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

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.

Keywords: Endoglin; Gene editing; Genetic diseases; Hereditary hemorrhagic telangiectasia; Induced pluripotent stem cells.

Fig. 1

Structure of ENG gene and position of target mutation. ENG c.360＋1G>A mutation. The ENG c.360＋1G>A mutation (red mark) is located at the splicing site in intron 3 and disrupts normal splicing.

Fig. 2

Strategy for prime editing to generate isogenic human induced pluripotent stem cells (hiPSCs) carrying clinically reported ENG mutation. (A) Scheme of the process for prime editing using hiPSCs. After choosing target mutation, engineered prime editing guide RNA (epegRNA) is designed using the PrimeDesign tool. The spacer sequence is preferably selected to be closest to the editing site, while reverse transcriptase template (RTT) and primer binding site (PBS) are typically chosen to be between 10 and 15 bp in length. To introduce designed sequences into epegRNA plasmid, annealed oligos for spacer, scaffold, extension including RTT and PBS are assembled into plasmid by golden gate cloning. Nucleofection is performed to introduce cloned epegRNA plasmid combined with PEmax plasmid and hMLH1dn plasmid, followed by isolating transfected cells via flow cytometry. Then, single-cell colonies are expended and sequenced to examine the genotype. (B) epegRNA design for targeting ENG c.360＋1G>A mutation. The spacer sequence was selected as the closest to the target, and an A was added to the RTT sequence to change the target base to the desired sequence (G>A). (C) Scheme of Golden gate cloning for epegRNA plasmid construction. Oligo sets corresponding to spacer, scaffold, extension (RTT and PBS) sequence are shown.

Fig. 3

Isolation of transfected human induced pluripotent stem cells (hiPSCs) and bulk sequencing for evaluating editing efficiency. (A) After 24 hours of nucleofection for prime editing, GFP-expressing cells were sorted by FACS to increase editing efficiency. (B) Isolated GFP-expressing cells were analyzed to examine efficiency. Bulk genomic DNA was extracted from sorted cells and the target sequence was amplified by polymerase chain reaction (PCR). Gel-extracted PCR product was sequenced and analyzed by EditR. The asterisk mark indicates the target base pair.

Fig. 4

Generation and characterization of an isogenic human induced pluripotent stem cells (hiPSC) line with intended ENG mutation by prime editing. (A) Single-cell colonies were formed and expanded. Genomic DNA samples of each colony were amplified by polymerase chain reaction (PCR). After sequencing the PCR products, the results were analyzed to investigate the genotype of each colony. Among 13 acquired clones, two clones possessed the intended mutation (ENG c.360＋1G>A) heterozygously. The asterisk mark indicates the target base pair. (B, C) Immunofluorescence staining and AP staining were performed to evaluate the pluripotency of generated isogenic mutant hiPSC line (scale bar=100 μm). (D) Chromosomal integrity was confirmed by karyotyping. (E) Teratoma assay was further conducted to verify in vivo pluripotency, resulting in the formation of all three germ layers including endoderm, mesoderm, and ectoderm (scale bar=50 μm).

Fig. 5

Endothelial organoid (EO) formation and proangiogenic marker analysis. (A) Differentiation schedule for EOs from isogenic control and hereditary hemorrhagic telangiectasia human induced pluripotent stem cell (HHT hiPSC) line. (B) mRNA expression levels of proangiogenic markers, including VEGFR2, ANGPT2, Tie2, and CD31, were analyzed by quantitative polymerase chain reaction. Statistical significance: *p<0.05, **p<0.01, ***p<0.001.