WNT10A调控恒牙胚发育的机制及基因转座子治疗先天性缺牙的研究

先天性缺牙可见于乳牙和恒牙。本课题组在国际上率先报道了WNT10A双等位基因失功能突变的患者乳牙个别缺失，而恒牙全部先天缺失这一独特表型，率先提出WNT10A是维持恒牙发育关键基因的科研假说。但是，已建立的Wnt10a基因敲除小鼠无法模拟恒牙缺失表型。为建立动物模型探索WNT10A对恒牙胚发育的关键作用和机制、以及先天性缺牙的基因治疗途径，本研究拟通过：1. 使用双牙列的比格犬，构建Wnt10a基因敲除犬模型，观察是否出现类似人类患者所有恒牙先天缺失的症状，验证Wnt10a基因对恒牙胚发育的作用，并通过牙胚RNA测序和原位杂交染色，探索可能的分子调控机制；2.探索应用基因转座子技术，变“改”为“补”，在Wnt10a基因敲除犬颌骨内局部注射补充外源性Wnt10a基因序列，研究体内局部恢复Wnt10a蛋白表达、挽救恒牙胚发育的基因疗法。为探索可转化应用的先天性缺牙基因治疗新技术提供基础。

In human, the tooth agenesis may happen to both primary and permanent dentition. Unlike the partial edentulous condition, we have reported that patient with bi-allelic WNT10A mutation lacked all their permanent teeth congenitally. And we have generated Wnt10a knockout mice, yet the mice have only one dentition and we failed to phenocopy tooth agenesis in murine model. Thus, due to the lack of animal model，the exact function of WNT10A is not clear. .In order to investigate the pivotal role of WNT10A for permanent tooth, and to explore applicable gene therapy for tooth agenesis, in this study, we propose: 1, by using diphyodont beagle dog, to generate Wnt10a gene knockout dog, and to assess whether it can phenocopy the congenital missing of permanent tooth as in human patient with bi-allelic WNT10A mutation, to confirm the pivotal role of Wnt10a for tooth development, and to explore the possible molecular mechanism by RNA-seq and in situ hybridization; 2, to apply local injection of exogenic Wnt10a gene segment into alveolar bone of Wnt10a knockout dog by employing transposon technique, in order to compensate the expression of Wnt10a and development of permanent tooth germ. Our research may help to elucidate the role of Wnt10a in permanent tooth development and to explore translational and feasible gene therapy for treatment of tooth agenesis with preclinical experimental evidence on animal model.