遗传性耳聋伴固醇代谢异常新型小型猪动物模型的建立与应用

Hearing loss (HL) is the most prevalent sensory deficit primarily originating from genetic etiologies. To find the effective methods to diagnose and treat the disease clinically, it is necessary to identify the genes that are responsible for the deafness and uncover their underlying mechanisms. Recently, a novel causative gene, OSBPL2 (encoding oxysterol binding protein-like protein 2), mutated in nonsyndromic hearing loss (NSHL) families was identified by our laboratory and German group. However, the underlying mechanism for the roles of mutated OSBPL2 in deafness with the sterol metabolic abnormalities remains unknown and OSBPL2 knock out animal models has not yet been reported. One useful approach to combat disorders is to generate genetically modifed animal models for studying pathogenesis, diagnosis, treatment, prognosis, and prevention. Although rodents, such as mice, have been used as model for significantly promoting the otological basic and clinical research, it is not a suitable model for the big differences of the morphologic and physiological formation in the inner ear between mice and humans. Oppositely, owing to the anatomic, physiologic, pathologic, and genetic similarities between pigs and humans, genetically modifed pig models will be the most ideal experimental animal models in otological research in the future. In the current project, the precision genome editing technology CRISPR/Cas9 will be used to target and disrupt OSBPL2 gene in Bama miniature pigs followed by somatic cell nuclear transfer (SCNT) and embryo transfer to produce the first genetically modifed pigs in the world. With this approach, the knock out pigs could be useful large animal models for providing further insight into pathogenesis and novel prevention and treatment applications of hereditary hearing loss with the sterol metabolic abnormalities.

耳聋是人类常见多涉及遗传因素的感觉缺陷性疾病，发现新的耳聋基因并探索其作用机制是寻找相应防治对策的重要基础。氧化固醇结合蛋白样蛋白2 (OSBPL2) 基因是新近申请者实验室和德国科研小组先后发现的新的耳聋致病基因，有关其致聋伴固醇代谢异常分子机制和基因敲除动物模型的构建迄今均未见报道。遗传修饰动物模型是有效地认识人类疾病发生、发展和防治措施的一个极重要的生物工具。作为啮齿类动物，特别是小鼠，虽较大地推动了耳科基础和临床应用研究，但因其在内耳结构及病理生理等与人类相去甚远。猪在内耳形成过程中其解剖、生理以及基因等与人类更为相似，普遍认为猪是未来耳科学研究领域最理想的实验动物模型。本项目以巴马小型猪为材料，拟结合CRISPR/Cas9技术制备OSBPL2基因敲除细胞系，后通过体细胞核移植和胚胎移植，首次创建OSBPL2基因敲除小型猪，为深入研究遗传性耳聋伴固醇代谢异常的发病机制及新的防治策略

基于以动物模型进行基因型-表型研究和功能探讨是研究听觉发生/障碍的重要实验手段。与其他模型动物相比，猪的内耳发育进程以及听器结构与人更为接近，很多猪/人同源基因突变的表型相似，因而猪作为耳聋动物模型更具优势。本项目在申请者课题组已发现的遗传性耳聋致病新基因-氧化固醇结合蛋白样蛋白2 (OSBPL2)的基础上，通过构建OSBPL2基因敲除(KO)的巴马小型猪模型，研究其基因型-表型的关系，并从在体水平进行听力学与内耳病理的表征，取得了预期的研究成果：1) CRISPR/Cas9介导的基因编辑技术、体细胞核移植和胚胎移植技术在国际上首次构建了OSBPL2-KO的巴马小型猪模型。2) OSBPL2-KO小型猪表现为渐进性听力损失，与人OSBPL2基因突变致聋(DFNA67)的临床表型一致，进一步明确了OSBPL2基因突变是导致DFNA67的分子病因。3) OSBPL2-KO敲除导致猪耳蜗外毛细胞数量减少，毛细胞静纤毛出现缺失或排列紊乱，且毛细胞区域出现凋亡。4) OSBPL2-KO小型猪呈现肥胖表型且血脂水平异常，高脂饮食干预可进一步促进总胆固醇(TC)与低密度脂蛋白胆固醇(LDL-C)的异常积累，显著加重和加速听力损失的水平和进展，并促进耳蜗组织的凋亡。5) 进一步从在体与离体水平探索了OSBPL2在听觉发生/障碍以及肥胖表型发生中的分子机制。6) 总结发表了7篇SCI期刊和2篇中文核心期刊收录的研究论文，另有1篇论文已被接收，申请国家发明专利1项。7）基于本项目的部分研究成果与技术支撑，本课题组2020年新获2项NSFC面上项目资助（批准号：32070587，82071052）。.本项目为深入理解OSBPL2生物学功能及其在听觉发生/障碍中的分子机制提供了理论依据和实验证据，并将为深入研究遗传性耳聋伴固醇代谢异常的发病机制及提出防治策略提供理想的大动物模型。