IL-7-dependent and -independent lineages of IL-7R-dependent human T cells.

Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T-B+NK+ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4+ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4+ and CD8+ T cells were high, indicating IL-7-independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ+ T cells and some TCRαß+ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4+ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R-binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7-independent pathway of human T cell development.

Dysregulation of RAS proteostasis by autosomal-dominant LZTR1 mutation induces Noonan syndrome-like phenotypes in mice.

Leucine-zipper-like post translational regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 AD mutations regulate the proteostasis of the RAS subfamily molecules or cause NS-like phenotypes in vivo. To elucidate the pathogenesis of LZTR1 mutations, we generated two novel LZTR1 mutation knock-in mice (Lztr1G245R/+ and Lztr1R409C/+), which correspond to the human p.G248R and p.R412C mutations, respectively. LZTR1-mutant male mice exhibit low birth weight, distinctive facial features, and cardiac hypertrophy. Cardiomyocyte size and the expression of RAS subfamily members, including MRAS and RIT1, were significantly increased in the left ventricles (LVs) of mutant male mice. LZTR1 AD mutants did not interact with RIT1 and functioned as dominant-negative forms of wild-type LZTR1. Multi-omics analysis revealed that the MAPK signaling pathway was activated in the LVs of mutant mice. Treatment with the MEK inhibitor trametinib ameliorated cardiac hypertrophy in mutant male mice. These results suggest that MEK/ERK pathway is a therapeutic target for NS-like phenotype resulting from dysfunction of RAS proteostasis by LZTR1 AD mutations.

Novel readthrough agent suppresses nonsense mutations and restores functional type VII collagen and laminin 332 in epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin ß3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the first study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.

Low-level mosaic GCK mutations in children with diazoxide-unresponsive congenital hyperinsulinism.

CONTEXT: Some children with diazoxide-unresponsive congenital hyperinsulinism (HI) lack any detectable disease-causing mutation in peripheral blood DNA. OBJECTIVE: To examine whether somatic post-zygotic mutations of known HI genes are responsible for disease in children with diazoxide-unresponsive HI requiring surgery with histology not classified as focal or Localized Islet Nuclear Enlargement (LINE), and without detectable mutations by standard genetic testing of peripheral blood DNA. METHODS: Next-generation sequencing (NGS) was performed on specimens of pancreas from 10 children with diazoxide-unresponsive HI. RESULTS: Four unique GCK mutations were identified at low levels of mosaicism ranging from 4.4-10.1% in pancreatic DNA from five of these 10 children. The GCK mutations were not detectable in peripheral blood DNA by NGS in three cases from which peripheral blood DNA was available for testing. All four GCK mutations have been previously published as activating HI mutations. The histology was consistent with diffuse-HI in four of the five cases with mosaic GCK mutations. In one of these, hypomethylation of IC2 on chromosome 11p was identified in pancreatic and peripheral blood DNA. Histology of the fifth case revealed minor islet abnormalities suggestive of Beckwith Wiedemann Spectrum (BWSp) although molecular analysis for 11pUPD was negative in pancreas. CONCLUSION: These results indicate that post-zygotic somatic GCK mutations are responsible for some cases of non-focal diazoxide-unresponsive hyperinsulinism.

Therapeutic development approaches to treat haploinsufficiency diseases: restoring protein levels.

Rare diseases affect one in ten people but only a small fraction of these diseases have an FDA-approved treatment. Haploinsufficiency, caused by a dominant loss-of-function mutation, is a unique rare disease group because patients have one normal allele of the affected gene. This makes rare haploinsufficiency diseases promising candidates for drug development by increasing expression of the normal gene allele, decreasing the target protein degradation and enhancing the target protein function. This review summarizes recent progresses and approaches used in the translational research of therapeutics to treat haploinsufficiency diseases including gene therapy, nucleotide-based therapeutics and small-molecule drug development. We hope that these drug development strategies will accelerate therapeutic development to treat haploinsufficiency diseases.

