PPi and dephosphorylating and inactivating OPN. This project is designed to provide new insights into local mineralization defects in dentoalveolar tissues using a mouse model of XLH, and to test novel treatment approaches to prevent and ameliorate those defects. We hypothesize that correction of OPN and/or PPi in XLH is required to effectively normalize dentoalveolar mineralization and improve oral health. We will test this hypothesis by three aims: (1) To establish the contribution of OPN to dentoalveolar mineralization defects in XLH; (2) To determine the pathogenic role of PPi in dentoalveolar mineralization defects in XLH; (3) To define effects of combined OPN and PPi reduction on Hyp mouse dentoalveolar heal...