Reversal of the Inflammatory Responses in Fabry Patient iPSC-Derived Cardiovascular Endothelial Cells by CRISPR/Cas9-Corrected Mutation

Int J Mol Sci. 2021 Feb 27;22(5):2381. doi: 10.3390/ijms22052381.

Abstract

The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.

Keywords: CRISPR/Cas9 gene editing; Fabry disease; induced pluripotent stem cells; inflammatory.

MeSH terms

  • CRISPR-Associated Protein 9
  • Endothelial Cells*
  • Fabry Disease / enzymology
  • Fabry Disease / genetics
  • Fabry Disease / pathology
  • Fabry Disease / therapy*
  • Gene Editing*
  • Humans
  • Induced Pluripotent Stem Cells*
  • Inflammation
  • Mutation*
  • Phenotype
  • alpha-Galactosidase / genetics*

Substances

  • CRISPR-Associated Protein 9
  • alpha-Galactosidase