腺相关病毒介导CRISPR-Cas9系统靶向修复Npc1基因突变治疗C1型尼曼-匹克病的研究

Niemann-Pick disease type C1 (NPC1) is a neurodegenerative disease caused by Npc1 gene mutation. Recently，drugs, stem cells, and transgenic therapy have been applied for NPC1 therapy. However, it is difficult to repair lesions in the nervous system. The previous studies indicated that CRISPR-Cas9 technology could modify the genome of neurons, and our previous study demonstrated that CRISPR-Cas9 technology could edit the mutated Npc1 gene in hippocampal neurons of Npc1-/- mice. Therefore, we speculated that CRISPR-Cas9 technology can be used on NPC1 thrapy. This project aimed to repair the genetic mutation in embryonic Npc1-/- mice using adeno-associated virus (AAV)-mediated CRISPR-Cas9 and in ovo transfection. Then, at the genome level, high-throughput sequencing was used to investigate the repair efficiency; at the protein level, immunofluorescence histochemical technique was used to detect the changes in neurons; finally, behavioral experiments were used to study the improvement of learning memory and movement impairment. Consequently, comprehensively evaluate the efficiency of NPC1 therapy using the CRISPR-Cas9 technology. This project will provide important theoretical basis and potential therapies for the non-drug NPC1 therapy.

C1型尼曼-匹克病（NPC1）是由Npc1基因突变引起的神经退行性疾病。目前，药物、干细胞和转基因治疗已应用到NPC1的治疗研究中，但难以修复神经系统的病变。已有研究表明，CRISPR-Cas9技术能够编辑神经细胞的基因组，而我们前期研究也证明该技术可有效编辑Npc1-/-小鼠海马神经元的Npc1突变基因，故推测，CRISPR-Cas9技术可用于NPC1的治疗研究。因此，本项目拟采用腺相关病毒介导的CRISPR-Cas9技术联合子宫内转染技术修复胚胎期Npc1-/-小鼠神经元的基因突变，进而在基因组水平通过高通量测序检测基因修复效率；然后，在蛋白质水平通过免疫荧光技术检测神经元的修复情况；最后，通过行为学实验评估小鼠学习记忆和运动障碍的改善情况，从而综合评价利用CRISPR-Cas9技术治疗NPC1的效果，这将为临床上使用非药物手段治疗NPC1提供重要的理论依据和潜在的治疗方法。

C1型尼曼-匹克病（NPC1）是由Npc1基因突变引起的神经退行性疾病。Npc1基因突变导致NPC1蛋白功能下降或缺失，从而丧失脂质转运功能，造成脂质在细胞内沉积，进而影响神经元树突的形成并导致神经元变性死亡。本课题首先利用CRISPR-Cas9技术成功筛选出有效编辑突变Npc1基因的sgRNA。然后构建了Npc1基因敲除的HEK 293T细胞系，证实了Npc1基因突变导致HEK 293T细胞中未酯化胆固醇的沉积；普通光学显微镜下观察细胞形态发现，细胞核增大、细胞聚集和突触缺失；鬼笔环肽染色显示细胞骨架蛋白丝状肌动蛋白（F-actin）变薄；CCK-8法检测细胞粘附增强，集落形成实验、流式细胞术和CCK-8法检测细胞增殖受到抑制，流式细胞术检测细胞凋亡增加。此外，初步探究了MenSCs治疗NPC1的作用，经小脑立体定位注射MenSCs后，通过免疫荧光技术对比检测小脑中Calbindin的表达发现，NPC1小鼠小脑浦肯野神经元数量增加；转棒实验表明NPC1小鼠运动协调能力改善；统计NPC1小鼠寿命有所延长，这为MenSCs在NPC1疾病治疗提供理论基础。