Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo
Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo
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通过 CRISPR 引导的胞苷脱氨酶进行治疗性外显子跳跃可挽救体内营养不良型心肌病
DOI：
10.1161/circulationaha.121.054628
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发表时间：
2021-10
期刊：
影响因子：
37.8
通讯作者：
Chang X
中科院分区：
文献类型：
--
作者：
Li J;Wang K;Zhang Y;Qi T;Yuan J;Zhang L;Qiu H;Wang J;Yang H.T;Dai Y;Song Y;Chang X
补充数字内容可在文本中找到。背景资料：肌营养不良蛋白的缺失导致杜氏肌营养不良症（DMD），其特征在于心脏和骨骼肌的进行性变性，以及青春期或年轻成年期的死亡率。虽然心力衰竭已上升为DMD患者死亡的主要原因，但有效的治疗干预措施仍不发达，部分原因是缺乏合适的临床前模型。研究方法：我们分析了一种新的DMD小鼠模型，该模型是通过将4-bp缺失引入外显子4（编码肌营养不良蛋白肌动蛋白结合结构域1的外显子之一）创建的（称为DmdE 4 * 小鼠）。进行超声心动图、微计算机断层扫描、肌力测量和组织学分析，以确定这些小鼠的心脏和骨骼肌缺陷。使用该模型，我们研究了使用胞苷碱基编辑器在体内安装外显子跳跃和拯救营养不良性心肌病的可行性。将基于AAV 9的CRISPR/Cas9-AID（eTAM）与AAV 9-sgRNA一起注射到新生DmdE 4 * 小鼠中，在治疗后2个月或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以及心脏和骨骼肌的表型改善的程度。结果如下：DmdE 4 * 小鼠重现了人类DMD的许多方面，包括寿命缩短（约50%）、进行性心肌病、脊柱后凸、肌肉力量严重丧失和肌细胞变性。AAV 9-eTAM的单剂量施用在Dmd转录物中建立了>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。因此，早期心室重塑得到预防，心脏和骨骼肌功能得到改善，导致DmdE 4 * 小鼠的寿命延长。尽管AAV载体和碱基编辑器的表达逐渐下降，肌营养不良症的肌营养不良蛋白恢复和病理生理学拯救持续了至少1年。结论：我们的研究证明了通过增强的TAM（eTAM）建立外显子跳跃用于治疗应用的可行性和有效性。
Supplemental Digital Content is available in the text. 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 DmdE4* 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 DmdE4* 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: DmdE4* 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 DmdE4* 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).
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影响因子：
14.9
作者：
Ritchie ME;Phipson B;Wu D;Hu Y;Law CW;Shi W;Smyth GK
通讯作者：
Smyth GK
影响因子：
20.1
作者：
Pinto AR;Ilinykh A;Ivey MJ;Kuwabara JT;D'Antoni ML;Debuque R;Chandran A;Wang L;Arora K;Rosenthal NA;Tallquist MD
通讯作者：
Tallquist MD
影响因子：
9.8
作者：
McQuin C;Goodman A;Chernyshev V;Kamentsky L;Cimini BA;Karhohs KW;Doan M;Ding L;Rafelski SM;Thirstrup D;Wiegraebe W;Singh S;Becker T;Caicedo JC;Carpenter AE
通讯作者：
Carpenter AE
影响因子：
2.6
作者：
Mavrogeni S;Papavasiliou A;Spargias K;Constandoulakis P;Papadopoulos G;Karanasios E;Georgakopoulos D;Kolovou G;Demerouti E;Polymeros S;Kaklamanis L;Magoutas A;Papadopoulou E;Markussis V;Cokkinos DV
通讯作者：
Cokkinos DV
影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR