PDF(Pubmed)
Abstract：
A single-nucleotide deletion in the stop codon of the nuclear import receptor transportin-3 (TNPO3), also involved in human immunodeficiency virus type 1 (HIV-1) infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by extending the wild-type protein. Here, we generated a patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNP O 3 mutation. The cell model reproduced critical molecular alterations seen in patients, such as TNP O 3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the TNP O 3 mutation via CRISPR-Cas9 editing caused a significant reversion of the pathological phenotypes in edited cells, including a complete absence of the mutant TNPO3 protein, as detected with a polyclonal antibody specific against the abnormal 15-aa peptide. Transcriptomic analyses found that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9-corrected cells showed that 44% of the alterations were rescued toward normal levels. MicroRNAs (miRNAs) analyses showed that around 50% of miRNAs with impaired expression because of the disease were recovered on the mutation edition. In summary, this work provides proof of concept of the potential of CRISPR-Cas9-mediated gene editing of TNP O 3 as a therapeutic approach and describes critical reagents in LGMDD2 research.
摘要：
核输入受体转运蛋白-3（TNPO3）的终止密码子中的单核苷酸缺失，还参与人类免疫缺陷病毒1型（HIV-1）感染，通过扩展野生型蛋白导致超常染色体显性遗传病肢带型肌营养不良D2（LGMDD2）。这里,我们建立了一个源自患者的LGMDD2体外模型,作为携带TNPO3突变的永生化成肌细胞.细胞模型再现了在患者中看到的关键分子改变，如TNPO3过表达，末端肌肉标记的缺陷，和自噬过度激活。通过CRISPR-Cas9编辑纠正TNPO3突变导致编辑细胞中病理表型的显著逆转，包括完全不存在突变的TNPO3蛋白，用对异常15-aa肽具有特异性的多克隆抗体检测。转录组分析发现，15％的转录组在模型肌管中差异表达。CRISPR-Cas9校正的细胞显示，44%的改变被挽救至正常水平。MicroRNAs（miRNA）分析显示，由于疾病而导致表达受损的大约50％的miRNA在突变版本中恢复。总之,这项工作证明了CRISPR-Cas9介导的TNPO3基因编辑作为治疗方法的潜力,并描述了LGMDD2研究中的关键试剂.
