*6.4. Targeting NPP1*

As most cases of GACI are caused by mutations in *ENPP1*, enzyme replacement therapy has been proposed as a potential strategy to counteract the clinical manifestations of the disease. NPP1 is a key enzyme in the calcification pathway, converting extracellular ATP into adenosine monophosphate and PPi. There has been success with this strategy in preclinical studies. Albright et al. demonstrated that replacement therapy with soluble recombinant human NPP1 (rhNPP1-Fc) successfully reduced ectopic mineralization, improved plasma PPi levels, and prevented mortality in a rodent GACI model [132]. Another study evaluating cardiac outcomes found recombinant NPP1 reduced not only aortic calcification, but also improved cardiovascular function and blood pressure [133].

GACI presents manifestations beyond calcification that can potentially be attributed to the influence of NPP1 enzyme activity on extracellular purinergic metabolism [89], which is consistent with what has been found for ABCC6 and PXE [124]. In a recent study, Nitschke and co-workers showed that nearly <sup>3</sup> 4 of GACI patients display arterial stenoses due to intimal proliferation of vascular smooth muscle cells (VSMC) [89]. This phenotype can be reversed effectively in culture and in vivo with rhNPP1-Fc or with adenosine. However, neither PPi nor the bisphosphonate etidronate (an off-label treatment for GACI) had an effect on VSMC proliferation. This is an important finding as it pertains directly to PXE. Indeed, NPP1, which is immediately downstream of ABCC6, is not only essential for the generation of extracellular PPi but also for the cleavage of extracellular ATP into adenosine via the action of CD73 (Figures 1 and 3). Remarkably, CALJA patients with *NT5E* mutations (and inactive CD73) develop similar vascular obstructions in the lower extremities in adulthood [10].

rhNPP1-Fc has yet to be tested in rodent models of PXE; however, there are some indications that it could potentially be used in this case as well. It has been observed, even with complete ABCC6 deficiency, that there are fairly high residual plasma levels of PPi (~40%), indicating that other sources of extracellular ATP exist [9,112]. However, it is possible that this strategy would require combination therapy to increase ATP release from cells other than hepatocytes and replace NPP1 in order to restore plasma PPi levels. However, preclinical studies are needed to demonstrate the viability of this therapeutic strategy for PXE.
