CRISPR/Cas9修复SMALED患者iPSC中DYNC1H1G807S杂合突变的基础研究

DYNC1H1 encodes the heavy chain of cytoplasmic dynein 1, a motor protein complex implicated in retrograde axonal transport, neuronal migration and other intracellular motility functions. Human DYNC1H1 mutations disrupt dynein complex assembly and function. The legs at odd angles (Loa) (DYNC1H1 F580Y) mouse model for spinal muscular atrophy with lower extremity predominance(SMA-LED) demonstrated that dynein dysfunction disrupted not only retrograde transport of neurotrophic receptors but also anterograde transport of synaptic vesicles. Dynein dysfunction also induced neuritic swelling, which is accompanied by a significant accumulation of neurofilaments. Recently, DYNC1H1G807S heterozygous mutation was found in a Chinese family with SMALED. Whole exome sequencing of the affected boy showed a novel de novo heterozygous missense mutation c.2419 G>A (p.G807S) in exon 8 of the DYNC1H1 gene encoding for cytoplasmic dynein heavy chain 1, causing a glycine to serine substitution. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the G807S mutation dominantly disrupted dynein complex stability and function. However, the consequences of DYNC1H1G807S heterozygous mutation on SMALED have not been explored. To identify the molecular mechanisms of DYNC1H1G807S heterozygous mutation, we are planning to generate the patient-specific induced pluripotent stem cells (iPSCs) from the SMALED patient with DYNC1H1G807S heterozygous mutation and subsequent correction of the disease-causing mutation may be a potential therapeutic strategy for this disease. After designing CRISPR/Cas9 to directly target the exon 8 mutation site p.G807S in the DYNC1H1 gene, we will observe the differences in the retrograde transport of neurotrophic receptors, anterograde transport of synaptic vesicles and accumulation of neurofilaments (NF) in the motor neurons before and after CRISPR/Cas9 targeting using Quantitative Real-time PCR, Living cell imaging with Mito-Tracker Green, Immunohistochemistry, Patch electrophysiology and Immunoprecipitation assays. In addition, more obvious CRISPR/Cas9 effects such as stem cell characteristics, self-renewal, and the potential to differentiate into motor neurons and astrocytes will be observed. Our results expand the set of pathological mutation in DYNC1H1 G807S, reinforce the role of cytoplasmic dynein in disorders of neuronal migration, and provide evidence for a syndrome including spinal nerve degeneration including SMA and amyotrophic lateral sclerosis (ALS). Using CRISPR/Cas9 to correct specific DYNC1H1 G807S mutation in patient-derived iPSCs will guide future applications of CRISPR/Cas9-based gene therapies in SMALED diseases.

胞浆动力蛋白在运动神经元（MN）发育过程中起重要作用。其核心组分-胞浆动力蛋白重链（DYNC1H1）突变导致下肢型脊髓性肌肉萎缩症（SMALED），但机制尚不明确。研究表明DYNC1H1突变造成动力蛋白受损，影响MN逆向运输及突触囊泡运输，并使神经丝聚集引起轴突末梢肿胀。我们发现DYNC1H1G807S杂合突变者细胞中动力蛋白表达减少。为揭示DYNC1H1G807S突变导致SMALED分子机制，我们拟以患者来源的诱导多能干细胞（iPSC）为模型，采用CRISPR/Cas9定点修复iPSC中DYNC1H1突变点并诱导为MN和星形胶质细胞。通过线粒体探针、免疫组化、神经电生理及免疫共沉淀等方法观察基因修复前后细胞形态、功能尤其是MN细胞内运输、突触囊泡转运及神经丝蛋白表达的变化。结果将揭示DYNC1H1G807S杂合突变导致SMALED的致病机制，为SMALED临床基因治疗提供实验依据。

胞浆动力蛋白在运动神经元（MN）发育过程中起重要作用。其核心组分-胞浆动力蛋白重链（DYNC1H1）突变导致下肢型脊髓性肌肉萎缩症（SMALED），但机制尚不明确。研究表明DYNC1H1突变造成动力蛋白受损，影响MN逆向运输及突触囊泡运输，并使神经丝聚集引起轴突末梢肿胀。我们发现DYNC1H1G807S杂合突变者细胞中动力蛋白表达减少。为揭示DYNC1H1G807S突变导致SMALED分子机制，我们拟以患者来源的诱导多能干细胞（iPSC）为模型，采用CRISPR/Cas9定点修复iPSC中DYNC1H1突变点并诱导为MN和星形胶质细胞。通过线粒体探针、免疫组化、神经电生理及免疫共沉淀等方法观察基因修复前后细胞形态、功能尤其是MN细胞内运输、突触囊泡转运及神经丝蛋白表达的变化。结果将揭示DYNC1H1G807S杂合突变导致SMALED的致病机制，为SMALED临床基因治疗提供实验依据。