Correction of DMD in human iPSC-derived cardiomyocytes by base-editing-induced exon skipping.
Correction of DMD in human iPSC-derived cardiomyocytes by base-editing-induced exon skipping.
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通过基础编辑引起的外显子跳过，对人IPSC衍生的心肌细胞的DMD进行校正。
DOI：
10.1016/j.omtm.2022.11.010
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发表时间：
2023-03-09
期刊：
影响因子：
--
通讯作者：
Han, Renzhi
中科院分区：
文献类型：
--
作者：
Wang, Peipei;Li, Haiwen;Zhu, Mandi;Han, Rena Y.;Guo, Shuliang;Han, Renzhi
杜氏肌营养不良症（DMD）是由DMD基因突变引起的。之前，我们证明了腺嘌呤碱基编辑（ABE）可以有效地纠正DMD小鼠模型中的无义点突变。在这里，我们探索了碱基编辑介导的外显子跳跃作为DMD治疗策略的可行性，使用来自人诱导多能干细胞（hiPSC）的心肌细胞。我们首先使用CRISPR-Cas9基因编辑产生了具有跨越外显子48至54（ΔE48-54）的大缺失的DMD hiPSC系。如通过RT-PCR、蛋白质印迹和免疫荧光染色所检查的，肌营养不良蛋白表达在DMD hiPSC衍生的心肌细胞（iCM）中被破坏。ABE和靶向剪接受体的指导RNA（gRNA）的转染导致AG向GG的有效转化（35.9% ± 5.7%），并使外显子55跳读成为可能。在DMD iCM中，单个克隆中的完全AG至GG转化恢复了肌营养不良蛋白表达（野生型[WT]的42.5% ± 11%）。此外，我们设计了gRNA以靶向突变热点中外显子6、7、8、43、44、46和53的剪接位点，并证明了它们在iCM中诱导外显子跳跃的效率。这些结果突出了ABE介导的外显子跳跃作为DMD的有希望的治疗方法的巨大前景。这项工作证明了通过诱导多能干细胞衍生的人类心肌细胞中腺嘌呤碱基编辑介导的外显子跳跃可以有效纠正杜氏肌营养不良症，并强调了碱基编辑治疗由DMD突变引起的遗传性心肌病的巨大前景。
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Previously, we showed that adenine base editing (ABE) can efficiently correct a nonsense point mutation in a DMD mouse model. Here, we explored the feasibility of base-editing-mediated exon skipping as a therapeutic strategy for DMD using cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs). We first generated a DMD hiPSC line with a large deletion spanning exon 48 through 54 (ΔE48–54) using CRISPR-Cas9 gene editing. Dystrophin expression was disrupted in DMD hiPSC-derived cardiomyocytes (iCMs) as examined by RT-PCR, western blot, and immunofluorescence staining. Transfection of ABE and a guide RNA (gRNA) targeting the splice acceptor led to efficient conversion of AG to GG (35.9% ± 5.7%) and enabled exon 55 skipping. Complete AG to GG conversion in a single clone restored dystrophin expression (42.5% ± 11% of wild type [WT]) in DMD iCMs. Moreover, we designed gRNAs to target the splice sites of exons 6, 7, 8, 43, 44, 46, and 53 in the mutational hotspots and demonstrated their efficiency to induce exon skipping in iCMs. These results highlight the great promise of ABE-mediated exon skipping as a promising therapeutic approach for DMD. This work demonstrates the efficient correction of Duchenne muscular dystrophy by adenine-base-editing-mediated exon skipping in human cardiomyocytes derived from induced pluripotent stem cells and highlights the great promise of therapeutic base editing for genetic cardiomyopathy caused by mutations in DMD.
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