{"title":"Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β<sup>654</sup>-Thalassemia Mouse Model.","authors":"Dan Lu, Xiuli Gong, Xinbing Guo, Qin Cai, Yanwen Chen, Yiwen Zhu, Xiao Sang, Hua Yang, Miao Xu, Yitao Zeng, Dali Li, Fanyi Zeng","doi":"10.1089/hum.2023.202","DOIUrl":null,"url":null,"abstract":"<p><p>β<sup>654</sup>-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 β<sup>654</sup> 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 β<sup>654</sup>-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β<sup>654</sup>-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 β<sup>654</sup> mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β<sup>654</sup> 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 β<sup>654</sup> mouse model.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11514127/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Human gene therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1089/hum.2023.202","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/13 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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.
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.