Background: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.Methods: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 Dmd^E4* 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 Dmd^E4* 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.Results:Dmd^E4* 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 Dmd 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 Dmd^E4* 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.Conclusions:Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).

中文翻译：

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通过 CRISPR 引导的胞苷脱氨酶跳过治疗性外显子可在体内挽救营养不良性心肌病

背景：肌营养不良蛋白的丢失会导致杜氏肌营养不良症（DMD），其特征是心肌和骨骼肌进行性退化，并在青春期或青年期死亡。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然不发达，部分原因是缺乏合适的临床前模型。方法：我们分析了一种新的 DMD 小鼠模型，该模型是通过引入 4 -bp 缺失到外显子 4，编码肌动蛋白肌动蛋白结合域 1 的外显子之一（称为Dmd ^E4*老鼠）。进行超声心动图、微型计算机断层扫描、肌肉力量测量和组织学分析以确定这些小鼠的心脏和骨骼肌缺陷。使用该模型，我们检查了使用胞苷碱基编辑器在体内安装外显子跳跃和挽救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Dmd ^E4*小鼠中，在治疗后 2 或 12 个月对其进行分析，以评估外显子跳跃、肌营养不良蛋白恢复和心脏表型改善的程度和骨骼肌。结果：Dmd ^E4*小鼠概括了人类 DMD 的许多方面，包括寿命缩短（约 50%）、进行性心肌病、脊柱后凸、肌肉力量的严重丧失和肌细胞退化。AAV9-eTAM 的单剂量给药在Dmd转录本中建立了 >50% 的靶向外显子跳跃，并在心脏中恢复了高达 90% 的肌营养不良蛋白。结果，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Dmd ^E4*的寿命老鼠。尽管 AAV 载体和碱基编辑器的表达逐渐下降，但肌营养不良的肌营养不良蛋白恢复和病理生理挽救至少持续了 1 年。结论：我们的研究证明了通过增强的 TAM (eTAM) 进行外显子跳跃治疗的可行性和有效性应用程序。