Gene therapy using recombinant AAV type 8 vector encoding TNAP-D10 improves the skeletal phenotypes in murine models of osteomalacia

Hypophosphatasia (HPP), caused by loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNAP), is characterized by skeletal and dental hypomineralization that can vary in severity from life-threatening to milder manifestations only in adulthood. PHOSPHO1 deficiency leads to early-onset scoliosis, osteomalacia, and fractures that mimic pseudo-HPP. Asfotase alfa, a life-saving enzyme replacement therapy approved for pediatric-onset HPP, requires subcutaneous injections 3-6 times/week. We recently showed that a single injection of an adeno-associated virus vector serotype 8 harboring TNAP-D10 (AAV8-TNAP-D10) effectively prevented skeletal disease and prolonged life in Alpl-/- mice phenocopying infantile HPP. Here, we aimed to determine the efficacy of AAV8-TNAP-D10 in improving the skeletal and dental phenotype in the AlplPrx1/Prx1 and Phospho1-/- mouse models of late-onset (adult) HPP and pseudo-HPP, respectively. A single dose of 3x1011 vector genomes per body (vg/b) was injected intramuscularly into 8-weeks-old AlplPrx1/Prx1 and WT littermates, or into 3-days old Phospho1-/- and WT mice, and treatment efficacy evaluated after 60 days for late-onset HPP mice and after 90 days for Phospho1-/- mice. Biochemical analysis showed sustained serum alkaline phosphatase activity, reduced plasma PPi levels and radiographic images, micro-CT analysis, and H&E staining showed improvements in the long bones in the late-onset HPP mice and corrected scoliosis in the Phospho1-/- mice. Micro-CT analysis of the dentoalveolar complex did not reveal significant changes in the phenotype of late-onset HPP and pseudo-HPP models. Moreover, alizarin red staining analysis showed that AAV8-TNAP-D10 treatment did not promote ectopic calcification of soft organs in adult HPP mice after 60 days of treatment, even after inducing chronic kidney disease. Overall, the AAV8-TNAP-D10 treatment improved the skeletal phenotype in both the adult HPP and pseudo-HPP mouse models. This pre-clinical study will contribute to the advancement of gene therapy for the improvement of skeletal disease in patients with heritable forms of osteomalacia.