ENPP1 enzyme replacement therapy improves ectopic calcification but does not rescue skeletal phenotype in a mouse model for craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a rare genetic bone disorder, characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. Craniofacial hyperostosis leads to the obstruction of neural foramina and neurological symptoms such as facial palsy, blindness, deafness, or severe headache. Mutations in ANKH (mouse ortholog ANK), a transporter of small molecules such as citrate and ATP, are responsible for autosomal dominant CMD. Knock-in (KI) mice carrying an ANK(F377del) mutation (Ank(KI/KI) ) replicate many features of human CMD. Pyrophosphate (PPi) levels in plasma are significantly reduced in Ank(KI/KI) mice. PPi is a potent inhibitor of mineralization. To examine the extent to which restoration of circulating PPi levels may prevent the development of a CMD-like phenotype, we treated Ank(KI/KI) mice with the recombinant human ENPP1-Fc protein IMA2a. ENPP1 hydrolyzes ATP into AMP and PPi. Male and female Ank(+/+) and Ank(KI/KI) mice (n ≥ 6/group) were subcutaneously injected with IMA2a or vehicle weekly for 12 wk, starting at the age of 1 wk. Plasma ENPP1 activity significantly increased in Ank(KI/KI) mice injected with IMA2a (Vehicle/IMA2a: 28.15 ± 1.65/482.7 ± 331.2 mOD/min; p <.01), which resulted in the successful restoration of plasma PPi levels (Ank(+/+) /Ank(KI/KI) vehicle treatment/Ank(KI/KI) IMA2a: 0.94 ± 0.5/0.43 ± 0.2/1.29 ± 0.8 μM; p <.01). We examined the skeletal phenotype by X-Ray imaging and μCT. IMA2a treatment of Ank(KI/KI) mice did not significantly correct CMD features such as the abnormal shape of femurs, increased bone mass of mandibles, hyperostotic craniofacial bones, or the narrowed foramen magnum. However, μCT imaging showed ectopic calcification near basioccipital bones at the level of the foramen magnum and on joints of Ank(KI/KI) mice. Interestingly, IMA2a treatment significantly reduced the volume of calcified nodules at both sites. Our data demonstrate that IMA2a is sufficient to restore plasma PPi levels and reduce ectopic calcification but fails to rescue skeletal abnormalities in Ank(KI/KI) mice under our treatment conditions.