Respiratory Distress in Sheep with Hypophosphatasia: Etiology, Functional Consequences and Rescue

Project Summary This new R21 application “Respiratory Distress in Sheep with Hypophosphatasia: Etiology, Functional Consequences and Rescue” will provide novel insights into the role of tissue non-specific alkaline phosphatase (TNSALP) on lung development and function. Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism in patients with loss of function mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL). This disorder is associated with high neonatal morbidity and mortality, impaired musculoskeletal development, and respiratory distress syndrome (RDS), yet the in utero origins of the disease are unknown and the ontogeny of development of lung pathology is poorly understood. The PI’s laboratory developed the first large animal model of HPP in sheep, creating an ALPL gene mutation in exon 10 (c.1077C>G) recapitulating the human HPP phenotype. As observed in human patients, but not in mice, neonatal lambs carrying a loss of function mutation in ALPL commonly have respiratory complications as neonates and have an increased incidence of pneumonia compared to wildtype control lambs throughout life. Preliminary findings in HPP sheep demonstrate severe lung pathology on Day 100 of gestation (GD100), suggesting programming of the postnatal lung phenotype during fetal development, and that the known postnatal respiratory complications result from more than insufficient rib cage mineralization. This sheep HPP model will be used to test the hypothesis that reduced TNSALP activity causes lung-specific developmental abnormalities, as well as deficiencies in diaphragm structure and respiratory function that contribute to RDS by the analyses of WT and HPP fetuses on GD130 and in lambs at 4 months of age. Specific Aim 1 will characterize the respiratory phenotype in WT and HPP sheep throughout development (GD130 and postnatal 4 months of age). Specific Aim 2 will determine the impact of HPP on resting lung function in 4 month old WT and HPP sheep by measuring arterial blood gases (pO2, pCO2), bicarbonate, pH, ammonia and lactate, and bronchoalveolar lavage (BAL). Specific Aim 3 will determine the efficacy of recombinant TNSALP therapy to rescue the developmental respiratory phenotype in HPP sheep using lentiviral GFP control or ALPL-GFP constructs injected into WT or HPP sheep zygotes. This interdisciplinary collaborative team includes a current clinical veterinary physiologist collaborator (an expert on respiratory diseases in ruminants) who will collect arterial blood and bronchoalveolar lavage fluids for assessment of lung pathophysiology. This study will provide the first investigations into the in utero developmental origins of the severe respiratory phenotype in sheep with HPP, and describe contributions of non-skeletal tissues (lung, lung-resident immune cells, diaphragm) to RDS in HPP. These findings will have immediate relevance to the management of RDS in human HPP.