Gene Editing for Environmentally-Responsive, Immune-Evasive Stem Cell-Derived Islets

About This Grant

PROJECT SUMMARY Despite the advances that have been made in the management type 1 diabetes (T1D), exogenous insulin administration with regular monitoring remains the primary treatment for T1D. Imprecise glucose control, which is commonplace, can lead to serious or even fatal complications. Consequently, the social and economic burden of T1D is substantial. Human pluripotent stem cell-derived (hSC) islet therapy has the potential to reduce these costs by providing a one-time, curative treatment that eliminates the need for continuous disease management. In response to NOSI NOT-AI-24-085, which calls for the development of somatic cell gene editing (SCGE) technologies to improve transplantation outcomes, we propose a multidisciplinary strategy to endow stem cell- derived islets (SC-islets) with graft context-based immune evasion, guided by real-time in vivo environment sensing and stimulus-responsive genetic rewiring. Our approach leverages prime editing, integrase-mediated DNA insertion, and machine learning-designed cis-regulatory elements (CREs) to reprogram endogenous loci to achieve these goals. We have assembled a multidisciplinary team, including leaders in T1D biology and stem cell models, genome engineering technologies, and computational genomics. In Aim 1, we will engineer molecular reporters in hiPSC lines to enable real-time, non-invasive monitoring of SC-islet graft inflammation, viability, and functional mass. We will leverage twin prime editing and optimized Bxb1 integrase technology to site-specifically integrate reporters quantifying β cell biomass, stress, apoptosis, and inflammation. We will validate these tools in vitro, and in vivo in humanized NSG MHC-DKO mice in the context of allorejection to establish a framework for high-resolution, longitudinal graft assessment in the context of inflammation and alloimmune attack. In Aim 2, we will rewire regulator elements within the endogenous CD47 locus to enable constitutive immunocloaking in SC-islets. We will design islet-specific synthetic enhancers using computational deep learning (Ledidi) and prime editing CRE interrogation approaches to achieve high levels of CD47 expression in β cells. These engineered SC-islets will be validated for protection in vitro against NK and T cell cytotoxicity, and in vivo in a PBMC-engrafted NSG MHC-DKO allorejection mouse model. In Aim 3, we will rewire the IL10 locus for inflammation-responsive immunosuppression. We will write cytokine-inducible regulatory elements into the regulatory logic of the endogenous IL10 locus to create inflammation adaptive SC-islets that secrete IL10 only in inflamed microenvironments, avoiding systemic effects. Functional assays will assess response kinetics and dynamic range of IL10 expression in response to inflammatory stimuli in vitro, and immunomodulatory capacity in vivo in alloresponse assays. Upon completion, this project will generate genetically engineered, immune-aware SC-islets with dynamic responsiveness to their environment, addressing a central challenge in β cell replacement therapy. If successful, these principles can be applied to other off-the- shelf cellular products to enhance safety, efficacy, and regulatory control of immunomodulation systems.