UBTF haploinsufficiency associated with UBTF-related global developmental delay and distinctive facial features without neuroregression.

BACKGROUND: The Upstream Binding Transcription Factor (UBTF) gene encodes two nucleolar proteins, UBTF1 and UBTF2. UBTF1 regulates rRNA transcription by RNA polymerase I, while UBTF2 regulates mRNA transcription by RNA polymerase II. A recurrent de novo dominant mutation c.628G>A (p.Glu210Lys) has been identified as a gain-of-function mutation associated with childhood onset neurodegeneration with brain atrophy (CONDBA). Evidence from large-scale population databases and Ubtf+/- mouse models indicates that UBTF haploinsufficiency is not tolerated. METHODS: Three unrelated patients with global developmental delay and distinctive facial features were recruited for the study. Whole exome sequencing (WES) was performed to identify potential genetic abnormalities. Additionally, copy number variation analysis was conducted based on the WES data. RESULTS: All three patients exhibited intellectual disabilities, social challenges and developmental delays in language and gross motor skills. Distinctive facial features included a wide forehead, sparse eyebrows, hypertelorism, narrow palpebral fissures, single-fold eyelids, a flat nasal bridge, anteverted nares, a long philtrum and a thin upper lip. Additionally, patient C presented with more severe language delay, recurrent hepatic dysfunction and an atrial septal defect. Patient A was found to have a nonsense variant, c.1327C>T (p.R443Ter), in the exon 13 of UBTF. Patients B and C both carried a heterozygous deletion encompassing the UBTF gene. CONCLUSION: In this study, we analysed the detailed phenotypes associated with UBTF haploinsufficiency, which, to our knowledge, have not been previously reported. We propose that UBTF haploinsufficiency-related global developmental delay and distinctive facial features, without neuroregression, constitute a new syndrome distinct from CONDBA.

Type 2 innate immunity promotes the development of pulmonary fibrosis in Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS), particularly in types 1 and 4, is characterized by progressive pulmonary fibrosis, a major cause of morbidity and mortality. However, the precise mechanisms driving pulmonary fibrosis in HPS are not fully elucidated. Our previous studies suggested that CHI3L1-driven fibroproliferation may be a notable factor in HPS-associated fibrosis. This study aimed to explore the role of CHI3L1-CRTH2 interaction on ILC2s and explored the potential contribution of ILC2-fibroblast crosstalk in the development of pulmonary fibrosis in HPS. We identified ILC2s in lung tissues from idiopathic pulmonary fibrosis (IPF) and HPS patients. Using bleomycin-challenged wild type (WT) and Hps1-/- mice we observed that ILC2s were recruited and appeared to contribute to fibrosis development in the Hps1-/- mice, with CRTH2 playing a notable role in ILC2 accumulation. We sorted ILC2s, profiled fibrosis-related genes and mediators, and conducted co-culture experiments with primary lung ILC2s and fibroblasts. Our findings suggest that ILC2s may directly stimulate the proliferation and differentiation of primary lung fibroblasts partially through Amphiregulin-EGFR-dependent mechanisms. Additionally, specific overexpression of CHI3L1 in the ILC2 population using the IL-7Rcre driver, which was associated with increased fibroproliferation, indicates that ILC2-mediated, CRTH2-dependent mechanisms might contribute to optimal CHI3L1-induced fibroproliferative repair in HPS-associated pulmonary fibrosis.

Prevalence of Individuals With Multiple Diagnosed Genetic Diseases in the Undiagnosed Diseases Network.

