Introduction and Objective: β-cell dysfunction and loss drive type 2 diabetes (T2D) progression. Glucagon-like peptide-1 (GLP-1) receptor agonists improve β-cell function and health but are limited by discontinuation and glycemic rebound. We developed a single-dose adeno-associated virus gene therapy encoding human GLP-1 driven by an engineered insulin promoter (AAV.INS.GLP1) for durable, β-cell-specific, nutrient-responsive expression and secretion. We have previously shown that AAV.INS.GLP1 durably improves glycemia in db/db mice. Here, we evaluated its effects on β-cell function and health in db/db islets and human β-cells. Methods: db/db mice received a dose of AAV.INS.GLP1 (7.8E11, 2.6E12, or 5.6E12 vector genomes) or vehicle. After 6-7 weeks, pancreata underwent histological and dual-label insulin/GLP-1 immunofluorescence analyses. Islets were isolated to assess GLP-1 content, insulin content, and glucose-stimulated insulin (GSIS) and GLP-1 secretion. Cellular viability, reactive oxygen species (ROS), and endoplasmic reticulum (ER) stress biomarkers (XBP1, ATF4, ATF6, CHOP, BiP, ERN/IRE1) were evaluated in AAV.INS.GLP1-transduced human EndoC-βH5 cells. Results: AAV.INS.GLP1 drove β-cell-restricted GLP-1 expression and reduced islet cell hyperplasia. GLP-1 and insulin content in islets increased dose-dependently (up to 3.4- and 2.7-fold; P0.0001). GSIS in islets from high-dose-treated mice improved by 2.4- to 6.4-fold at 2.8 mM (P0.01), 16.7 mM (P0.01), and 16.7 mM glucose with 3-isobutyl-1-methylxanthine (P0.05). GLP-1 secretion was glucose-responsive, correlating with insulin secretion (r=0.66, P0.001). In human β-cells, AAV.INS.GLP1 maintained viability without increasing ROS or ER stress biomarkers. Conclusion: Single-dose, β-cell-specific, pancreatic gene therapy improves β-cell secretory function and reduces cellular stress, supporting its potential as a durable, disease-modifying therapy for T2D. Disclosure A.L. Fitzpatrick: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. L. Quek: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. C. Lubaczeuski: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. J. Sabadell-Basallote: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. S. Wang: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. R. Reese: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. J. Von Stetina: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. L.A. Schulman: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. K. Ishida: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. N. Picard: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. A. Alhamood: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. Z. Siddiquee: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. E. Horowitz: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. E. Cozzi: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. S. Slomovic: None. J. Caplan: Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. H. Rajagopalan: Board Member; Current; Fractyl Health, Inc. Employee; Current; Fractyl Health, Inc. Stock/Shareholder; Current; Fractyl Health, Inc. Funding Fractyl Health, Inc.
Fitzpatrick et al. (Sat,) studied this question.