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

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.