ORF14-05 - Targeting ARX to Turn Human Α-Cells into Insulin Producers

Background:
Diabetes results from an absolute or relative deficiency of insulin-producing β-cells, and strategies that restore endogenous β-cell mass are urgently needed. Pancreatic α-cells exhibit notable plasticity and can be reprogrammed toward insulin production. Current reprogramming approaches rely on viral overexpression of β-cell transcription factors and yield hybrid cells retaining α-cell identity. ARX is a key transcription factor maintaining α-cell fate. This study aimed to determine whether ARX deletion in primary α-cells facilitates the acquisition of β-identity.

Methods:
Human primary α-cells were isolated from four male donors’ islets by FACS and edited using Cas9 ribonucleoprotein complexes targeting ARX exons. Edited cells were reaggregated into pseudoislets for 7 days and analyzed by bulk RNA sequencing to assess transcriptional changes. To characterize chromatin remodeling and cellular heterogeneity, single-nucleus multiome profiling (snRNA-seq and snATAC-seq) was performed on edited and control reaggregated cells from one donor. Differential gene expression, chromatin accessibility, lineage signature enrichment, and transcription factor motif analyses were conducted to evaluate identity shifts following ARX deletion.

Results:
ARX was efficiently deleted across multiple donors, with an average transcript-level editing efficiency of about 90%. ARX deletion led to reduced expression of α-cell genes, including GCG, and induction of β- and δ-cell identity genes, such as PDX1 and HHEX. Gene set enrichment analysis showed attenuation of α-cell identity programs and activation of β- and δ-lineage signatures. In this line, snATAC-seq revealed coordinated chromatin remodeling, with decreased accessibility at α-enriched regulatory regions and increased accessibility at β- and δ-related loci. Notably, regions that became more accessible following ARX deletion were enriched in ARX DNA-binding motifs, suggesting that ARX normally represses these loci in α-cells. Newly accessible regions were enriched for motifs of key β-cell transcription factors, including PDX1, PAX4, and NKX6.1, consistent with derepression and priming of β-cell regulatory programs.

Conclusion:
CRISPR-Cas9-mediated deletion of ARX in human primary α-cells destabilizes α-cell identity and induces transcriptional and epigenomic changes consistent with acquisition of β- and δ-lineage traits. These findings establish ARX as a potential barrier to α-cell reprogramming and identify its targeting as a promising strategy to enhance the human α-cell conversion into insulin producers.