基于单碱基编辑系统的遗传性痉挛性截瘫CYP7B1基因点突变修复研究

Hereditary spastic paraplegia (HSP or SPG) is a group of neurodegenerative diseases, which mainly involved in the corticospinal tract. Unfortunately, there are no effective treatments for this disease. In recent years, the establishment and improvement of base editing technology, BE3 and ABEs, have brought new hope for treating HSP disease. We conducted a mutation screening of autosomal recessive HSP（ARHSP） families in Fujian province and found that spastic paraplegia type 5 (SPG 5) is the most common subtype of ARHSP. The proportion of SPG5 in Chinese ARHSP (up to 34.6%) is significantly higher than that in Europe and the United States (7%). In addition, up to 86.1% of SPG5 patients were found to carry the hotspot mutation c.334 C> T (p.R112*) of CYP7B1 gene. Therefore, the precise correction targeted on this hotspot mutation is of great significance. Besides, we have established fibroblast cell lines from the patient carrying hotspot mutation, and optimized platforms for iPSCs reprogramming and glutamatergic neurons differentiation. In this project, we will firstly use ABEs system to correct the genome of SPG5 iPSCs, which carrying homozygous mutation c.334 C> T (p.R112*) of CYP7B1 gene. Then, we will evaluate the expression of CYP7B1 protein, the neuron morphology, and the electrophysiological property of glutamatergic neurons derived from the corrected SPG5 iPSCs. Above all, our research will provide a new direction for the clinical treatment of SPG5.

遗传性痉挛性截瘫（HSP或SPG）是一类主要累及皮质脊髓束的神经退行性疾病，临床缺乏有效治疗手段。近年来，单碱基基因编辑技术BE3和ABEs的建立和完善为本病治疗带来了希望。我们前期对福建地区常染色体隐性HSP家系进行基因突变筛查，发现痉挛性截瘫5型（SPG5）是其最常见的亚型，比例高达34.6%，明显高于欧美人群7%的比例。同时，还发现高达86.1% 的SPG5患者携带CYP7B1基因热点突变c.334 C>T（p.R112*）。因此，基于该突变位点的精准修复具有重要意义。此外，我们还建立了上述热点突变患者的成纤维细胞系，优化了iPS重编程和谷氨酸能神经元诱导分化等平台。本项目将应用ABEs系统对携带热点突变的iPS细胞进行原位修复；进一步在修复后iPS细胞分化来源的谷氨酸能神经元水平，从CYP7B1蛋白表达、神经元形态及功能等多个层次探讨基因修复效果，为本病临床治疗提供新的方向。

遗传性痉挛性截瘫（HSP）是一类主要累及皮质脊髓束的神经变性病。痉挛性截瘫5型（SPG5）作为中国南方人群中最为常见的HSP亚型，致病基因CYP7B1突变可导致羟基胆固醇在外周的蓄积，但对神经退行性变的具体作用机制尚不清楚。本项目以SPG5 诱导性多能干细胞（IPS）来源的皮层神经元为细胞模型，初步阐明了CYP7B1基因突变影响神经突起生长是神经变性的可能机制。此外，HSP明显的遗传异质性，给临床诊断带来了困难。截至目前，超过半数的痉挛性截瘫患者未发现已知致病基因。因此，本项目还建立了遗传性痉挛性截瘫国际注册队列（NCT04006418），通过全外显子测序、多重连接探针扩增技术等，报道了多个临床罕见亚型，丰富了疾病表型谱和基因突变谱。更为重要的是，我们首个发现并命名了遗传性痉挛性截瘫新的亚型SPG80，并初步阐明内体转运障碍是引起神经细胞凋亡的可能原因。这将有效提高患者的临床诊断率，并将来的治疗提供干预靶点。以上相关结果，本项目申请人以共同第一或通讯身份在《Brain》等国外专业期刊上发表6篇论著。