The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

Bone is a common site for metastasis in breast cancer patients and is associated with a series of complications that significantly compromise patient survival, partially due to the advanced stage of disease at the time of detection. Currently, no clinically-approved biomarkers can identify or predict the development of bone metastasis. We recently identified a unique peptide fragment of parathyroid hormone-related protein (PTHrP), PTHrP(12-48), as a validated serum biomarker in breast cancer patients that correlates with and predicts the presence of bone metastases. In this study, the biological activity and mode of action of PTHrP(12-48) was investigated. Sequence-based and structure-based bioinformatics techniques predicted that the PTHrP(12-48) fragment formed an alpha helical core followed by an unstructured region after residue 40 or 42. Thereafter, detailed structure alignment and molecular docking simulations predicted a lack of interaction between PTHrP(12-48) and the cognate PTH1 receptor (PTHR1). The in silico prediction was confirmed by the lack of PTHrP(12-48)-stimulated cAMP accumulation in PTHR1-expressing human SaOS2 cells. Using a specific human PTHrP(12-48) antibody that we developed, PTHrP(12-48) was immunolocalized in primary and bone metastatic human breast cancer cells, as well as within human osteoclasts (OCLs) in bone metastasis biopsies, with little or no localization in other resident bone or bone marrow cells. In vitro, PTHrP(12-48) was internalized into cultured primary human OCLs and their precursors within 60 min. Interestingly, PTHrP(12-48) treatment dose-dependently suppressed osteoclastogenesis, via the induction of apoptosis in both OCL precursors as well as in mature OCLs, as measured by the activation of cleaved caspase 3. Collectively, these data suggest that PTHrP(12-48) is a bioactive breast cancer–derived peptide that locally regulates the differentiation of hematopoietic cells and the activity of osteoclasts within the tumor–bone marrow microenvironment, perhaps to facilitate tumor control of bone.

Down syndrome (DS), characterized by trisomy of human chromosome 21, is associated with a variety of endocrine disorders as well as profound skeletal abnormalities. The low bone mass phenotype in DS is defined by low bone turnover due to decreased osteoclast and osteoblast activity, decreasing the utility of antiresorptive agents in people with DS. Sclerostin antibody (SclAb) is a therapeutic candidate currently being evaluated as a bone anabolic agent. Scl, the product of the sclerostin gene (SOST), inhibits bone formation through its inhibition of Wnt signaling. SclAb increases bone mass by suppressing the action of the endogenous inhibitor of bone formation, Scl. To examine the effects of SclAb on the DS bone phenotype, 8‐week‐old male wild‐type (WT) andTs65Dn DS mice were treated with 4 weekly iv injections of 100 mg/kg SclAb. Dual‐energy X‐ray absorptiometry (DXA), microCT, and dynamic histomorphometry analyses revealed that SclAb had a significant anabolic effect on both age‐matched WT littermate controls and Ts65Dn DS mice that was osteoblast mediated, without significant changes in osteoclast parameters. SclAb treatment significantly increased both cortical and trabecular bone mass at multiple sites; SclAb treatment resulted in the normalization of Ts65Dn bone mineral density (BMD) to WT levels in the proximal tibia, distal femur, and whole body. Ex vivo bone marrow cultures demonstrated that SclAb increased the recruitment of the mesenchymal progenitors into the osteoblast lineage, as indicated by increased alkaline phosphatase–positive colonies, with no effect on osteoclast differentiation. Together, in the setting of a murine model of DS and decreased bone turnover, SclAb had a potent anabolic effect. SclAb stimulated bone formation and increased osteoblastogenesis without affecting osteoclastogenesis or bone resorption. These data suggest that SclAb is a promising new therapy to improve bone mass and reduce fracture risk in the face of the low bone mass and turnover prevalent in the DS population. © 2017 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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