Patient-specific Fabry disease cell models as a tool to evaluate the amenability to chaperone therapy

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology

AJP Renal Physiology ◽

10.1152/ajprenal.00283.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. F1015-F1024 ◽

Cited By ~ 6

Author(s):

Ester M. Pereira ◽

Anatália Labilloy ◽

Megan L. Eshbach ◽

Ankita Roy ◽

Arohan R. Subramanya ◽

...

Keyword(s):

Fabry Disease ◽

Human Kidney ◽

Premature Death ◽

Antibody Array ◽

Cell Models ◽

Lysosomal Storage ◽

Protein Levels ◽

Gla Gene ◽

Fabry Nephropathy ◽

And Control

Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.

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Sortilin expression and uptake of α-galactosidase A: A general mechanism of endocytosis in Fabry disease cell types

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2015.12.439 ◽

2016 ◽

Vol 117 (2) ◽

pp. S106

Author(s):

Jin-Song Shen ◽

Taniqua S. Day ◽

Xing-Li Meng ◽

Zhi-Ping Liu ◽

Raphael Schiffmann

Keyword(s):

Fabry Disease ◽

Cell Types ◽

General Mechanism ◽

Disease Cell

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Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome

New England Journal of Medicine ◽

10.1056/nejmoa0908679 ◽

2010 ◽

Vol 363 (15) ◽

pp. 1397-1409 ◽

Cited By ~ 813

Author(s):

Alessandra Moretti ◽

Milena Bellin ◽

Andrea Welling ◽

Christian Billy Jung ◽

Jason T. Lam ◽

...

Keyword(s):

Stem Cell ◽

Long Qt Syndrome ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Cell Models ◽

Long Qt ◽

Qt Syndrome ◽

Induced Pluripotent ◽

Stem Cell Models

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Unsaturated mannuronate oligosaccharide ameliorates β‐amyloid pathology through autophagy in Alzheimer’s disease cell models

Carbohydrate Polymers ◽

10.1016/j.carbpol.2020.117124 ◽

2021 ◽

Vol 251 ◽

pp. 117124

Author(s):

Decheng Bi ◽

Lijun Yao ◽

Zhijian Lin ◽

Lianli Chi ◽

Hui Li ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cell Models ◽

Amyloid Pathology ◽

Β Amyloid ◽

Disease Cell

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Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106005 ◽

2019 ◽

Vol 56 (8) ◽

pp. 548-556 ◽

Cited By ~ 17

Author(s):

Malte Lenders ◽

Franciska Stappers ◽

Christoph Niemietz ◽

Boris Schmitz ◽

Michel Boutin ◽

...

Keyword(s):

Fabry Disease ◽

Cell Model ◽

White Blood Cells ◽

Disease Patient ◽

Patient Specific ◽

Specific Cell ◽

Missense Mutations ◽

Activity Measurements

BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.MethodsSince current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.ResultsUnder treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells.ConclusionWe conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

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