The Construction Of X-linked Hypophosphatemia Model In Rabbits

Hypophosphatemic rickets(HR) is a kind of disorders accompanied by a defect in bone mineralization and hypophosphatemia caused by a renal phosphate wasting. X-linked hypophosphatemia(XLH) is the most common genetic disorder of phosphate homeostasis, with an incidence of 1 in 20,000 in human, which showed hypophosphatemia, low serum of 1, 25-dihydroxyvitamin D, increased alkaline phosphatase, short stature, and also the abnormal development of the bone and tooth, etc.Previously studies have showed that the gene mutation of PHEX could cause the human X-linked Hypophosphatemia(XLH). To date, more than 368 mutations of the PHEX, including missense, frameshift, nonsense and splicing mutation have been reported in the XLH patients(http://www.phe-xdb.mcgill.ca). The PHEX is located in X chromosome, with 22 exons and encodes 749-amino acid glycoprotein, which is also belongs to the M13 membrane bound zinc metalloendopeptidase family member. It is postulated that the PHEX has been identified as an important factor involved in the regulation of phosphate homeostasis, bone mineralization, renal inorganic phosphate handing and vitamin D metabolism. Therefore, the function of PHEX will beneficial on the physiopathologic mechanism of XLH, such as the bone mineralization, kidney phosphorus transport and activated vitamin D production, etc.Recently, it have been demonstrated that many diseases are related to gene mutation, while the exact pathogenesis mechanism is unknown, and the lack of effective treatment measures. Therefore, it is extremely urgent to establish an effective animal model which can simulate the key symptoms of human disease, could be utilized as a drug screening model. Currently, mice models have been widely used to model human diseases for investigating pathogenesis and developing therapeutic strategies. However, these mouse models cannot fully recapitulate the human phenotypes due to the different physiological traits, gene expression between mice and human. Rabbits are a classic animal model species, which has many similarities with human beings in terms of physiology, anatomy and genetics than mice and rats. They also require low cost maintenance and short pregnancy period compared to pigs and monkey. Therefore, the rabbit were used as an animal model, which will provide a reliable and ideal animal model for human metabolic diseases in this study.At present, the clustered regularly interspaced short repeats(CRISPR)-associated(Cas), CRISPR/Cas9 system has been emerged as a powerful genome engineering tool. The technology is simple in design, easy to use and can accurately modify the target genes, which shown highly efficiency in the genome engineering of many species, including mice, pigs, rats, zebrafish, and goats. The wide application of this technology will helpful for the establishment of the human disease animal model.Although the PHEX mutation mice model has been reported and showed an advance in understanding the pathogenesis of XLH, there is no adequate and proper animal model for the safety assessment of physiotherapy and drug screening. In attempt to establish a novel animal model to recapitulate human XLH, a XLH model with PHEX gene knockout(KO) was generated via co-injection of Cas9/sg RNA m RNA into rabbit zygotes in this study. The results showed that the typical phenotypes of growth retardation, hypophosphatemia, elevated serum FGF23 levels and bone mineralization were observed in PHEX KO rabbit, which compared to those normal controls. In conclusion, for the first time, we have successfully obtained PHEX KO rabbits recapitulating human XLH using CRISPR/Cas9 system. This novel XLH rabbit model could be utilized as a drug screening model for XLH prevention and treatment.