Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China (J.L., K.W., Y.Z., T.Q., L.Z., H.Q., X.C.).
Chinese Academy of Sciences, China. Joint Research Center of Hangzhou First Hospital Group and Westlake University, Zhejiang, China (J.L., K.W., Y.Z., T.Q., X.C.).
Circulation. 2021 Nov 30;144(22):1760-1776. doi: 10.1161/CIRCULATIONAHA.121.054628. Epub 2021 Oct 26.
Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model.
We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles.
mice recapitulated many aspects of human DMD, including shortened life span (by ≈50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year.
Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).
肌营养不良蛋白的缺失导致杜氏肌营养不良症（DMD），其特征是心脏和骨骼肌进行性退化，并在青少年或成年早期死亡。尽管心力衰竭已成为 DMD 患者死亡的主要原因，但有效的治疗干预措施仍未得到发展，部分原因是缺乏合适的临床前模型。
我们分析了一种通过在编码肌营养不良蛋白肌动蛋白结合域 1 的外显子 4 中引入 4 个碱基缺失而创建的新型 DMD 小鼠模型（简称 小鼠）。通过超声心动图、微计算机断层扫描、肌肉力量测量和组织学分析来确定这些小鼠的心脏和骨骼肌缺陷。利用该模型，我们研究了使用胞嘧啶碱基编辑器在体内进行外显子跳跃并修复肌营养不良性心肌病的可行性。将基于 AAV9 的 CRISPR/Cas9-AID（eTAM）与 AAV9-sgRNA 一起注射到新生 小鼠中，在治疗后 2 或 12 个月分析这些小鼠，以评估外显子跳跃、肌营养不良蛋白恢复以及心脏和骨骼肌表型改善的程度。
小鼠重现了许多人类 DMD 的特征，包括寿命缩短（约 50%）、进行性心肌病、脊柱后凸、肌肉力量严重丧失和肌细胞退化。单次给予 AAV9-eTAM 可使 转录物中的靶向外显子跳跃超过 50%，并使心脏中的肌营养不良蛋白恢复到 90%。因此，预防了早期心室重构，改善了心脏和骨骼肌功能，从而延长了 小鼠的寿命。尽管 AAV 载体和碱基编辑器的表达逐渐下降，但肌营养不良的肌蛋白恢复和病理生理学修复至少持续了 1 年。
我们的研究表明，通过增强 TAM（eTAM）进行外显子跳跃用于治疗具有可行性和疗效。