Report the prevalence of multiple genetic diseases in the Undiagnosed Diseases Network (UDN) cohort in the post-exome-sequencing era. UDN subjects underwent genome sequencing before inclusion in the cohort. Records of all UDN subjects until January 2024 were analyzed. The number of diagnoses, proportion of molecular versus nonmolecular (i.e., not attributable to a discretely identifiable genetic change) diagnoses, and the inheritance patterns of the genetic diagnoses were determined. Of 2799 subjects, 766 (27.4%) had diagnoses. Of these 766, 95.4% had one diagnosis, 4.0% had two diagnoses, and 0.5% had three diagnoses. Of the diagnosed subjects, 93.4% had a genetic disease, and 6.5% had a nonmolecular disease. Of subjects with two diagnoses, both diagnoses were molecular in 90.3%, while 9.7% had one molecular and one nonmolecular diagnosis. All four subjects with three diagnoses had three molecular diagnoses. 4.2% of diagnosed subjects in the UDN had more than one molecular diagnosis, with four individuals having three concurrent Mendelian diagnoses. Additionally, three subjects had concurrent molecular and nonmolecular diagnoses. Given that numerous UDN subjects had a negative genome sequence prior to UDN enrollment, multiple molecular diagnoses may contribute to diagnostic uncertainty even with genome sequencing, as may concurrent nonmolecular disease.

A novel pathogenic variant in the KCTD7 gene in a patient with neuronal ceroid lipofuscinosis (CLN14): a case report and review of the literature.

BACKGROUND: Neuronal ceroid lipofuscinosis (NCL) is a heterogeneous group of 13 rare, progressive neurodegenerative diseases of the brain and retina. CLN14 is a very rare subtype of NCL caused by pathogenic variants in the KCTD7 gene. Only four cases of this subtype have been reported in the literature. CASE PRESENTATION: A nine-month-old, previously healthy male who was firstborn to first-cousin parents presented with progressive psychomotor regression, dysmorphic facial features, myoclonus, and vision loss. Neurological examination showed generalized hypotonia and brisk reflexes. He continued to deteriorate until age 18 months, when he developed his first generalized tonic-clonic seizure. An ophthalmological examination showed a hypopigmented fundus and slight temporal disc pallor. Brain MRI showed mild generalized brain atrophy and white matter disease. EEG revealed a severely abnormal trace marked by generalized, high amplitude, sharply contoured, polymorphic delta slowing intermixed with theta slowing and some alpha activity, with disorganized and scattered spikes and sharp waves. The patient continued to have uncontrolled seizures and further psychomotor regression until he died of status epilepticus and pneumonia at the age of 44 months. WES identified a novel homozygous variant c.413T > C, p.(Leu138Pro) in the KCTD7 gene, causing an amino acid transition from leucine to proline at position 138. Both parents were carriers of the same variant. CONCLUSIONS: We present the fifth known case of CLN14 in the literature and report the clinical course and a novel underlying likely causative variant in the KCTD7 gene. The improving accessibility and affordability of genetic testing will likely uncover more NCL cases and further expand the disease's genotypic and phenotypic spectrum.

The Role of Patient Organizations in Shaping Research, Health Policies, and Health Services for Rare Genetic Diseases: The Dutch Experience.

In 2023, the genetics scientific community celebrated two special anniversaries: the discovery of the double helix structure of DNA was published in 1953 and in 2003 the Human Genome Project was declared completed and made publicly available. To this day, genetics and genomics research is continuing to evolve at high pace and is identifying a steadily increasing number of genes as causal for distinct genetic diseases. The success story of genetics and genomics would not be complete without taking due account of the role of patient advocacy organizations in this process. This paper is based on the personal narrative (oral history) of a father whose daughter was born with a rare genetic disease (RGD) in the 1960s. The first-hand experience of living as a family with an RGD in those days made him a leading pioneer not only in the foundation of patient organizations at national, pan-European, and international levels but also in the development of multi-stakeholder co-operation and networking. Today, patient advocacy organizations play an active role in shaping health and research policies at national, EU, and international levels to ensure that their needs in regard to advancing RGD diagnostics, care, and treatment are addressed.

Elucidating disparities in sunscreen coverage among state Medicaid preferred drug lists.

