SUMMARY/ABSTRACT Hypophosphatasia (HPP) is a rare inherited disorder that affects the development of bones and teeth the cause of which is mutations in the tissue-nonspecific alkaline phosphatase (TNSALP/TNAP) gene (ALPL). In addition, dental hard tissues have been hypothesized to be the most sensitive to HPP, as premature loss of anterior teeth in toddlers is often the first sign of disease in mild cases of HPP, and some individuals fall within the subtype odonto-HPP, where dental defects are the only apparent manifestation. Although useful for modeling many important features of HPP, murine models harboring ALPL mutations do not faithfully represent the broad spectrum of human HPP clinical tooth and bone abnormalities. Therefore, in order to gather important insights into the molecular and natural history of the profound tooth and bone phenotypes of HPP, it is essential to develop new models. In this light, the size and basic anatomy of the sheep skeleton and Haversian remodeling is comparable to humans, and sheep are born with baby teeth that are lost naturally before being replaced by permanent teeth. Recent advances in mammalian gene editing using the CRISPR/Cas9 system have made disease-specific point mutation knock-in accessible in a variety of species, including sheep. Since the sheep TNAP protein sequence is highly conserved compared to humans (89% identity), two distinct mutations were targeted in the human ALPL gene that have previously been characterized, [Alanine (GCC)?Threonine (ACC) in exon 5 (c.346 G>A) and Isoleucine (ATC)?Methionine (ATG) in exon 10 (c.1077 C>G). Capitalizing on the advances in gene editing, a sheep model of HPP has been generated, for the first time, that accurately recapitulates the tooth phenotype of the exon 10 (c.1077 C>G) human point mutation. In Aim 1, demonstration of the validity of the ALPL knock-in sheep as a model for dental manifestations of HPP in both deciduous and permanent teeth will be accomplished. In Aim 2, the efficacy of a bone/tooth-targeted recombinant TNAP therapy to reverse the dental manifestations of HPP and improve the mineralization for of all dental tissues will be determined. If successful, these studies will represent a new platform for the accurate evaluation of the phenotype of HPP but also define the sheep as an amenable animal platform to model other human bone and mineral disorders.

R21DE028076

2018-08-01

2020-07-31