美国华盛顿大学医学院Jeffrey S. Chamberlain和Hichem Tasfaout共同合作，近期取得重要工作进展。他们研究通过分裂内含肽介导的蛋白反式剪接来表达大型肌营养不良蛋白。相关研究成果2024年7月17日在线发表于《自然》杂志上。

据介绍，使用腺相关病毒（AAV）载体进行基因替换一种有前景的基因疾病治疗方法。然而，这种治疗方式受到AAV（约4.7千碱基）包装能力的挑战，限制了其在涉及大编码序列的疾病中的应用，如具有14千碱基mRNA的杜氏肌营养不良症。

研究人员开发了一种利用分裂内含肽介导的蛋白质反式剪接机制来表达大型肌营养不良蛋白的新方法。研究人员鉴定了几对高效连接两段或三段片段的分裂内含肽，可以生成中型肌营养不良蛋白或全长蛋白。研究人员发现，向患肌营养不良症的小鼠体内递送两种或三种腺相关病毒（AAV）能够显著表达大型肌营养不良蛋白，并与微型肌营养不良蛋白相比表现出显著的生理改善。

此外，利用强效的肌肉向性AAVMYO，研究人员证明低总剂量（2 × 10¹³病毒基因组）足以在全身的横纹肌中表达大型肌营养不良蛋白，并在患肌营养不良症的小鼠中实现显著的生理校正。

总之，这一研究数据清楚地显示，大型肌营养不良蛋白在功能上明显优于目前正在临床试验中的微型肌营养不良蛋白。这种方法可使许多杜氏或贝克氏肌营养不良症患者受益，还可以适用于由超过AAV容量的大基因突变引起的许多其他疾病。

附：英文原文

Title: Split intein-mediated protein trans-splicing to express large dystrophins

Author: Tasfaout, Hichem, Halbert, Christine L., McMillen, Timothy S., Allen, James M., Reyes, Theodore R., Flint, Galina V., Grimm, Dirk, Hauschka, Stephen D., Regnier, Michael, Chamberlain, Jeffrey S.

Issue&Volume: 2024-07-17

Abstract: Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2×1013viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

DOI: 10.1038/s41586-024-07710-8

Source: https://www.nature.com/articles/s41586-024-07710-8