将神经退行性疾病病人的皮肤细胞高效转分化为功能性神经元用于疾病模拟、药物筛选和毒理测试是再生医学精准治疗的前提。本实验室前期研究发现, 利用两步法—先用shRNA敲低PTB之后再高表达Brn2, 可快速高效的将人成纤维细胞转分化为成熟的神经元, 若同时敲低PTB和高表达Brn2则难以得到成熟的神经元, 说明了循序敲除PTB和过表达Brn2的重要性。为简化试验流程加大临床应用潜能, 本课题根据PTB调控可变剪接的位置效应, 构建受PTB调控的Brn2表达质粒，以实现PTB敲低和Brn2逐步高表达的AAV单质粒重编程系统。在此基础上研究与渐冻人密切相关的RNA结合蛋白EWSR1基因的突变, 利用CRISPR/Cas9技术编辑IMR90细胞, 建立EWSR1基因点突变的isogenic疾病模型, 利用发展的AAV系统解析渐冻人症的发病机理并探索临床应用的前景，为精准医学治疗提供理论基础。

Efficiently convert the skin cells derived from neurodegenerative diseases into functional neurons emerging as a valuable tool for disease modeling, drug screening and toxicology tests, which is the basis of precision medicine. Our previous data revealed a two-step process that firstly reduced the expression of PTB by shRNA and then overexpress Brn2 protein, which is able to quickly and efficiently convert the human fibroblasts into mature neurons. However, it is difficult to get functional neuronal cells if knockdown PTB and overexpress Brn2 simultaneously, suggesting that the sequential event is essential for neuronal conversion. In order to further simplify the process for future clinical application, here we proposed to make a single AAV plasmid for induced expression of Brn2, which is based on the positional effect of PTB in splicing regulation. The new reprogramming system will be used to study amyotrophic lateral sclerosis (ALS) related point mutations in RNA binding protein EWSR1 gene. We planned to first make an isogenic mutation cell line in IMR90 cell by CRISPR/Cas9 technology, and then to directly convert both wild type and mutation cell lines into functional neurons for disease modeling, the research may decipher the pathological mechanisms caused by EWSR1 mutation and provide a basis for precision medical treatment.