Hichem Tasfaout, Christine L. Halbert, Timothy S. McMillen, James M. Allen, Theodore R. Reyes, Galina V. Flint, Dirk Grimm, Stephen D. Hauschka, Michael Regnier, Jeffrey S. Chamberlain
{"title":"分裂内含蛋白介导的蛋白质转接,以表达大型肌营养不良症蛋白。","authors":"Hichem Tasfaout, Christine L. Halbert, Timothy S. McMillen, James M. Allen, Theodore R. Reyes, Galina V. Flint, Dirk Grimm, Stephen D. Hauschka, Michael Regnier, Jeffrey S. Chamberlain","doi":"10.1038/s41586-024-07710-8","DOIUrl":null,"url":null,"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 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase 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 × 1013 viral 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. A method is developed for expressing large dystrophins to enhance muscle function in mouse models of muscular dystrophy, with potential clinical benefits for numerous disorders caused by mutations in large genes that exceed the adeno-associated virus capacity.","PeriodicalId":18787,"journal":{"name":"Nature","volume":null,"pages":null},"PeriodicalIF":50.5000,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Split intein-mediated protein trans-splicing to express large dystrophins\",\"authors\":\"Hichem Tasfaout, Christine L. Halbert, Timothy S. McMillen, James M. Allen, Theodore R. Reyes, Galina V. Flint, Dirk Grimm, Stephen D. Hauschka, Michael Regnier, Jeffrey S. Chamberlain\",\"doi\":\"10.1038/s41586-024-07710-8\",\"DOIUrl\":null,\"url\":null,\"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 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase 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 × 1013 viral 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. 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Split intein-mediated protein trans-splicing to express large dystrophins
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 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase 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 × 1013 viral 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. A method is developed for expressing large dystrophins to enhance muscle function in mouse models of muscular dystrophy, with potential clinical benefits for numerous disorders caused by mutations in large genes that exceed the adeno-associated virus capacity.
期刊介绍:
Nature is a prestigious international journal that publishes peer-reviewed research in various scientific and technological fields. The selection of articles is based on criteria such as originality, importance, interdisciplinary relevance, timeliness, accessibility, elegance, and surprising conclusions. In addition to showcasing significant scientific advances, Nature delivers rapid, authoritative, insightful news, and interpretation of current and upcoming trends impacting science, scientists, and the broader public. The journal serves a dual purpose: firstly, to promptly share noteworthy scientific advances and foster discussions among scientists, and secondly, to ensure the swift dissemination of scientific results globally, emphasizing their significance for knowledge, culture, and daily life.