ABSTRACT Pseudoxanthoma elasticum (PXE) is a genetic disorder of ectopic calcification with considerable morbidity and mortality due to deposition of hydroxyapatite crystals in the connective tissues. Though ABCC6 was identified as the causative gene for PXE 20 years ago, the disease mechanism was just recently unveiled and there is currently still no effective or specific treatment for the pathologic calcification. We have previously developed and characterized mouse models for PXE, and these mice provide the platform for preclinical development of therapeutics for this currently intractable condition. A critical pathological characteristic in PXE is the reduction in circulating levels of inorganic pyrophosphate (PPi), a key endogenous inhibitor of calcification. Therefore, the goal of the research we propose herein is to use our mouse models in preclinical studies to develop safe and effective treatments that can prevent ectopic calcification in PXE by normalization of extracellular PPi levels. We have identified ENPP1 and TNAP proteins as key regulators of PPi homeostasis. ENPP1 and TNAP have opposing actions in maintaining extracellular PPi concentrations, the former generating PPi and the latter hydrolyzing PPi. We have generated a recombinant ENPP1 enzyme biologic and our strong preliminary data demonstrate that this therapeutic biologic raised plasma PPi levels in a mouse model of PXE, and its circulating half-life can be extended by pharmacologic inhibition of TNAP. Based upon these findings and the known enzymatic activities of ENPP1 and TNAP, we propose that modulation of plasma PPi, either using a recombinant ENPP1 enzyme, TNAP inhibitors, or a combination of both approaches, represents an innovative strategy to prevent the ectopic calcification that arises as a consequence of PPi deficiency. To test this hypothesis, we propose to utilize genetic and pharmacologic approaches to define mechanisms by which inhibition of TNAP extends the plasma half-life of PPi from ENPP1 enzyme supplementation, and subsequently prevents and/or diminishes the ectopic calcification in a mouse model of PXE. Our team has the requisite research expertise in the ENPP1-PPi-TNAP axis and appropriate mouse models to complete these studies. Collectively, we anticipate that the proposed studies will provide critical translational information from preclinical approaches that will allow development of novel treatments for ectopic calcification in patients with PXE. If successful, our findings will advance clinical management of ectopic calcification broadly, as PPi deficiency plays an important role in development of ectopic calcification in other genetic and acquired disorders.