CRISPR/Cas9-mediated A4GALT suppression rescues Fabry disease phenotypes in a kidney organoid model

Sheng, Chang; Fang, Xianying; Lee, Hanbi; Eum, Sang Hun; Ko, Eun Jeong; Lim, Sun Woo; Shin, Eunah; Lee, Kang In; Lee, Jae Young; Lee, Chae Bin; Bae, Soo Kyung; Yang, Chul Woo; Chung, Byung Ha

doi:10.1016/j.trsl.2023.02.005

Cited by 11 publications

(13 citation statements)

References 54 publications

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“…Previous studies including our have shown signi cant alterations in gene expression in differentiated cells or tissues derived from GLAmutant hiPSCs. [13,33,46,47] The current study also revealed notable changes in the transcriptome of endothelial cells (ECs) upon GLA-KO. In an analysis of gene sets associated with proper vascular function such as angiogenesis, cellular stress response, and also gene sets associated with Fb vasculopathy such as aging, cell death, ECM formation (Figure S4A-4C), GLA-KO hiPSC-EC exhibited distinct expression patterns compared to WT hiPSC-ECs.…”

Section: Discussionsupporting

confidence: 51%

“…[12] We have already proven that CRISPR/Cas9 mediated suppression of A4GALT can rescue FD phenotype in GLA-mutant hiPSCs and kidney organoid system. [13] As a result, A4GLAT-KO successfully rescued the FD phenotype in terms of Gb-3 deposition. It increased the expression of EC markers, enabled successful interconnecting branching point networks, and rescued the migration ability.…”

Section: Discussionmentioning

confidence: 90%

“…Patient derived and gene-edited induced pluripotent stem cells GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002) and A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) were already as described previously. [13] Methods for generating patient-derived hiPSCs and genetically modifying hiPSCs using CRISPR/Cas9 technique have been presented previously. [13,14,16,17] Brie y, we generated hiPSCs using peripheral blood mononuclear cells of FD patients.…”

Section: Methodsmentioning

confidence: 99%

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CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a Fabry disease vasculopathy model derived from human induced pluripotent stem cells

Shin,

Chae,

Lee

et al. 2023

Preprint

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Backgrounds: The objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). Methods: We differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing. Results: GLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays with, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated whereas SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs. Conclusions: CRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

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Section: Discussionsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

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CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a Fabry disease vasculopathy model derived from human induced pluripotent stem cells

Shin,

Chae,

Lee

et al. 2023

Preprint

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“…They show a native genomic context and cell-type heterogeneity within the kidney [ 18 , 19 , 20 ]. In addition, kidney organoids from genetically modified or patient-derived hiPSCs can successfully recapitulate the phenotype of various genetic kidney diseases [ 18 , 19 , 21 , 22 , 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

“…For this, we created FAN1 -mutant hiPSCs using KIN patient-derived peripheral blood mononuclear cells and performed FAN1 gene editing using CRISPR/Cas9 in wild-type hiPSCs. We then differentiated them into kidney organoids using a well-established protocol [ 21 , 22 , 23 , 24 , 25 ]. After that, we investigated whether the disease phenotype of KIN was recapitulated in FAN1 -mutant kidney organoids in comparison with wild-type kidney organoids.…”

Section: Introductionmentioning

confidence: 99%

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System

Lim,

Na,

Lee

et al. 2023

Cells

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Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/−), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.

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Organoids as Sophisticated Tools for Renal Cancer Research: Extensive Applications and Promising Prospects

Huang,

Wang,

et al. 2024

Cel. Mol. Bioeng.

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CRISPR/Cas9-mediated A4GALT suppression rescues Fabry disease phenotypes in a kidney organoid model

Cited by 11 publications

References 54 publications

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a Fabry disease vasculopathy model derived from human induced pluripotent stem cells

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a Fabry disease vasculopathy model derived from human induced pluripotent stem cells

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System

Organoids as Sophisticated Tools for Renal Cancer Research: Extensive Applications and Promising Prospects

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