Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β654-Thalassemia Mouse Model.

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Human gene therapy Pub Date : 2024-10-01 Epub Date: 2024-08-13 DOI:10.1089/hum.2023.202
Dan Lu, Xiuli Gong, Xinbing Guo, Qin Cai, Yanwen Chen, Yiwen Zhu, Xiao Sang, Hua Yang, Miao Xu, Yitao Zeng, Dali Li, Fanyi Zeng
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引用次数: 0

Abstract

β654-thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β654 mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β654-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β654-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β654 mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β654 allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β654 mouse model.

对内源性隐性 3'剪接位点进行基因编辑可纠正β654-地中海贫血小鼠模型的 RNA 剪接缺陷。
β-654-地中海贫血症是由β-球蛋白基因第二个内含子(IVS-II)中的一个点突变引起的,该突变激活了一个隐性 3'剪接位点,从而导致不正确的 RNA 剪接。我们之前的研究表明,当直接删除β654突变序列或IVS-II中的隐性3'剪接位点时,β-球蛋白mRNA的正确剪接可以恢复。在此,我们进行了深入分析,以探索一种更精确的基因编辑方法来治疗β-654-地中海贫血症。我们利用 CRISPR-Cas9 介导的同源定向修复(HDR)技术,在β654-地中海贫血小鼠模型的基因组中引入了一个单碱基替换的隐性 3' 接受剪接位点。所有经过 HDR 编辑的小鼠都能检测到正确剪接的 β-球蛋白 mRNA。与未编辑的β654小鼠相比,病理变化得到了改善。这使得携带β654等位基因的小鼠断奶后的存活率提高了两倍多。这种基因编辑策略的治疗效果表明,当 HDR 频率超过 20% 时,典型的 β-地中海贫血表型可以通过剂量依赖的方式得到改善。我们的研究为在β654小鼠模型中通过基因编辑反应性剪接受体位点来纠正剪接缺陷提供了一种独特而有效的方法。
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来源期刊
Human gene therapy
Human gene therapy 医学-生物工程与应用微生物
CiteScore
6.50
自引率
4.80%
发文量
131
审稿时长
4-8 weeks
期刊介绍: Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.
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