Generation of an Isogenic Hereditary Hemorrhagic Telangiectasia Model via Prime Editing in Human Induced Pluripotent Stem Cells.

IF 2.5 4区 医学 Q3 CELL & TISSUE ENGINEERING
International journal of stem cells Pub Date : 2024-11-30 Epub Date: 2024-09-06 DOI:10.15283/ijsc24084
Min Woo Kim, Kyu Sik Jeong, Jin Kim, Seul-Gi Lee, C-Yoon Kim, Hyung Min Chung
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Abstract

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.

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。
主编辑(PE)是最近开发的一种基因组编辑技术,可进行多功能编辑。尽管PE具有灵活性和潜力,但在人类诱导多能干细胞(hiPSCs)中应用PE的问题尚未得到广泛讨论。利用源自患者的 hiPSCs 建立的遗传疾病模型已被用于研究疾病机理和药物疗效。然而,患者和对照细胞之间的基因差异被认为是疾病模型不准确的原因,这凸显了异源 hiPSC 模型的重要性。遗传性出血性毛细血管扩张症1(HHT1)是一种由内皮素(ENG)常染色体显性突变引起的遗传性疾病。虽然以前使用过小鼠和 HUVEC 的 HHT 模型,但这些模型并不能充分阐明 HHT 基因型与疾病表型之间的关系,因此需要更多反映人类遗传学的临床相关模型。因此,在本研究中,我们利用 PE 提出了一种建立同源 hiPSC 株系的方法。我们选择了临床报道的ENG目标突变,并设计了PE的策略。克隆ENGineered PE guide RNA后,用PEmax和hMLH1dn质粒对hiPSC进行核感染。结果,获得了具有预期突变的 hiPSC 克隆,这些克隆的多能性和基因完整性没有发生变化。此外,在内皮类器官分化过程中,引入ENG突变增加了促血管生成标志物的表达。因此,我们的研究结果表明,PE 有可能成为建立同源系的工具包,从而在 hiPSC 衍生的疾病相关细胞或器官组织的基础上建立疾病模型。这种方法有望促进遗传疾病的机理和治疗研究。
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来源期刊
International journal of stem cells
International journal of stem cells Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
5.10
自引率
4.30%
发文量
38
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.
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