CRISPR/Cas9-mediated suppression of A4GALT rescue endothelial cell dysfunction in a human induced pluripotent stem cell derived fabry disease vasculopathy model

This study investigated the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). 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 the reactive oxygen species (ROS) formation, and transcriptome profiles through RNA sequencing. GLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression, 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 the expression of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, and migration of cells into the scratched wound area were significantly decreased. In contrast, A4GALT suppression improved tube formation and cell migration capacity. In GLA-KO hiPSC-ECs, western blot analysis revealed downregulated MAPK, and AKT phosphorylation, while SOD and catalase were upregulated. 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, and they were effectively restored in GLA/A4GALT-KO hiPSC-ECs. CRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.