EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells

Dordevic, Marija, Stepper, Peter, Feuerstein-Akgoz, Clarissa, Gerhauser, Clarissa, Paunovic, Verica, Tolic, Anja, Rajic, Jovana, Dinic, Svetlana, Uskokovic, Aleksandra, Grdovic, Nevena, Mihailovic, Mirjana, Jurkowska, Renata Z. ORCID: https://orcid.org/0000-0002-4507-2222, Jurkowski, Tomasz P. ORCID: https://orcid.org/0000-0002-2012-0240, Jovanovic, Jelena Arambasic and Vidakovic, Melita 2023. EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells. Frontiers in Endocrinology 14 , 1134478. 10.3389/fendo.2023.1134478

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Abstract

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. 1 Introduction

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	Frontiers Media
ISSN:	1664-2392
Date of First Compliant Deposit:	16 March 2023
Date of Acceptance:	24 February 2023
Last Modified:	05 May 2023 15:14
URI:	https://orca.cardiff.ac.uk/id/eprint/157757

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