대한신장학회

논문분류	춘계학술대회 초록집
제목	Development of CRISPR/Cas9 therapeutics targeting A4GALT using patient derived kidney organoids model of fabry disease
저자	Sheng Cui,Han Bi Lee,Sang Hun Eum,Yoo Jin Shin,Eun Jeong Ko,Sun Woo Lim,Kang In Lee,Jea Young Lee,Chunl Woo Yang,Byung Ha Chung
출판정보	2022; 2022(1):
키워드
초록	Objectives: Deposition of globotriaosylceramide (Gb3) is a critical step for the progression of fabry disease nephropathy (FdN). In this study, we investigated whether knock-out (K/O) of A4GALT gene (Gb3 synthase gene) by CRISPR/Cas9 technology can reduce the deposition of Gb3; hence to appraise its potential as a therapeutic approach for FdN using kidney organoids. Methods: First, we generated patient-specific hiPSC (human induced pluripotent stem cells) (CMC-Fb02) using PBMCs from a patient with FdN. Second, we generated disease-specific hiPSC (GLA-KO-WTC-11) by K/O of GLA gene in wild-type WTC-11-hiPSC. Third, we performed A4GALT K/O by CRISPR/Cas9 technology in both CMC-Fb02 and GLA-KO-WTC-11), so generated CMC-Fb02-Gb3-KO and GLA-Gb3-KO-WTC-11 hiPSCs. Lastly, we generated kidney organoids using wild-type-WTC-11, CMC-Fb02 (patient-specific), GLA-KO-WTC-11 (disease-specific), and their corresponding A4GALT K/O hiPSCs (CMC-Fb2-Gb3-KO, GLA-Gb3-KO-WTC-11). We compared alpha-galactosidase-A enzyme (α-GLA) activity, Gb-3 deposition by LC_MS/MS and immunofluorescent (IF) staining, zebra body formation under electron microscopy (EM). Results: All of 5 hiPSCs showed typical pluripotency markers and normal karyotyping. All of generated kidney organoids from those hiPSCs showed the expression of typical nephron markers such as PODXL (Podocyte), LTL (Proximal tubule), and E-cad (distal tubule) under IF staining. In the kidney organoids derived from CMC-Fb02 and GLA-KO-WTC-11, α-GLA activity was significantly decreased compared to kidney organoids from wild-type WTC-11. In contrast, deposition of Gb3 measured by LC-MS/MS and IF staining was significantly increased in the CMC-Fb-2 and GLA-KO-WTC-11 kidney organoids. In both A4GALT KO kidney organoids (CMC-Fb2-Gb3-KO and GLA-Gb3-KO-WTC-11), Gb3 deposition by LC-MC/MC and IF significantly decreased in comparison with CMC-Fb2 and GLA-KO-WTC-11 kidney organoids respectively. In EM, typical zebra body formations were detected in CMC-Fb2 and GLA-KO-WTC-11 kidney organoids, but they disappeared in CMC-Fb2-Gb3-KO and GLA-Gb3-KO-WTC-11 kidney organoids Conclusions: Our results suggest that A4GALT targeting therapy can be proposed as an innovative approach for the treatment of FdN.
원문(PDF)	PDF 원문보기

간행물 검색

대한신장학회 회원관리 규정