Abstract
Restoring insulin production through replacement of pancreatic β-cells presents a promising strategy for treating individuals with type 1 diabetes. However, current methods involving induced pluripotent stem cell differentiation are often time-consuming, multi-stage, and limited by safety and efficiency concerns. To overcome these challenges, we developed a simplified and direct strategy to convert human somatic cells into insulin-producing cells using an epigenetic activation system. This system combines a multiplex epigenetic engineering vector composed of dCas9.P300core and guide RNAs targeting five key β-cell genes: PDX1, NKX6.1, MAFA, Insulin, and glucose transporter type 2 (Glut2). The resulting Glut2⁺ cells exhibited glucose-responsive insulin secretion and expressed essential β-cell transcription factors including NKX2.2, along with insulin-processing and secretory machinery genes (Cav1.3, GSK3β, KCNJ11, SLC30A8). Absence of α-cell markers (aristaless-related homeobox or glucagon) confirmed lineage specificity and functional fidelity. This reprogramming approach eliminates the need for pluripotent intermediates and significantly reduces the time required to generate functional β-like cells. Our platform offers a rapid, non-integrative, and scalable method for producing insulin-secreting cells, with potential applications in personalized cell therapy, disease modeling, and high-throughput drug screening for diabetes research.