小柯机器人

德国慕尼黑工业大学C. Kupatt、A. Moretti等研究人员合作利用体细胞基因编辑改善了猪和人杜氏肌营养不良症模型中的骨骼与心肌功能衰竭。相关论文于2020年1月27日在线发表于国际学术期刊《自然—医学》。

研究人员表示，DMD基因（编码抗肌萎缩蛋白）的移码突变会导致杜氏肌营养不良症（DMD），从而引起患者出现终末肌肉和心力衰竭。通过序列特异性核酸酶进行的体细胞基因编辑为恢复DMD阅读框提供了新的选择，从而表达缩短但功能强大的抗肌萎缩蛋白。

研究人员在缺少DMD外显子52（DMDΔ52）的DMD猪模型以及相应的患者来源的多能干细胞模型中验证了这种方法。在DMDΔ52猪中，肌内注射血清型9的腺相关病毒载体，该载体带有一个intein-split Cas9和一对靶向外显子51两侧序列的向导RNA（AAV9-Cas9-gE51），从而诱导了缩短的抗肌萎缩蛋白（DMDΔ51–52）的表达并改善了骨骼肌功能。此外，AAV9-Cas9-gE51的全身应用导致抗肌萎缩蛋白在肌肉中的广泛表达（包括横隔膜和心脏），从而延长了存活时间并减少了心律失常的易感性。类似地，在缺乏DMDΔ52患者的诱导性多能干细胞来源的成肌细胞和心肌细胞中，AAV6-Cas9-g51介导的外显子51切除可恢复抗肌萎缩蛋白的表达并减轻骨骼肌肌管的形成，以及异常的心肌Ca²⁺信号和心律失常的易感性。在这些转化模型中，Cas9介导的外显子切除能够改善DMD的病理，并为这种严重疾病患者的新治疗方法铺平了道路。

附：英文原文

Title: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy

Author: A. Moretti, L. Fonteyne, F. Giesert, P. Hoppmann, A. B. Meier, T. Bozoglu, A. Baehr, C. M. Schneider, D. Sinnecker, K. Klett, T. Frhlich, F. Abdel Rahman, T. Haufe, S. Sun, V. Jurisch, B. Kessler, R. Hinkel, R. Dirschinger, E. Martens, C. Jilek, A. Graf, S. Krebs, G. Santamaria, M. Kurome, V. Zakhartchenko, B. Campbell, K. Voelse, A. Wolf, T. Ziegler, S. Reichert, S. Lee, F. Flenkenthaler, T. Dorn, I. Jeremias, H. Blum, A. Dendorfer, A. Schnieke, S. Krause, M. C. Walter, N. Klymiuk, K. L. Laugwitz, E. Wolf, W. Wurst, C. Kupatt

Issue&Volume: 2020-01-27

Abstract: Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51–52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

DOI: 10.1038/s41591-019-0738-2

Source: https://www.nature.com/articles/s41591-019-0738-2

Nature Medicine：《自然—医学》，创刊于1995年。隶属于施普林格·自然出版集团，最新IF：87.241
官方网址：https://www.nature.com/nm/
投稿链接：https://mts-nmed.nature.com/cgi-bin/main.plex