EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells
2023
Authors:
Đorđević, MarijaStepper, Peter
Feuerstein-Akgoz, Clarissa
Gerhauser, Clarissa
Paunović, Verica
Tolić, Anja
Rajić, Jovana
Dinić, Svetlana
Uskoković, Aleksandra
Grdović, Nevena
Mihailović, Mirjana
Jurkowska, Renata
Jurkowski, Tomasz
Arambašić Jovanović, Jelena
Vidaković, Melita
Document Type:
Article (Published version)
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© 2023 Đorđević, Stepper, Feuerstein-Akgoz, Gerhauser, Paunović, Tolić, Rajić, Dinić, Uskoković, Grdović, Mihailović, Jurkowska, Jurkowski, Jovanović and Vidaković
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Show full item recordAbstract:
Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity.
Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells.
Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.
Keywords:
Arx gene; CRISPR/dCas9; diabetes; pancreatic alpha cells; epigenetic editing; targeted DNA methylationSource:
Frontiers in Endocrinology, Section - Diabetes: Molecular Mechanisms, 2023, 14, 1134478-Funding / projects:
- European Foundation for the Study of Diabetes (EFSD), European Diabetes Research Programme in Cellular Plasticity Underlying the Pathophysiology of Type 2 Diabetes, research grant from Astra Zeneca
- European Cooperation in Science and Technology (COST)
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200007 (University of Belgrade, Institute for Biological Research 'Siniša Stanković') (RS-MESTD-inst-2020-200007)
Related info:
- Referenced by
https://radar.ibiss.bg.ac.rs/handle/123456789/5669
DOI: 10.3389/fendo.2023.1134478
ISSN: 1664-2392
PubMed: 37008919