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

• Published in: Translational research : the journal of laboratory and clinical medicine 2023-08, Vol.258, p.35-46
• Main Authors: Cui, Sheng, Shin, Yoo Jin, Fang, Xianying, Lee, Hanbi, Eum, Sang Hun, Ko, Eun Jeong, Lim, Sun Woo, Shin, Eunji, Lee, Kang In, Lee, Jae Young, Lee, Chae Bin, Bae, Soo Kyung, Yang, Chul Woo, Chung, Byung Ha
• Format: Article
• Language: English
• Subjects: A4GALT; CRISPR/Cas9; Fabry disease; Gb-3; GLA
• ISSN: 1931-5244; 1878-1810
• DOI: 10.1016/j.trsl.2023.02.005

The objective of this study was to investigate whether CRISPR/Cas9-mediated suppression of A4GALT could rescue phenotype of Fabry disease nephropathy (FDN) using human induced pluripotent stem cells (hiPSCs) derived kidney organoid system. We generated FDN patient-derived hiPSC (CMC-Fb-002) and FD-specific hiPSCs (GLA-KO) by knock-out (KO) of GLA in wild-type (WT) hiPSCs using CRISPR/Cas9. We then performed A4GALT KO in both CMC-Fb-002 and GLA-KO to make Fb-002-A4GALT-KO and GLA/A4GALT-KO, respectively. Using these hiPSCs, we generated kidney organoids and compared alpha-galactosidase-A enzyme (α-GalA) activity, globotriaosylceramide (Gb-3) deposition, and zebra body formation under electron microscopy (EM). We also compared mRNA expression levels using RNA-seq and qPCR. Generated hiPSCs showed typical pluripotency markers without chromosomal disruption. Expression levels of GLA in CMC-Fb-002 and GLA-KO and expression levels of A4GALT in Fb-002-A4GALT-KO and GLA/A4GALT-KO were successfully decreased compared to those in WT-hiPSCs, respectively. Generated kidney organoids using these hiPSCs expressed typical nephron markers. In CMC-Fb-002 and GLA-KO organoids, α-GalA activity was significantly decreased along with increased deposition of Gb-3 in comparison with WT organoids. Intralysosomal inclusion body was also detected under EM. However, these disease phenotypes were rescued by KO of A4GALT in both GLA/A4GALT-KO and Fb-002-A4GALT-KO kidney organoids. RNA-seq showed increased expression levels of genes related to FDN progression in both GLA-mutant organoids compared to those in WT. Such increases were rescued in GLA/A4GALT-KO or Fb-002-A4GALT-KO organoids. CRISPR/Cas9 mediated suppression of A4GALT could rescue FDN phenotype. Hence, it can be proposed as a therapeutic approach to treat FDN.