ABSTRACT
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.
Keywords
Abbreviations:
A4GALT (alpha 1,4-galactosyltransferase), α-GalA (α-galactosidase A), EM (Electron microscopy), ERT (enzyme replacement therapy), FD (fabry disease), FDN (fabry disease nephropathy), Gb-3 (globotriaosylceramide), GlcCer (glucosylceramide), GSL (glycosphingolipids), hiPSCs (human induced pluripotent stem cells), KO (knock-out), LacCer (lactosylceramide), LC-MS/MS (liquid chromatography-tandem mass spectrometry), sgRNA (Single guide RNA), WT (wild-type)To read this article in full you will need to make a payment
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Article info
Publication history
Published online: February 17, 2023
Accepted:
February 13,
2023
Received in revised form:
January 28,
2023
Received:
September 17,
2022
Publication stage
In Press Journal Pre-ProofIdentification
Copyright
© 2023 Elsevier Inc. All rights reserved.
