2425-P: GMP-Compatible Engineering of iPSC-Derived Islet-Like Clusters with an Improved Immune-Shielding/ Kill-Switch Combination for Type 1 Diabetes Cell Therapy

Introduction and Objective: Islet replacement provides a safe, effective T1D therapy without the shortcomings of insulin injections. However, treatment with allogeneic donor islets or non-modified stem cell-derived islets requires life-long immunosuppression, associated with significant side effects and impairing islet function. Here, we report on the generation of novel off-the-shelf iPSC-derived islet-like clusters (ILCs), genetically shielded from allorejection/autoimmunity, harboring a safety kill-switch, and performing like WT islets as next-generation T1D therapy. Methods: We used a targeted, GMP-compatible gene engineering strategy to introduce a drug-inducible, proliferation-dependent kill-switch and immune-shielding edits against T and NK cell rejection into a GMP-compatible iPSC line. After FACS-enrichment, engineered cells were subjected to automated clonal line generation. We validated iPSC clones for correct editing and genetic integrity prior to ILC differentiation in a scalable 3D process. Edit functionality was tested in drug-induced killing and primary immune cell co-culture in vitro assays. Results: Kill-switch and immune-shielded iPSC clones displayed normal growth rates and optimal pluripotency. They efficiently differentiated into ILCs showing activity equivalent to WT controls in C-peptide release assays. After drug induction, kill-switch iPSC but not non-proliferative ILCs were killed potently. Immune-shielded ILCs were efficiently protected against T and NK cell killing and did not trigger cytokine release with lymphocytes from multiple donors. Conclusion: We have established a GMP-compatible strategy for targeted engineering of kill-switch and immune-shielded iPSC clones with sustained genetic integrity, pluripotency and ILC differentiation capacity, and demonstrated edit functionality in vitro. Our results pave the way towards GMP manufacturing of next-generation genetically enhanced stem cell-derived therapeutics for T1D. Disclosure K. Ridders: Employee; Current; Evotec International GmbH. P. Hublitz: Employee; Current; Evotec International GmbH. C. Poussereau Pomie: Employee; Current; Evotec (France) SAS. E. Zanfrini: Employee; Current; Evotec International GmbH. T. Schneider: Employee; Current; Evotec International GmbH. L. Fillol-Bruguera: Employee; Current; Evotec International GmbH. D. Omarova: Employee; Current; Evotec International GmbH. A. Cossu: Employee; Current; Evotec International GmbH. I. Jugovic: Employee; Current; Evotec International GmbH. H.S. Pies: Employee; Current; Evotec International GmbH. L. Kallenbach: Employee; Current; Evotec International GmbH. N. Duran Jacobs: Employee; Current; Evotec International GmbH. D. Hartung: Employee; Current; Evotec International GmbH. K. Pingris: Employee; Current; Evotec (France) SAS. F. Hermann: Employee; Current; Evotec International GmbH. M. Austen: Employee; Current; Evotec International GmbH. Stock/Shareholder; Current; Evotec International GmbH.