Regular application of over-the-counter (OTC) sunscreen is considered the foundation of skin cancer prevention, yet OTC sunscreen is not eligible for reimbursement in almost all state Medicaid benefit plans. On review of 111 Medicaid preferred drug lists (PDLs) across 50 states and the District of Columbia (DC), only five plans were identified that incorporate coverage of sunscreen. Thus, many recipients of Medicaid, the majority of whom are individuals and families of lower socioeconomic status, may encounter financial difficulty and thus forego utilizing sun protective measures due to financial constraints. Here, we compare current Medicaid coverage of OTC sunscreen and discuss calculated and theoretical annual costs of this skin cancer prevention method.

Cell-Penetrating Peptides and CRISPR-Cas9: A Combined Strategy for Human Genetic Disease Therapy.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has revolutionized the field of genetic engineering, offering unprecedented potential for the targeted manipulation of DNA sequences. Advances in the mechanism of action of the CRISPR-Cas9 system allowed potential applicability for the treatment of genetic diseases. CRISPR-Cas9's mechanism of action involves the use of an RNA guide molecule to target-specific DNA sequences and the Cas9 enzyme to induce precise DNA cleavage. In the context of the CRISPR-Cas9 system, this review covers nonviral delivery methods for gene editing based on peptide internalization. Here, we describe critical areas of discussion such as immunogenicity, emphasizing the importance of safety, efficiency, and cost-effectiveness, particularly in the context of treating single-mutation genetic diseases using advanced editing techniques genetics as prime editor and base editor. The text discusses the versatility of cell-penetrating peptides (CPPs) in forming complexes for delivering biomolecules, particularly ribonucleoprotein for genome editing with CRISPR-Cas9 in human cells. In addition, it emphasizes the promise of combining CPPs with DNA base editing and prime editing systems. These systems, known for their simplicity and precision, hold great potential for correcting point mutations in human genetic diseases. In summary, the text provides a clear overview of the advantages of using CPPs for genome editing with CRISPR-Cas9, particularly in conjunction with advanced editing systems, highlighting their potential impact on clinical applications in the treatment of single-mutation genetic diseases. [Figure: see text].

Saturation mutagenesis-reinforced functional assays for disease-related genes.

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods are obstacles for achieving genome-wide resolution of variants in disease-related genes. Our framework, saturation mutagenesis-reinforced functional assays (SMuRF), offers simple and cost-effective saturation mutagenesis paired with streamlined functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for all possible coding single-nucleotide variants, which aid in resolving clinically reported variants of uncertain significance. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Overall, our approach enables variant-to-function insights for disease genes in a cost-effective manner that can be broadly implemented by standard research laboratories.

Facial Sebaceous Hyperplasia in an Adolescent With Hypohidrotic Ectodermal Dysplasia.

We report on a 13-year-old boy diagnosed with hypohidrotic ectodermal dysplasia (HED) due to a pathogenic variant in ectodysplasin A (EDA). He exhibited multiple whitish, millimetric papules clustered on the nasal ala, forehead, temporal, and zygomatic arch areas. Histological examination revealed numerous hyperplastic sebaceous lobules within the upper dermis. The occurrence of sebaceous papules in this distribution among HED patients has rarely been reported. An association with the blockage of the Wnt/ß-catenin pathway due to EDA malfunction has been speculated.

Modeling primary microcephaly with human brain organoids reveals fundamental roles of CIT kinase activity.

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic CIT missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it does not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lose cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupt polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

A novel pathogenic germline chromosome 3 inversion in von Hippel-Lindau disease.

von Hippel-Lindau (VHL) is an autosomal-dominant hereditary tumour susceptibility disease associated with pathogenic germline variants in the VHL tumour suppressor gene. VHL patients are at increased risk of developing multiple benign and malignant tumours. Current CLIA-based genetic tests demonstrate a very high detection rate of germline VHL variants in patients with clinical manifestations of VHL. In this report, we describe a large family with canonical VHL manifestations, for which no germline alteration had been detected by conventional germline testing. We identified a novel 291 kb chromosomal inversion involving chromosome 3p in affected family members. This inversion disrupts the VHL gene between exon 2 and exon 3 and is thereby responsible for the disease observed in this family.

