Min Woo Kim, Kyu Sik Jeong, Jin Kim, Seul-Gi Lee, C-Yoon Kim, Hyung Min Chung
{"title":"通过在人类诱导多能干细胞中进行主基因编辑,生成异源遗传性出血性远端血管扩张症模型。","authors":"Min Woo Kim, Kyu Sik Jeong, Jin Kim, Seul-Gi Lee, C-Yoon Kim, Hyung Min Chung","doi":"10.15283/ijsc24084","DOIUrl":null,"url":null,"abstract":"<p><p>Prime editing (PE) is a recently developed genome-editing technique that enables versatile editing. Despite its flexibility and potential, applying PE in human induced pluripotent stem cells (hiPSCs) has not been extensively addressed. Genetic disease models using patient-derived hiPSCs have been used to study mechanisms and drug efficacy. However, genetic differences between patient and control cells have been attributed to the inaccuracy of the disease model, highlighting the significance of isogenic hiPSC models. Hereditary hemorrhagic telangiectasia 1 (HHT1) is a genetic disorder caused by an autosomal dominant mutation in endoglin (<i>ENG</i>). Although previous HHT models using mice and HUVEC have been used, these models did not sufficiently elucidate the relationship between the genotype and disease phenotype in HHT, demanding more clinically relevant models that reflect human genetics. Therefore, in this study, we used PE to propose a method for establishing an isogenic hiPSC line. Clinically reported target mutation in <i>ENG</i> was selected, and a strategy for PE was designed. After cloning the engineered PE guide RNA, hiPSCs were nucleofected along with PEmax and hMLH1dn plasmids. As a result, hiPSC clones with the intended mutation were obtained, which showed no changes in pluripotency or genetic integrity. Furthermore, introducing the <i>ENG</i> mutation increased the expression of proangiogenic markers during endothelial organoid differentiation. Consequently, our results suggest the potential of PE as a toolkit for establishing isogenic lines, enabling disease modeling based on hiPSC-derived disease-related cells or organoids. This approach is expected to stimulate mechanistic and therapeutic studies on genetic diseases.</p>","PeriodicalId":14392,"journal":{"name":"International journal of stem cells","volume":" ","pages":"397-406"},"PeriodicalIF":2.5000,"publicationDate":"2024-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11612218/pdf/","citationCount":"0","resultStr":"{\"title\":\"Generation of an Isogenic Hereditary Hemorrhagic Telangiectasia Model via Prime Editing in Human Induced Pluripotent Stem Cells.\",\"authors\":\"Min Woo Kim, Kyu Sik Jeong, Jin Kim, Seul-Gi Lee, C-Yoon Kim, Hyung Min Chung\",\"doi\":\"10.15283/ijsc24084\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Prime editing (PE) is a recently developed genome-editing technique that enables versatile editing. Despite its flexibility and potential, applying PE in human induced pluripotent stem cells (hiPSCs) has not been extensively addressed. Genetic disease models using patient-derived hiPSCs have been used to study mechanisms and drug efficacy. However, genetic differences between patient and control cells have been attributed to the inaccuracy of the disease model, highlighting the significance of isogenic hiPSC models. Hereditary hemorrhagic telangiectasia 1 (HHT1) is a genetic disorder caused by an autosomal dominant mutation in endoglin (<i>ENG</i>). Although previous HHT models using mice and HUVEC have been used, these models did not sufficiently elucidate the relationship between the genotype and disease phenotype in HHT, demanding more clinically relevant models that reflect human genetics. Therefore, in this study, we used PE to propose a method for establishing an isogenic hiPSC line. Clinically reported target mutation in <i>ENG</i> was selected, and a strategy for PE was designed. After cloning the engineered PE guide RNA, hiPSCs were nucleofected along with PEmax and hMLH1dn plasmids. As a result, hiPSC clones with the intended mutation were obtained, which showed no changes in pluripotency or genetic integrity. Furthermore, introducing the <i>ENG</i> mutation increased the expression of proangiogenic markers during endothelial organoid differentiation. Consequently, our results suggest the potential of PE as a toolkit for establishing isogenic lines, enabling disease modeling based on hiPSC-derived disease-related cells or organoids. 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Generation of an Isogenic Hereditary Hemorrhagic Telangiectasia Model via Prime Editing in Human Induced Pluripotent Stem Cells.
Prime editing (PE) is a recently developed genome-editing technique that enables versatile editing. Despite its flexibility and potential, applying PE in human induced pluripotent stem cells (hiPSCs) has not been extensively addressed. Genetic disease models using patient-derived hiPSCs have been used to study mechanisms and drug efficacy. However, genetic differences between patient and control cells have been attributed to the inaccuracy of the disease model, highlighting the significance of isogenic hiPSC models. Hereditary hemorrhagic telangiectasia 1 (HHT1) is a genetic disorder caused by an autosomal dominant mutation in endoglin (ENG). Although previous HHT models using mice and HUVEC have been used, these models did not sufficiently elucidate the relationship between the genotype and disease phenotype in HHT, demanding more clinically relevant models that reflect human genetics. Therefore, in this study, we used PE to propose a method for establishing an isogenic hiPSC line. Clinically reported target mutation in ENG was selected, and a strategy for PE was designed. After cloning the engineered PE guide RNA, hiPSCs were nucleofected along with PEmax and hMLH1dn plasmids. As a result, hiPSC clones with the intended mutation were obtained, which showed no changes in pluripotency or genetic integrity. Furthermore, introducing the ENG mutation increased the expression of proangiogenic markers during endothelial organoid differentiation. Consequently, our results suggest the potential of PE as a toolkit for establishing isogenic lines, enabling disease modeling based on hiPSC-derived disease-related cells or organoids. This approach is expected to stimulate mechanistic and therapeutic studies on genetic diseases.
期刊介绍:
International Journal of Stem Cells (Int J Stem Cells), a peer-reviewed open access journal, principally aims to provide a forum for investigators in the field of stem cell biology to present their research findings and share their visions and opinions. Int J Stem Cells covers all aspects of stem cell biology including basic, clinical and translational research on genetics, biochemistry, and physiology of various types of stem cells including embryonic, adult and induced stem cells. Reports on epigenetics, genomics, proteomics, metabolomics of stem cells are welcome as well. Int J Stem Cells also publishes review articles, technical reports and treatise on ethical issues.