应用非天然氨基酸系统修复肌肉干细胞及干性维持的调控机制

Genetic nonsense mutation will form a stop codon and result in a truncated protein. This will cause an inherited disease, called premature termination codons (PTC)disease. PTC disease accounted for 11.2% of single-gene genetic diseases. However, there is no effective treatment for this disease so far. Our previous 973 project established a system, which inserts an unnatural amino acid into a UAG stop codon and endows protein new physical and chemical properties via introducing unnatural amino acids into random sites of a target protein. To find new therapies of the PTC diseases, our project intends to expand UAG-encoded unnatural amino acid technology and establish a new read- through system covering the UAA and UGA stop codons. Generally, protein translation terminates at one of the three stop codons. However, occasional stop codon read-through by homologous suppressor or near-cognate tRNAs carrying canonical amino acids has long been observed in both prokaryotic and eukaryotic organisms. Recently, reassignment of stop codons has been utilized to incorporate unnatural amino acids (UAAs) with novel chemical or physical properties at the introduced amber codon into proteins of interest using an orthogonal tRNA-synthetase/ tRNA pair. The technology to incorporate UAAs into proteins at specifically chosen sites has become an extremely powerful tool for protein engineering. However, a major drawback of this technology is that the UAA incorporation efficiency varies dramatically from one incorporation site to another, due to the codon context effect, which limits the biological significance and potential applications of genetic code expansion. Based on muscle stem cells and iPS cells from Duchenne muscular dystrophy patients, we insert unnatural amino acids at the endogenous nonsense mutation sites to achieve full-length protein and repair the mutant protein. Moreover, we trace the differentiation, and migration of the repaired muscle stem cell in vivo via using functional group-containing unnatural amino acids with fluorescence, NMR, and photo-crosslinking markers. We establish a high-throughput and universal gene code expansion platform of stem cell to provide a new chemical biology method for the PTC disease therapy and stem cell research，to finally achieve depth development of genetic diseases treatment.

终止密码子病（PTC病）是由于基因编码框发生无义突变，形成终止密码子导致截短蛋白的遗传性疾病，其发病率占单基因遗传病的11.2%，至今无任何治疗手段。我们前期973项目建立了以终止密码子UAG编码非天然氨基酸，实现在目标蛋白位点引入非天然氨基酸从而赋予其新的理化性质。本课题针对PTC病，拟拓展UAG编码非天然氨基酸技术，建立涵盖终止密码子UAA和UGA的新型通读体系。我们基于杜氏肌营养不良患者来源的肌肉干细胞和iPS细胞，在内源性任意无义突变位点插入非天然氨基酸，实现全长蛋白的通读，修复受损突变蛋白；进而利用含有特殊官能团的非天然氨基酸，发展荧光、核磁、光交联和光转化的蛋白质标记技术，实现示踪移植肌肉干细胞在体的增殖、分化和迁移过程监控，并建立高通量和普适性兼具的干细胞基因密码子拓展平台，为PTC病治疗和干细胞研究提供全新的化学生物学方法，实现向遗传性疾病治疗的深度跨越。

单基因遗传病（monogenic diseases）为单个基因突变引起的遗传性疾病，无义突变为单基因遗传病主要基因突变类型之一，该突变致使蛋白翻译提前终止形成截短型蛋白，进而导致提前终止密码子（Premature termination codons，PTC）疾病。PTC疾病突变类型简单，发病比例较高，是单基因遗传病治疗的极佳切入点。基因密码子扩展技术是由终止密码子编码的（UAG），由正交的tRNA，tRNA合成酶和非天然氨基酸组成，能够特异性识别终止密码子，进行翻译延伸步骤在特性位点插入非天然氨基酸的技术。.本课题利用基因密码子扩展中氨酰化tRNA能够特异性识别终止密码子并恢复蛋白表达，从而达到疾病治疗的目的。主要实验结果如下：.1.首先扩展了UAG编码系统，包括利用UAA和UAG编码非天然氨基酸NAEK，获得分别识别UAG, UAA和UGA终止密码子的三套系统。.2.以杜氏肌营养不良小鼠（mdx）为实验模型，通过基因密码子扩展技术通读无义突变TAA位点，并考察肌肉功能恢复。利用胫前肌（TA）电转质粒的方法，使PylRS/tRNA在mdx小鼠TAA处表达，并肌肉注射NAEK溶液，成功实现抗肌萎缩蛋白和mRNA上调，部分恢复肌肉功能。.3.利用非天然氨基酸NAEK具有叠氮环功能侧链，通过click反应连接荧光基团Alex488，特异性荧光标记并示踪Dystrophin蛋白。.4.首次成功制备PylRS/PyltRNA正交对的转基因小鼠，验证基因密码子扩展系统的安全性，并进一步观察其对PylRS/PyltRNA-mdx小鼠的实验性治疗效果。.此项研究验证了基因密码子扩展技术能够通读内源性无义突变位点，恢复抗肌萎缩蛋白的表达从而缓解疾病症状，提出了PTC疾病治疗和致病蛋白机制研究的新策略。

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