MYH9-related inherited thrombocytopenia: the genetic spectrum, underlying mechanisms, clinical phenotypes, diagnosis, and management approaches.

Inherited thrombocytopenias have been considered exceedingly rare for a long time, but recent advances have facilitated diagnosis and greatly enabled the discovery of new causative genes. MYH9-related disease (MYH9-RD) represents one of the most frequent forms of inherited thrombocytopenia, usually presenting with nonspecific clinical manifestations, which renders it difficult to establish an accurate diagnosis. MYH9-RD is an autosomal dominant-inherited thrombocytopenia caused by deleterious variants in the MYH9 gene encoding the heavy chain of nonmuscle myosin IIA. Patients with MYH9-RD usually present with thrombocytopenia and platelet macrocytosis at birth or in infancy, and most of them may develop one or more extrahematologic manifestations of progressive nephritis, sensorial hearing loss, presenile cataracts, and elevated liver enzymatic levels during childhood and adult life. Here, we have reviewed recent advances in the study of MYH9-RD, which aims to provide an updated and comprehensive summary of the current knowledge and improve our understanding of the genetic spectrum, underlying mechanisms, clinical phenotypes, diagnosis, and management approaches of this rare disease. Importantly, our goal is to enable physicians to better understand this rare disease and highlight the critical role of genetic etiologic analysis in ensuring accurate diagnosis, clinical management, and genetic counseling while avoiding ineffective and potentially harmful therapies for MYH9-RD patients.

Proteogenomic analysis integrated with electronic health records data reveals disease-associated variants in Black Americans.

BACKGROUND: Most genome wide association studies (GWAS) of plasma proteomics have focused on White individuals of European ancestry, limiting biological insight from other ancestry enriched protein quantitative loci (pQTL). METHODS: We conducted a discovery GWAS of ~3,000 plasma proteins measured by the antibody based Olink platform in 1,054 Black adults from the Jackson Heart Study (JHS), and validated our findings in the Multi-Ethnic Study of Atherosclerosis (MESA). The genetic architecture of identified pQTLs were further explored through fine mapping and admixture association analysis. Finally, using our pQTL findings, we performed a phenome wide association study (PheWAS) across two large multi-ethnic electronic health record (EHR) systems in All of Us and BioMe. RESULTS: We identified 1002 pQTLs for 925 proteins. Fine mapping and admixture analyses suggested allelic heterogeneity of the plasma proteome across diverse populations. We identified associations for variants enriched in African ancestry, many in diseases that lack precise biomarkers, including cis-pQTLs for Cathepsin L (CTSL) and Siglec-9 that were linked with sarcoidosis and non-Hodgkin's lymphoma, respectively. We found concordant associations across clinical diagnoses and laboratory measurements, elucidating disease pathways, including a cis-pQTL associated with circulating CD58, white blood cell count, and multiple sclerosis. CONCLUSIONS: Our findings emphasize the value of leveraging diverse populations to enhance biological insights from proteomics GWAS, and we have made this resource readily available as an interactive web portal.

Development of mRNA Lipid Nanoparticles: Targeting and Therapeutic Aspects.

Lipid nanoparticles (LNPs) have emerged as leading non-viral carriers for messenger RNA (mRNA) delivery in clinical applications. Overcoming challenges in safe and effective mRNA delivery to target tissues and cells, along with controlling release from the delivery vehicle, remains pivotal in mRNA-based therapies. This review elucidates the structure of LNPs, the mechanism for mRNA delivery, and the targeted delivery of LNPs to various cells and tissues, including leukocytes, T-cells, dendritic cells, Kupffer cells, hepatic endothelial cells, and hepatic and extrahepatic tissues. Here, we discuss the applications of mRNA-LNP vaccines for the prevention of infectious diseases and for the treatment of cancer and various genetic diseases. Although challenges remain in terms of delivery efficiency, specific tissue targeting, toxicity, and storage stability, mRNA-LNP technology holds extensive potential for the treatment of diseases.

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.a.