自闭症患者SHANK2突变对神经元结构与功能发育的影响及其基因编辑修复研究

Autism Spectrum Disorders (ASDs) are childhood neurodevelopment disorders with a strong genetic basis. Candidate genes, such as SHANKs family gene SHANK2, are well known to be associated with susceptibility to ASDs. However, our understandings of how SHANK2 influences neuronal function and contributes to ASDs are still limited, yet mouse models did not provide much insights. We obtained preliminary data that SHANK2 knockdown result in neurodevelopmental defects in human induced pluripotent stem cell (iPSC) models. We collected an autism pedigree with a de novo SHANK2 nonsense mutation in the proband, and we have generated iPSCs from peripheral blood mononuclear cell of the proband by reprogramming. We hypothesize that (1) the SHANK2 nonsense mutation leads to developmental defects of the neurons and synaptic dysfunction, which is responsible for the phenotypes in the proband, (2) CRISPR/Cas9 based genome editing that corrects the nonsense mutation may reverse the neurodevelopmental defects. This project proposes to test these hypothesis using patient-derived iPSCs, and construct mouse models with SHANK2 nonsense mutations for further validation. Successful completion of the study will contribute to our understanding of the molecular mechanism of autism pathogenesis and facilitate the development of potential therapeutic approaches.

自闭症具有遗传相关性，SHANK2等易感基因复杂遗传变异是自闭症表型多样的生物学基础。基于SHANK2动物模型，既往关于SHANK2遗传变异影响神经元形态与功能，导致自闭症相关表型的机制研究尚无明确结论，提示动物模型研究结果对人源遗传变异诊疗探索的参考性有限。我们前期基于人源诱导多能干细胞来源神经发育模型，发现SHANK2基因敲减导致神经元突起发育异常。申请人前期已收集SHANK2突变自闭症家系，重编程建立了该家系诱导多能干细胞研究模型，并提出以下科学假设：1自闭症患者SHANK2突变导致神经元突起等形态发育异常，继而引起神经元功能发育异常，从而导致自闭症相关临床表型；2 基因编辑修复SHANK2突变能逆转患者神经元形态与功能表型异常。本项目拟基于患者来源神经发育模型验证上述假设，同时构建SHANK2突变动物模型在体内予以验证。本研究将为探索自闭症发病机制和治疗策略奠定理论基础。

自闭症具有遗传相关性，SHANK2等易感基因复杂遗传变异是自闭症表型多样的生物学基础。基于SHANK2动物模型，既往关于SHANK2遗传变异影响神经元形态与功能，导致自闭症相关表型的机制研究尚无明确结论，提示动物模型研究结果对人源遗传变异诊疗探索的参考性有限。本项目主要研究内容：1）该研究建立了携带SHANK2特定遗传变异的自闭症患者来源诱导多能干细胞的神经发育模型（后文简称hiPSC-iNeuron模型）；2）基于该hiPSC-iNeuron模型，利用病毒稀疏标记、神经元三维结构重建和电生理检测技术，探索特定遗传变异是否影响神经元的形态与功能发育；3）利用基因编辑技术修复该遗传变异，探索发育异常是否得到逆转；4）该遗传变异基因敲入野生小鼠模型，监测小鼠模型社交行为改变及其机制。本项目研究结果：1）成功建立携带SHANK2特定遗传变异的自闭症患者来源hiPSC-iNeuron模型；2）该SHANK2特定遗传变异影响患者hiPSC-iNeuron模型模型中多巴胺神经元形态与功能发育，基因编辑修复该遗传变异，发育异常得到部分逆转；3）该遗传变异基因敲入野生小鼠模型，小鼠模型出现社交异常等自闭样行为，且该小鼠模型运动功能未见受损。本项目基于患者来源神经发育模型提出特定遗传变异与特定发育表型之间的联系，并提出，同一突变对不同多巴胺通路有不同影响（高级情绪情感与运动功能），本项目同时构建SHANK2突变动物模型在体内予以验证。本研究将为探索自闭症发病机制和治疗策略奠定理论基础。

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